U.S. patent number 10,385,359 [Application Number 15/410,252] was granted by the patent office on 2019-08-20 for targeted modification of rat genome.
This patent grant is currently assigned to Regeneron Pharmaceuticals, Inc.. The grantee listed for this patent is Regeneron Pharmaceuticals, Inc.. Invention is credited to Wojtek Auerbach, Ka-Man Venus Lai, Jeffrey D. Lee, Alexander O. Mujica, David M. Valenzuela, George D. Yancopoulos.
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United States Patent |
10,385,359 |
Lee , et al. |
August 20, 2019 |
Targeted modification of rat genome
Abstract
Compositions and methods are provided for modifying a rat
genomic locus of interest using a large targeting vector (LTVEC)
comprising various endogenous or exogenous nucleic acid sequences
as described herein. Compositions and methods for generating a
genetically modified rat comprising one or more targeted genetic
modifications in their germline are also provided. Compositions and
methods are provided which comprise a genetically modified rat or
rat cell comprising a targeted genetic modification in the rat
interleukin-2 receptor gamma locus, the rat ApoE locus, the rat
Rag2 locus, the rat Rag1 locus and/or the rat Rag2/Rag1 locus. The
various methods and compositions provided herein allows for these
modified loci to be transmitted through the germline.
Inventors: |
Lee; Jeffrey D. (New York,
NY), Mujica; Alexander O. (Elmsford, NY), Auerbach;
Wojtek (Ridgewood, NJ), Lai; Ka-Man Venus (Tarrytown,
NY), Valenzuela; David M. (Yorktown Heights, NY),
Yancopoulos; George D. (Yorktown Heights, NY) |
Applicant: |
Name |
City |
State |
Country |
Type |
Regeneron Pharmaceuticals, Inc. |
Tarrytown |
NY |
US |
|
|
Assignee: |
Regeneron Pharmaceuticals, Inc.
(Tarrytown, NY)
|
Family
ID: |
51687240 |
Appl.
No.: |
15/410,252 |
Filed: |
January 19, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
|
US 20170204430 A1 |
Jul 20, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14254715 |
Apr 16, 2014 |
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61812319 |
Apr 16, 2013 |
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61914768 |
Dec 11, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/907 (20130101); C12N 15/8509 (20130101); A61D
19/04 (20130101); C07K 14/775 (20130101); A01K
67/0276 (20130101); A01K 67/0278 (20130101); C07K
14/7155 (20130101); A01K 2217/07 (20130101); A01K
2267/0362 (20130101); A01K 2227/105 (20130101); C12N
2800/30 (20130101); C12N 2015/8527 (20130101); C12N
2810/00 (20130101); A01K 2267/0381 (20130101) |
Current International
Class: |
C12N
15/85 (20060101); C07K 14/715 (20060101); C12N
15/90 (20060101); C07K 14/775 (20060101); A01K
67/027 (20060101); A61D 19/04 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
1 726 640 |
|
Nov 2005 |
|
EP |
|
2336329 |
|
Jun 2011 |
|
EP |
|
2152880 |
|
Aug 2011 |
|
EP |
|
1360287 |
|
Sep 2012 |
|
EP |
|
2508595 |
|
Oct 2012 |
|
EP |
|
2602323 |
|
Jun 2013 |
|
EP |
|
3009511 |
|
Apr 2016 |
|
EP |
|
3064585 |
|
Sep 2016 |
|
EP |
|
2 436 737 |
|
Oct 2007 |
|
GB |
|
WO 97/30151 |
|
Aug 1997 |
|
WO |
|
WO 1997/030151 |
|
Aug 1997 |
|
WO |
|
WO 2002/036789 |
|
May 2002 |
|
WO |
|
WO 2002/066630 |
|
Aug 2002 |
|
WO |
|
WO 2003/087341 |
|
Oct 2003 |
|
WO |
|
WO 2006/028723 |
|
Mar 2006 |
|
WO |
|
WO 2006/044962 |
|
Apr 2006 |
|
WO |
|
WO 2007/113505 |
|
Oct 2007 |
|
WO |
|
WO 2007/117410 |
|
Oct 2007 |
|
WO |
|
WO 2008/015418 |
|
Feb 2008 |
|
WO |
|
WO 2008/151081 |
|
Dec 2008 |
|
WO |
|
WO 2010/077955 |
|
Jul 2010 |
|
WO |
|
WO 2011/020005 |
|
Feb 2011 |
|
WO |
|
WO 2011/051390 |
|
May 2011 |
|
WO |
|
WO 2011/078665 |
|
Jun 2011 |
|
WO |
|
WO 2011/146121 |
|
Nov 2011 |
|
WO |
|
WO 2011/154927 |
|
Dec 2011 |
|
WO |
|
WO 2012/012667 |
|
Jan 2012 |
|
WO |
|
WO 2012/018726 |
|
Sep 2012 |
|
WO |
|
WO 2012/129198 |
|
Sep 2012 |
|
WO |
|
WO 2012/168307 |
|
Dec 2012 |
|
WO |
|
WO 2013/063361 |
|
May 2013 |
|
WO |
|
WO 2013/141680 |
|
Sep 2013 |
|
WO |
|
WO 2013/142578 |
|
Sep 2013 |
|
WO |
|
WO 2013/163394 |
|
Oct 2013 |
|
WO |
|
WO 2013/169802 |
|
Nov 2013 |
|
WO |
|
WO 2013/176772 |
|
Nov 2013 |
|
WO |
|
WO 2013/188522 |
|
Dec 2013 |
|
WO |
|
WO 2014/018423 |
|
Jan 2014 |
|
WO |
|
WO 2014/065596 |
|
May 2014 |
|
WO |
|
WO 2014/089290 |
|
Jun 2014 |
|
WO |
|
WO 2014/093479 |
|
Jun 2014 |
|
WO |
|
WO 2014/093595 |
|
Jun 2014 |
|
WO |
|
WO 2014/093622 |
|
Jun 2014 |
|
WO |
|
WO 2014/093635 |
|
Jun 2014 |
|
WO |
|
WO 2014/093655 |
|
Jun 2014 |
|
WO |
|
WO 2014/093661 |
|
Jun 2014 |
|
WO |
|
WO 2014/093694 |
|
Jun 2014 |
|
WO |
|
WO 2014/093701 |
|
Jun 2014 |
|
WO |
|
WO 2014/093709 |
|
Jun 2014 |
|
WO |
|
WO 2014/093712 |
|
Jun 2014 |
|
WO |
|
WO 2014/093718 |
|
Jun 2014 |
|
WO |
|
WO 2014/099744 |
|
Jun 2014 |
|
WO |
|
WO 2014/099750 |
|
Jun 2014 |
|
WO |
|
WO 2014/104878 |
|
Jul 2014 |
|
WO |
|
WO 2014/127287 |
|
Aug 2014 |
|
WO |
|
WO 2014/130706 |
|
Aug 2014 |
|
WO |
|
WO 2014/130955 |
|
Aug 2014 |
|
WO |
|
WO 2014/131833 |
|
Sep 2014 |
|
WO |
|
WO 2014/143381 |
|
Sep 2014 |
|
WO |
|
WO 2014/150624 |
|
Sep 2014 |
|
WO |
|
WO 2014/165825 |
|
Oct 2014 |
|
WO |
|
WO 2014/172458 |
|
Oct 2014 |
|
WO |
|
WO 2014/172470 |
|
Oct 2014 |
|
WO |
|
WO 2014/172489 |
|
Oct 2014 |
|
WO |
|
WO 2014/173955 |
|
Oct 2014 |
|
WO |
|
WO 2014/182700 |
|
Nov 2014 |
|
WO |
|
WO 2014/186585 |
|
Nov 2014 |
|
WO |
|
WO 2014/191128 |
|
Dec 2014 |
|
WO |
|
WO 2014/191518 |
|
Dec 2014 |
|
WO |
|
WO 2014/201015 |
|
Dec 2014 |
|
WO |
|
WO 2014/204723 |
|
Dec 2014 |
|
WO |
|
WO 2014/204724 |
|
Dec 2014 |
|
WO |
|
WO 2014/204725 |
|
Dec 2014 |
|
WO |
|
WO 2014/204726 |
|
Dec 2014 |
|
WO |
|
WO 2014/204728 |
|
Dec 2014 |
|
WO |
|
WO 2014/204729 |
|
Dec 2014 |
|
WO |
|
WO 2014/205192 |
|
Dec 2014 |
|
WO |
|
WO 2015/006290 |
|
Jan 2015 |
|
WO |
|
WO 2015/006294 |
|
Jan 2015 |
|
WO |
|
WO 2015/006498 |
|
Jan 2015 |
|
WO |
|
WO 2015/010114 |
|
Jan 2015 |
|
WO |
|
WO 2015/013583 |
|
Jan 2015 |
|
WO |
|
WO 2015/026885 |
|
Feb 2015 |
|
WO |
|
WO 2015/035139 |
|
Mar 2015 |
|
WO |
|
WO 2015/035162 |
|
Mar 2015 |
|
WO |
|
WO 2015/040402 |
|
Mar 2015 |
|
WO |
|
WO 2015/048690 |
|
Apr 2015 |
|
WO |
|
WO 2015/052231 |
|
Apr 2015 |
|
WO |
|
WO 2015/057980 |
|
Apr 2015 |
|
WO |
|
WO 2015/070083 |
|
May 2015 |
|
WO |
|
WO 2015/077290 |
|
May 2015 |
|
WO |
|
WO 2015/079057 |
|
Jun 2015 |
|
WO |
|
WO 2015/086798 |
|
Jun 2015 |
|
WO |
|
WO 2015/088643 |
|
Jun 2015 |
|
WO |
|
WO 2015/089351 |
|
Jun 2015 |
|
WO |
|
WO 2015/089427 |
|
Jun 2015 |
|
WO |
|
WO 2015/089462 |
|
Jun 2015 |
|
WO |
|
WO 2015/089465 |
|
Jun 2015 |
|
WO |
|
WO 2015/089473 |
|
Jun 2015 |
|
WO |
|
WO 2015/095804 |
|
Jun 2015 |
|
WO |
|
WO 2015/105928 |
|
Jul 2015 |
|
WO |
|
WO 2015/112790 |
|
Jul 2015 |
|
WO |
|
WO 2015/116969 |
|
Aug 2015 |
|
WO |
|
WO 2015/117041 |
|
Aug 2015 |
|
WO |
|
WO 2015/123339 |
|
Aug 2015 |
|
WO |
|
WO 2015/127439 |
|
Aug 2015 |
|
WO |
|
WO 2015/134812 |
|
Sep 2015 |
|
WO |
|
WO 2015/138510 |
|
Sep 2015 |
|
WO |
|
WO 2015/138739 |
|
Sep 2015 |
|
WO |
|
WO 2015/139008 |
|
Sep 2015 |
|
WO |
|
WO 2015/143046 |
|
Sep 2015 |
|
WO |
|
WO 2015/148761 |
|
Oct 2015 |
|
WO |
|
WO 2015/159086 |
|
Oct 2015 |
|
WO |
|
WO 2015/163733 |
|
Oct 2015 |
|
WO |
|
WO 2015/166272 |
|
Nov 2015 |
|
WO |
|
WO 2015/173436 |
|
Nov 2015 |
|
WO |
|
WO 2015/184259 |
|
Dec 2015 |
|
WO |
|
WO 2015/184262 |
|
Dec 2015 |
|
WO |
|
WO 2015/184268 |
|
Dec 2015 |
|
WO |
|
WO 2015/188109 |
|
Dec 2015 |
|
WO |
|
WO 2015/200805 |
|
Dec 2015 |
|
WO |
|
WO 2016/011210 |
|
Jan 2016 |
|
WO |
|
WO 2016/011428 |
|
Jan 2016 |
|
WO |
|
WO 2016/033246 |
|
Mar 2016 |
|
WO |
|
WO 2016/033315 |
|
Mar 2016 |
|
WO |
|
WO 2016/036754 |
|
Mar 2016 |
|
WO |
|
WO 2016/037161 |
|
Mar 2016 |
|
WO |
|
WO 2016/037162 |
|
Mar 2016 |
|
WO |
|
WO 2016/049024 |
|
Mar 2016 |
|
WO |
|
WO 2016/049163 |
|
Mar 2016 |
|
WO |
|
WO 2016/049258 |
|
Mar 2016 |
|
WO |
|
WO 2016/054032 |
|
Apr 2016 |
|
WO |
|
WO 2016/054326 |
|
Apr 2016 |
|
WO |
|
WO 2016/057821 |
|
Apr 2016 |
|
WO |
|
WO 2016/057961 |
|
Apr 2016 |
|
WO |
|
WO 2016/061073 |
|
Apr 2016 |
|
WO |
|
WO 2016/061374 |
|
Apr 2016 |
|
WO |
|
WO 2016/061481 |
|
Apr 2016 |
|
WO |
|
WO 2016/061523 |
|
Apr 2016 |
|
WO |
|
WO 2016/065364 |
|
Apr 2016 |
|
WO |
|
WO 2016/073955 |
|
May 2016 |
|
WO |
|
WO 2016/073990 |
|
May 2016 |
|
WO |
|
WO 2016/081923 |
|
May 2016 |
|
WO |
|
WO 2016/084084 |
|
Jun 2016 |
|
WO |
|
WO 2016/089866 |
|
Jun 2016 |
|
WO |
|
WO 2016/094872 |
|
Jun 2016 |
|
WO |
|
WO 2016/094874 |
|
Jun 2016 |
|
WO |
|
WO 2016/094880 |
|
Jun 2016 |
|
WO |
|
WO 2016/097751 |
|
Jun 2016 |
|
WO |
|
WO 2016/100819 |
|
Jun 2016 |
|
WO |
|
WO 2016/106338 |
|
Jun 2016 |
|
WO |
|
WO 2016/108926 |
|
Jul 2016 |
|
WO |
|
WO 2016/112242 |
|
Jul 2016 |
|
WO |
|
WO 2016/112351 |
|
Jul 2016 |
|
WO |
|
WO 2016/114972 |
|
Jul 2016 |
|
WO |
|
WO 2016/115326 |
|
Jul 2016 |
|
WO |
|
WO 2016/130697 |
|
Aug 2016 |
|
WO |
|
WO 2016/135557 |
|
Sep 2016 |
|
WO |
|
WO 2016/135558 |
|
Sep 2016 |
|
WO |
|
WO 2016/135559 |
|
Sep 2016 |
|
WO |
|
WO 2016/142719 |
|
Sep 2016 |
|
WO |
|
WO 2016/154579 |
|
Sep 2016 |
|
WO |
|
WO 2016/138574 |
|
Apr 2017 |
|
WO |
|
Other References
Valenzuela et al., Nature Biotechnology 23(6): 652-659, 2003. cited
by examiner .
Zhang et al., "The CRISPR/Cas9 system produces specific and
homozygous targeted gene editing in rice in one generation," Plant
Biotechnol. J., vol. 12(6), pp. 797-807, May 23, 2014. cited by
applicant .
"Stem Cells: Scientific Progress and Future Research Directions,"
National Institute of Health, Department of Health and Human
Services, (2001). cited by applicant .
Abudayyeh et al., "C2c2 is a single-component programmable
RNA-guided RNA-targeting CRISPR effector," Science, vol. 353(6299),
Jun. 2, 2016. cited by applicant .
Aitman et al., "Progress and prospects in rat genetics: a community
view," Nature Genetics, vol. 40(5), pp. 516-522, May 2008. cited by
applicant .
Auerbach et al., "Establishment and chimera analysis of 129/SvEv-
and C57BL/6-derived mouse embryonic stem cell lines,"
Biotechniques, vol. 29(5), pp. 1024-1028, 1030, 1032, Nov. 2000.
cited by applicant .
Baker, M., "Gene editing at CRISPR speed," Nature Biotechnology
(2014), vol. 32(4), p. 309-312. cited by applicant .
Barrangou, "RNA-mediated programmable DNA cleavage," Nature
Biotechnology, 2012, vol. 30(9), pp. 836-838. cited by applicant
.
Bassett, A.R., et al., "CRISPR/Cas9 and Genome Editing in
Drosophila," Journal of Genetics and Genomics (2014), vol. 41, pp.
7-19. cited by applicant .
Benders et al., "Cloning whole bacterial genomes in yeast," Nucleic
Acids Res., vol. 38(8), pp. 2558-2569, Mar. 7, 2010. cited by
applicant .
Berdien, et al., "TALEN-mediated editing of endogenous T-cell
receptors facilitates efficient reprogramming of T lymphocytes by
lentiviral gene transfer," Gene Therapy, 21, 539-548 (2014). cited
by applicant .
Bernardini et al., "Site-specific genetic engineering of the
Anopheles gambiae Y chromosome," Proc. Natl. Acad. Sci. USA, vol.
111(21), pp. 7600-7605, May 12, 2014. cited by applicant .
Beumer et al., "Donor DNA Utilization During Gene Targeting with
Zinc-Finger Nucleases," Genes Genomes Genetics, vol. 3, pp.
657-664, Apr. 2013. cited by applicant .
Blair et al., "Culture parameters for stable expansion, genetic
modification and germline transmission of rat pluripotent stem
cells," Biol. Open, vol. 1(1): pp. 58-65, Nov. 1, 2011. cited by
applicant .
Brouns, S.J.J., "A Swiss Army Knife of Immunity," Science (2012),
vol. 337, pp. 808-809. cited by applicant .
Buehr et al., "Capture of Authentic Embryonic Stem Cells from Rat
Blastocysts," Cell, vol. 135, pp. 1287-1298, 2008. cited by
applicant .
Buehr, M., et al., "Capture of Authentic Embryonic Stem Cells from
Rat Blastocysts," Cell, 2008, vol. 135(7), pp. 1287-1298. cited by
applicant .
Byrne et al., "Multi-kilobase homozygous targeted gene replacement
in human induced pluripotent stem cells," Nucleic Acids Research,
Vo. 43(3), p. e21, 2014 (epub Nov. 20, 2014). cited by applicant
.
Carlson, et al., "Targeting DNA with Fingers and TALENs," Molecular
Therapy-Nucleic Acids, 2012, 1:e3. cited by applicant .
Carroll, "Staying on target with CRISPR-Cas," Nature Biotechnology,
2013, vol. 31(9), pp. 807-809. cited by applicant .
Carroll, "Zinc-Finger Nucleases as Gene Therapy Agents", Gene
Ther., Nov. 15, 2008:(22):1463-1468. cited by applicant .
Cartwright et al., "LIF/STAT3 controls ES cell self-renewal and
pluripotency by a Myc-dependent mechanism," Development, vol.
132(5), pp. 885-896, 2005. cited by applicant .
Casanova et al., "Cross-Species Genome Wide Expression Analysis
during Pluripotent Cell Determination in Mouse and Rat
Preimplantation Embryos," PLoS One, vol. 7(10), e47107, 2012. cited
by applicant .
Cathomen, et al., "Zinc-finger nucleases: the next generation
emerges," Molecular Therapy, 208, vol. 16(7), pp. 1200-1207 (Jul.
2008). cited by applicant .
Chang et al., "Genome editing with RNA-guided Cas9 nuclease in
Zebrafish embryos," Cell Research, vol. 23, pp. 465-472, 2013.
(published Mar. 2013). cited by applicant .
Chari et al., "Unraveling CRISPR-Cas9 genome engineering parameters
via a library-on-library approach," Nature Methods, vol. 12(9):
823-826 plus Supplementary Figures, Jul. 13, 2015. cited by
applicant .
Chen et al., "High-frequency genome editing using ssDNA
oligonucleotides with zinc-finger nucleases," Nature Methods, vol.
8(9), pp. 753-755, 2011. (Jul. 17, 2011). cited by applicant .
Chen et al., "Highly Efficient Mouse Genome Editing by CRISPR
Ribonucleoprotein Electroporation of Zygoes," J. Biol. Chem., May
5, 2016. cited by applicant .
Cho, et al., "Targeting genome engineering in human cells with the
Cas9 RNA-guided endonuclease," Nature Biotechnology, 2013, vol.
31(3):233-239. cited by applicant .
Choulika et al., "Induction of Homologous Recombination in
Mammalian Chromosomes by Using the I-Scel System of Saccharomyces
cerevisiae," Mol. Cell. Biol., vol. 15(4), pp. 1968-1973, 1995.
cited by applicant .
Christian M., et al., "Targeting DNA Double-Strand Breaks with TAL
Effector Nucleases," Genetics (2010), vol. 186, pp. 757-761. cited
by applicant .
Christian, et al., "Targeting DNA double-strand breaks with TAL
effector nucleases," Genetics, 2010, vol. 186, pp. 757-761. cited
by applicant .
Chu et al., "Increasing the efficiency of homology-directed repair
for CRISPR-Cas9-induced precise gene editing in mammalian cells,"
Nature Biotech., vol. 33(5), pp. 543-548, 2015. (published Mar.
2015). cited by applicant .
Cobb and Zhao, "Direct cloning of large genomic sequences," Nature
Biotechnology, 2012, vol. 30(5), pp. 405-406. cited by applicant
.
Cong et al., "Multiplex Genome Engineering Using CRISPR/Cas
Systems," Science, vol. 339(6121), pp. 819-823 plus Supplemental
Materials, Jan. 3, 2013. cited by applicant .
Cong, et al., "Multiplex genome engineering using CRISPR/Cas
systems," Science, 2013, vol. 339(6121), pp. 819-823. cited by
applicant .
Cui, et al., "Targeted integration in rat and mouse embryos with
zinc-finger nucleases," Nature Biotechnology, vol. 29, No. 1, 64-68
(Dec. 12, 2010). cited by applicant .
DeChiara, T.M., et al., "VelociMouse: Fully ES Cell-Derived
FO-Generation Mice Obtained from the Injection of ES Cells into
Eight-Cell-Stage Embryos," Jan. 1, 2009, Methods in Molecular
Biology, 530(16): 311-324. cited by applicant .
Ding et al., "Enhanced Efficiency of Human Pluripotent Stem Cell
Genome Editing through Replacing TALENs with CRISPRs," Cell Stem
Cell, vol. 12, pp. 393-394 plus supplemental materials, 2013 (Apr.
4, 2013). cited by applicant .
Ding, et al., "A TALEN genome-editing system for generating human
stem cell-based disease models," Cell Stem Cell, 2013, vol. 12, pp.
238-251. cited by applicant .
Donoho et al., "Analysis of Gene Targeting and Intrachromosomal
Homologous Recombination Stimulated by Genomic Double-Strand Breaks
in Mouse Embryonic Stem Cells," Mol. Cell. Biol., vol. 18(7), pp.
4070-4078, 1998. cited by applicant .
Doudna et al., "The new frontier of geome engineering with
CRISPR-Cas9," Science, vol. 346(6213), pp. 1258096-1-1258096-9,
Nov. 28, 2014. cited by applicant .
EMD-Millipore, Certificate of Analysis for Recombinant Human
Leukemia Inhibitory Factor, retrieved from internet on Apr. 25,
2015 at
<http://www.emdmillipore.com/US/en/product/Leukemia-Inhibitory-Factor--
Protein%2C-Recombinant-human,MM_NF-LIF1010#documen. cited by
applicant .
EMD-Millipore, "Product Information sheet for Rat ESGRO," retrieved
from internet on Apr. 25, 2015 at <
http://www.emdmillipore.com/US/en/product/Rat-ESGRO%C2%AE%2C-1-million-un-
its1-ml,MM_NF-ESG2206#anchor_COA>. cited by applicant .
EP Application No. 14754746.7, Extended European Search Report
dated Jun. 13, 2016. cited by applicant .
EP Application No. 14784879.0, Extended European Search Report
dated Sep. 19, 2016. cited by applicant .
Evers et al, "CRISPR knockout screening outperforms shRNA and
CRISPRi in identifying essential genes," Nature Biotechnology, vol.
24(6), pp. 631-633, Apr. 25, 2016. cited by applicant .
Fan et al, "107 Genetic Inactivation of the Sry Gene in Argali Wild
and Romney Domestic Sheep with CRISPR/Cas Systems for Producing
Sex-Reversed Female Animals," Reproduction Fertility and
Development, vol. 26(1), p. 167, Dec. 5, 2013. cited by applicant
.
Flisikowska, et al., "Efficient Immunoglobulin Gene Disruption and
Targeted Replacement in Rabbit Using Zinc Finger Nucleases," Plos
one, vol. 6 Issue 6 (Jun. 2011). cited by applicant .
Frendewey, "VelociGene: Large-scale Modification of Rodent
Genomes," Wellcome Trust Advanced Course: Genetic Engineering of
Mammalian Stem Cells, Mar. 13, 2014. cited by applicant .
Frendewey, "VelociGene: Large-scale Modification of Rodent
Genomes," Wellcome Trust Advanced Course: Genetic Engineering of
Mammalian Stem Cells, Feb. 20, 2015. cited by applicant .
Frokjaer-Jensen C., "Exciting Prospects for Precise Engineering of
Caenorhabditis elegans Genomes with CRISPR/Cas9," Genetics (2013),
vol. 195, pp. 635-642. cited by applicant .
Fu, et al., "High-frequency off-target mutagenesis induced by
CRISPR-Cas nucleases in human cells," Nature Biotechnology, 2013,
vol. 31(9), pp. 822-826. cited by applicant .
Fujii et al., "Efficient generation of genome-modified mice via
offset-nicking by CRISPR/Cas system," Biochemical and Biophysical
Research Communications, vol. 445(4), pp. 791-794 plus
Supplementary Information, Jan. 31, 2014. cited by applicant .
Fujii W., et al., "Efficient generation of large-scale
genome-modified mice using gRNA and CAS9 endonuclease," Nucleic
Acids Research (2013), vol. 41(20), p. e187. cited by applicant
.
Gaj et al., "ZFN, TALEN, and CRISPR/Cas-based methods for genome
engineering," Trends Biotech., vol. 31(7), pp. 397-405, 2013. cited
by applicant .
Gao et al., "DNA-guided genome editing using the Natronobacterium
gregoryi Argonuate," Nature Biotechnology, vol. 34(7), pp. 768-773,
May 2, 2016. cited by applicant .
Garg, A., et al., "Engineering synthetic TAL effectors with
orthogonal target sites," Nucleic Acids Research (2012), vol.
40(15), p. 7584-7595. cited by applicant .
Gasiunas, G., et al., "Cas9-crRNA ribonucleoprotein complex
mediates specific DNA cleavage for adaptive immunity in bacteria,"
PNAS, 2012, vol. 108, pp. 10098-10103. cited by applicant .
Gennequin, et al., "CRISPR/Cas-induced double-strand breaks boost
the frequency of gene replacements for humanizing the mouse Cnr2
gene," Biochem. Biophys. Res. Commun., (2013),
http://dx.doi.org/10.1016/j.bbrc.2013.10.138. cited by applicant
.
Gibco Data Sheet for "Recombinant Mouse Leukemia Inhibitory Factor
(LIF), PMC4054," pp. 1-2, 2011. cited by applicant .
Gibson, D., et al., "Enzymatic assembly of DNA molecules up to
several hundred kilobases," Nature Methods, 2009, vol. 6(5), pp.
343-345. cited by applicant .
Gibson, Daniel G., "Enzymatic Assembly of Overlapping DNA
Fragments," Methods in Enzymology, 2011, vol. 498, pp. 349-361.
cited by applicant .
Graf et al., "The Role of the Leukemia Inhibitory Factor
(LIF)--Pathway in Derivation and Maintenance of Murine Pluripotent
Stem Cells," Genes, vol. 2, pp. 280-297, 2011. cited by applicant
.
Gratz et al., "Genome Engineering of Drosophila with the CRISPR
RNA-Guided Cas9 Nuclease," Genetics, vol. 194, pp. 1029-1035, 2013.
(published May 2013). cited by applicant .
Gratz et al., "Highly Specific and Efficient CRISPR/Cas9-Catalyzed
Homology-Directed Repair in Drosophila," Genetics, vol. 196(4), pp.
961-971 plus Supporting Information, Jan. 29, 2014. cited by
applicant .
Harrison, M.M., et al., "A CRISPR view of development," Genes &
Development (2014), vol. 28(17), pp. 1859-1872. cited by applicant
.
Hirabayashi et al., "Establishment of Rat Embryonic Stem Cell Lines
That Can Participate in Germline Chimerae at High Efficiency," Mol.
Reprod. Dev., vol. 77, p. 94, 2010. cited by applicant .
Hirano et al., "Human and Mouse Induced Pluripotent Stem Cells Are
Differentially Reprogrammed in Response to Kinase Inhibitors," Stem
Cells and Development, vol. 21(8), pp. 1287-1298, May 20, 2012.
cited by applicant .
Horvath, P., et al., "CRISPR/Cas, the Immune System of Bacteria and
Archaea," Science, 2010, vol. 327, pp. 167-170. cited by applicant
.
Hsu, et al., "DNA targeting specificity of RNA-guided Cas9
nucleases," Nature Biotechnology, 2013, vol. 31(9), pp. 827-832.
cited by applicant .
Hsu, P.D., et al., "Development and Applications of CRISPR-Cas9 for
Genome Engineering," Cell (2014), vol. 157, pp. 1262-1278. cited by
applicant .
Hwang et al., "Efficient In Vivo Genome Editing Using RNA-Guided
Nucleases," Nat. Biotechnol., vol. 31(3), pp. 227-229 (plus
supplemental materials), 2013. cited by applicant .
Hwang, W.Y., et al., "Efficient In Vivo Genome Editing Using
RNA-Guided Nucleases," Nature Biotechnology (2013), vol. 31(3), pp.
227-229. cited by applicant .
Iannaccone, P., et al., "Pluripotent Embryonic Stem Cells from the
Rat Are Capable of Producing Chimeras," Developmental Biology,
1994, vol. 163, pp. 288-292. cited by applicant .
Jallepalli et al., "Securin is required for chromosomal stability
in human cells," Cell, vol. 105(4), pp. 445-457, May 18, 2001.
cited by applicant .
Jao et al., "Efficient multiplex biallelic zebrafish genome editing
using a CRISPR nuclease system," Proc. Natl. Acad. Sci. U.S.A.,
vol. 110(34), pp. 13904-13909 plus Supporting Information, Aug. 5,
2013. cited by applicant .
Jasin, et al., "Repair of Strand Breaks by Homologous
Recombination," Cold Spring Harb. Perspect. Biol., vol. 5(11), p.
a012740, Nov. 1, 2013. cited by applicant .
Jiang, et al., "RNA-guided editing of bacterial genomes using
CRISPR-Cas systems," Nature Biotechnology, 2013, vol. 31(3), pp.
233-239. cited by applicant .
Jinek et al., "A Programmable Dual-RNA-Guided DNA Endonuclease in
Adaptive Bacterial Immunity," Science, vol. 337, pp. 816-821 plus
Supplemental Materials, Jun. 28, 2012. cited by applicant .
Jinek, M., et al., "A Programmable Dual-RNA-Guided DNA Endonuclease
in Adaptive Bacterial Immunity," Science, 2012, vol. 337, pp.
816-821. cited by applicant .
Johnson, et al., "A rapid assay to quantify the cleavage efficiency
of custom-designed nucleases in planta," Plant Mol Biol, 82:207-221
(2013). cited by applicant .
Kashimada et al., "Sry: the master switch in mammalian sex
determination," Development, vol. 137(23), pp. 3921-3930, Dec. 2,
2010. cited by applicant .
Kato et al., "Production of Sry knockout mouse using TALEN via
oocyte injection," Scientific Reports, vol. 3, p. 3136, 2013
(published Nov. 5, 2013). cited by applicant .
Kawamata et al., "Two distinct knockout approaches highlight a
critical role for p53 in rat development," Sci. Rep., vol. 2, p.
945, 2012. cited by applicant .
Kawamata, et al., "Generation of genetically modified rats from
embryonic stem cells," PNAS, vol. 107, No. 32, pp. 14223-14228
(Aug. 10, 2010). cited by applicant .
Kawamata, M., et al., "Establishment of Embryonic Stem Cells from
Rat Blastocysts," Methods Mol. Biol., 2010, vol. 597, pp. 169-177.
cited by applicant .
Kawamata, M., et al., "Generation of genetically modified rats from
embryonic stem cells," PNAS, 2010, vol. 107(32), pp. 14223-14228.
cited by applicant .
Kim et al., "Targeted genome editing in human cells with zinc
finger nucleases constructed via modular assembly," Genome Res.,
vol. 19(7), pp. 1279-1288, 2009. cited by applicant .
Kobayashi et al., "Identification of Rat Rosa26 Locus Enables
Generation of Knock-In Rat Lines Ubiquitously Expressing tdTomato,"
Stem Cells and Development, vol. 21(16), pp. 2981-2986, May 7,
2012. cited by applicant .
Komor et al, "Programmable editing of a target base in genomic DNA
without double-stranded cleavage," Nature, vol. 533(7603), pp.
420-424, Apr. 20, 2016. cited by applicant .
Kondo, et al., "Highly Improved Gene Targeting by Germline-Specific
Cas9 Expression in Drosophila," Genetics, vol. 195, pp. 715-721,
Sep. 3, 2013. cited by applicant .
Konermann et al., "Genome-scale transcriptional activation by an
engineered CRISPR-Cas9 complex," Nature, vol. 517(7536), pp.
583-588, published online Dec. 10, 2014. cited by applicant .
Krivokharchenko, A., et al., "In Vitro Formation of Tetraploid Rat
Blastocysts After Fusion of Two-Cell Embryos," Molecular
Reproduction and Development, 2002, vol. 61, pp. 460-465. cited by
applicant .
Kuijpers et al., "One-step assembly and targeted integration of
multigene constructs assisted by the I-Scel meganuclease in
Saccharomyces cerevisiae," FEMS Yeast Res., vol. 13(8), pp.
769-781, Oct. 7, 2013. cited by applicant .
Kuno et al., "Generation of fertile and fecund F0 XY female mice
from XY ES cells," Transgenic Research, vol. 24(1), pp. 19-29, 2014
(epub Aug. 3, 2014). cited by applicant .
Kuroiwa, et al., "Sequential targeting of the genes encoding
immunoglobulin-.mu. and prion protein in cattle," Nature Genetics,
vol. 36, No. 7, (Jul. 2004). cited by applicant .
Li et al., "Derivation of Germline Competent Rat Embryonic Stem
Cells from DA Rats," J. Genet. Genomics, vol. 39, pp. 603-606,
2012. cited by applicant .
Li et al., "Germline Competent Embryonic Stem Cells Derived from
Rat Blastocysts," Cell, vol. 135, pp. 1299-1310, 2008. cited by
applicant .
Li et al., "Genetic modification and screening in rat using haploid
embryonic stem cells," Cell Stem Cell, vol. 14(3): pp. 404-414,
2013 (epub Dec. 19, 2013). cited by applicant .
Li et al., "Optimization of Genome Engineering Approaches with the
CRISPR/Cas9 System," PLoS One, vol. 9(8), p. e105779, Aug. 28,
2014. cited by applicant .
Li, D., et al., "Heritable gene targeting in the mouse and rat
using a CRISPR-Cas system," Nature Biotechnology, 2013, vol. 31(8),
pp. 681-683. cited by applicant .
Li, et al., "Modularly assembled designer TAL effector nucleases
for targeted gene knockout and gene replacement in eukaryotes,"
Nucleic Acids Research, 2011, vol. 39(14), pp. 6315-6325. cited by
applicant .
Li, M., et al., "A Cut above the Rest: Targeted Genome Editing
Technologies in Human Pluripotent Stem Cells," Journal of
Biological Chemistry (2014), vol. 289(8), pp. 4594-4599. cited by
applicant .
Li, P., et al., "Germline competent embryonic stem cells derived
from rat blastocysts," Cell, 2008, vol. 135(7), pp. 1299-1310.
cited by applicant .
Li, T., et al., "Derivation of Germline Competent Rat Embryonic
Stem Cells from DA Rats," Journal of Genetics and Genomics, 2012,
vol. 39, pp. 603-606. cited by applicant .
Lin et al., "Enhanced homology-directed human genome engineering by
controlled timing of CRISPR/Cas9 delivery," eLife, vol. 3, e04766,
2014. (published Dec. 15, 2014). cited by applicant .
Lin, S.-C., et al., "Strategies for gene disruption in Drosophila,"
Cell & Bioscience (2014), vol. 4(1), p. 63. cited by applicant
.
Liu et al., "A one-step cloning method for the construction of
somatic cell gene targeting vectors: application to production of
human knockout cell lines," BMC Biotechnol., vol. 12, p. 71, Oct.
9, 2012. cited by applicant .
Lombardo, et al., "Gene editing in human stem cells using zinc
finger nucleases and integrase-defective lentiviral vector
delivery," Nature Biotechnology, 2007, vol. 25(11), pp. 1298-1306.
cited by applicant .
Ma et al., "Heritable multiplex genetic engineering in rats using
CRISPR/Cas9," PLoS One, vol. 9(3), p. e89413, Mar. 5, 2014. cited
by applicant .
MacDonald, et al., "Precise and in situ genetic humanization of 6
Mb of mouse immunoglobulin genes," PNAS, vol. 111, No. 14:
5147-5152, (Apr. 8, 2014). cited by applicant .
Mali et al., "RNA-Guided Human Genome Engineering via Cas9,"
Science, vol. 339(6121), pp. 823-826 plus Supplemental Materials,
Jan. 3, 2013. cited by applicant .
Mali et al., "CAS9 transcriptional activators for target
specificity screening and paired nickases for cooperative genome
engineering," Nature Biotechnology, vol. 31(9), pp. 833-838 plus
Supplementary Information, Aug. 1, 2013. cited by applicant .
Mali, et al., "Cas9 as a versatile tool for engineering biology,"
Nature Methods, vol. 10(10), pp. 957-963, 2013. cited by applicant
.
Mali, et al., "CAS9 transcriptional activators for target
specificity screening and paired nickases for cooperative genome
engineering," Nature Biotechnology, 2013, vol. 31(9), pp. 833-838.
cited by applicant .
Mali, et al., "RNA-guided human genome engineering via Cas9,"
Science, 2013, vol. 339(6121), pp. 823-826. cited by applicant
.
Manjunath., et al., "Newer Gene Editing Technologies toward HIV
Gene Therapy," Viruses (2013), vol. 5, pp. 2748-2766. cited by
applicant .
Maruyama et al., "Increasing the efficiency of precise genome
editing with CRISPR-Cas9 by inhibition of nonhomologous end
joining," Nature Biotech., vol. 33(5), pp. 538-542, 2015.
(published Mar. 2015). cited by applicant .
Mashiko et al., "Generation of mutant mice by pronuclear injection
of circular plasmid expressing Cas9 and single guided RNA," Sci.
Rep., vol. 3, p. 3355, Nov. 27, 2013. cited by applicant .
Mashimo et al., "Generation of knockout rats with X-linked sever
combined immunodeficiency (X-SCID) using zinc finger nucleases,"
PLoS One, vol. 5(1), p. e8870, 2010. cited by applicant .
Matsuda et al., "STAT3 activation is sufficient to maintain an
undifferentiated state of mouse embryonic stem cells," The EMBO
Journal, vol. 18(15), pp. 4261-4269, 1999. cited by applicant .
Meek et al., "Efficient Gene Targeting by Homologous Recombination
in Rat Embryonic Stem Cells," PLoS One, vol. 5(12), p. e14225,
2010. cited by applicant .
Meek, S., et al., "Efficient Gene Targeting by Homologous
Recombination in Rat Embryonic Stem Cells," PloS One, 2010, vol.
5(12) pp. 1-6. cited by applicant .
Men et al., "Germline Transmission of a Novel Rat Embryonic Stem
Cell Line Derived from Transgenic Rats," Stem Cells Dev., vol.
21(14), pp. 2606-2612, 2012. cited by applicant .
Miller, et al., "A TALE nuclease architecture for efficient genome
editing," Nature Biotechnology, 2011, vol. 29(2), pp. 143-148.
cited by applicant .
Moehle et al., "Targeted gene addition into a specified location in
the human genome using designed zinc finger nucleases," Proc. Natl.
Acad. Sci. U.S.A., vol. 104(9), pp. 3055-3060, 2007 (epub Feb. 20,
2007). cited by applicant .
Morgens et al., "Systemic comparison of CRISPR/Cas9 and RNAi
screens for essential genes," Nature Biotechnology, vol. 34(6), pp.
634-636, May 9, 2016. cited by applicant .
Moscou and Bogdanove, "A simple cipher governs DNA recognition by
TAL effectors," Science, 2009, vol. 326, p. 1501. cited by
applicant .
Musser, "Rodent," Brittanica. Retrieved from the Internet May 31,
2016: http://www.brittanica.com/animal/rodent. cited by applicant
.
Mussolino, et al., "TALE nucleases: tailored genome engineering
made easy," Curr. Opin. Biotechnol., vol. 23(5), pp. 644-650, 2012.
(epub Feb. 17, 2012). cited by applicant .
Narsinh, et al., "Gene Correction in Human Embryonic and Induced
Pluripotent Stem Cells: Promise and Challenges Ahead", Molecular
Therapy, vol. 18, No. 6, pp. 1061-1063, (Jun. 2010). cited by
applicant .
News Feature "Move over ZFNs," Nature Biotechnology, 2011, vol.
29(8), pp. 681-684. cited by applicant .
Ota, S., et al., "Zebrafish: A model vertebrate suitable for the
analysis of human genetic disorders," Congenital Anomalies (2014),
vol. 54, pp. 8-11. cited by applicant .
Parikh et al., "Detailed Phenotypic and Molecular Analyses of
Genetically Modified Mice Generated by CRISPR-Cas9-Mediated
Editing," PLoS One, vol. 10(1), p. e0116484, Jan. 14, 2015. cited
by applicant .
Pattanayak, et al., "High-throughput profiling of off-target DNA
cleavage reveals RNA-programmed Cas9 nuclease specificity," Nature
Biotechnology, 2013, vol. 31(9), pp. 839-843. cited by applicant
.
PCT International Preliminary Report on Patentability for
application PCT/US2013/038165 dated Oct. 28, 2014. cited by
applicant .
PCT International Preliminary Report on Patentability for
application PCT/US2014/017452 dated Aug. 25, 2015. cited by
applicant .
PCT International Preliminary Report on Patentability for
application PCT/US2014/034412 dated Oct. 30, 2015. cited by
applicant .
PCT International Preliminary Report on Patentability for
application PCT/US2014/060788 dated Jun. 23, 2016. cited by
applicant .
PCT International Search Report and Written Opinion of the
International Searching Authority for application PCT/US2014/060788
dated Jan. 26, 2015. cited by applicant .
PCT International Search Report and Written Opinion of the
International Searching Authority for application PCT/US2015/038001
dated Feb. 25, 2016. cited by applicant .
PCT International Search Report and Written Opinion of the
International Searching Authority for application PCT/US2015/062023
dated May 13, 2016. cited by applicant .
PCT International Search Report and Written Opinion of the
International Searching Authority for application PCT/US2015/066681
dated Mar. 29, 2016. cited by applicant .
PCT International Search Report for application PCT/US2015/034503
dated Sep. 8, 2015. cited by applicant .
PCT Written Opinion of the International Searching Authority for
application PCT/US2015/034503 dated Sep. 8, 2015. cited by
applicant .
PCT/US2013/038165 International Search Report and Written Opinion
dated Jul. 12, 2013. cited by applicant .
PCT/US2014/017452 International Search Report and Written Opinion
of the Searching Authority dated May 14, 2014. cited by applicant
.
PCT/US2014/034412 International Search Report and Written Opinion
of the Searching Authority dated Oct. 9, 2014. cited by applicant
.
PCT/US2015/038001 Invitation of Pay Additional Fees dated Nov. 13,
2015. cited by applicant .
PCT/US2015/062023 Invitation of Pay Additional Fees dated Feb. 8,
2016. cited by applicant .
Peng, Y., et al., "Making designer mutants in model organisms,"
Development (2014), vol. 141, pp. 4042-4054. cited by applicant
.
Pennisi, E., "Beyond TALENs," Science (2012), vol. 338, p. 1411.
cited by applicant .
Perez et al., "Establishment of HIV-1 resistance in CD4+ T cells by
genome editing using zinc finger nucleases," Nature Biotech., vol.
26(7), pp. 808-816, 2008. cited by applicant .
Piatkevich et al., "Guide to Red Fluorescent Proteins and
Biosensors for Flow Cytometry," Methods Cell Biol., vol. 102, pp.
431-461, 2011. cited by applicant .
Platt et al., "CRISPR-Cas9 Knockin Mice for Genome Editing and
Cancer Modeling," Cell, vol. 159, pp. 1-16, Sep. 25, 2014. cited by
applicant .
Port et al., "Optimized CRISPR/Cas tools for efficient germline and
somatic genome engineering in Drosophila," Proc. Natl. Acad. Sci.
U.S.A., vol. 111(29), pp. E2967-E2976 plus Supporting Information,
Jul. 7, 2014. cited by applicant .
Porteus, et al,, "Gene targeting using zinc finger nucleases,"
Nature Biotechnology, vol. 23(8), pp. 967-973, 2005. cited by
applicant .
Poueymirou et al., "F0 generation mice fully derived from
gene-targeted embryonic stem cells allowing immediate analysis,"
Nature Biotechnology, Epub Dec. 24, 2006, vol. 25(1):91-99. cited
by applicant .
Qi, et al., "Repurposing CRISPR as an RNA-guided platform for
sequence-specific control of gene expression," Cell, 2013, vol.
152, pp. 1173-1183. cited by applicant .
Quinn et al., "A Site-Specific, Single-Copy Transgenesis Stragety
to Identify 5' Regulatory Sequences of the Mouse Testis-Determining
Gene Sry," PLoS One, vol. 9(4), p. e94813, Apr. 2014. cited by
applicant .
Ramirez et al., "Engineered zinc finger nickases induce
homology-directed repair with reduced mutagenic effects," Nucleic
Acids Research, vol. 40(12), pp. 5560-5568, 2012. (published Feb.
2012). cited by applicant .
Ran et al., "Double nicking by RNA-guided CRISPR Cas9 for enhanced
genome editing specificity," Cell, vol. 154, pp. 1380-1389, 2013.
cited by applicant .
Ran et al., "Genome engineering using the CRISPR-Cas9 system,"
Nature Protocols, vol. 8(11), pp. 2281-2308, Oct. 24, 2013. cited
by applicant .
Ran et al., "In vivo genome editing using Staphylococcus aureus
Cas9," Nature, vol. 520(7546), pp. 186-191, Apr. 1, 2015. cited by
applicant .
Rathjen et al., "Differentiation Inhibiting Activity Is Produced in
Matrix-Associated and Diffusible Forms That Are Generated by
Alternate Promoter Usage," Cell, vol. 62, pp. 1105-1114, 1990.
cited by applicant .
Ruhnke, M., et al., "Long-Term Culture and Differentiation of Rat
Embryonic Stem Cell-Like Cells into Neuronal, Glial, Endothelial,
and Hepatic Lineages," Stem Cells, 2003, vol. 21, pp. 428-436.
cited by applicant .
Sadhu et al., "CRISPR-directed mitotic recombination enables
genetic mapping without crosses," Science, vol. 352(6289):
1113-1116, May 5, 2016. cited by applicant .
Scharenberg, A.M., et al., "Genome Engineering with TAL-Effector
Nucleases and Alternative Modular Nuclease Technologies," Current
Gene Therapy (2013), vol. 13, pp. 291-303. cited by applicant .
Schwank, G., et al., "Functional Repair of CFTR by CRISPR/Cas9 in
Intestinal Stem Cell Organoids of Cystic Fibrosis Patients," Cell
Stem Cell (2013), vol. 13, pp. 653-658. cited by applicant .
Shan et al., "Targeted genome modification of crop plants using a
CRISPR-Cas system," Nature Biotechnology, vol. 31(8), pp. 686-688,
Aug. 1, 2013. cited by applicant .
Shao et al., "CRISPR/Cas-mediated genome editing in the rat via
direct injection of one-cell embryos," Nature Protocols, vol.
9(10), pp. 2493-2512, 2014 (epub Sep. 25, 2014). cited by applicant
.
Shen, H, et al., "The heterogeneity and dynamic equilibrium of rat
embryonic stem cells," Cell Research (2011), vol. 21, pp.
1143-1147. cited by applicant .
Siao et al., "Single-step homozygous humanization induced by dual
CRISPR/Cas9 cleavage," Oct. 28, 2015. cited by applicant .
Sigma-Aldrich, "Product Information sheet for Leukemia Inhibitory
Factor human," retrieved from internet on Apr. 25, 2015 at
<http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Data-
sheet/4/I5283dat.pdf>. cited by applicant .
Singh et al., "A Mouse Geneticist's Practical Guide to CRISPR
Applications," Genetics, vol. 199, pp. 1-15, (2015). cited by
applicant .
Stemgent Product Specification Sheet, PD0325901, pp. 1-2 (2012).
cited by applicant .
Straub A., et al., "Zinc Fingers, TAL Effectors, or Cas9-Based DNA
Binding Proteins: What's Best for Targeting Desired Genome Loci?,"
Molecular Plant (2013), vol. 6(5), pp. 1384-1387. cited by
applicant .
Tong et al., "Production of p53 gene knockout rats by homologous
recombination in embryonic stem cells," Nature, vol. 467(7312), pp.
211-213, 2010. cited by applicant .
Tong et al., "Generating gene knockout rats by homologous
recombination in embryonic stem cells," Nature Protocols, vol.
6(6), pp. 827-844, 2011 (epub May 26, 2011). cited by applicant
.
Tong, C., et al., "Production of p53 gene knockout rats by
homologous recombination in embryonic stem cells," Nature, 2010,
vol. 467(7312), pp. 211-213. cited by applicant .
U.S. Appl. No. 13/870,280 Final Rejection dated Oct. 15, 2015.
cited by applicant .
U.S. Appl. No. 13/870,280, Requirement for Restriction/Election
dated Jul. 22, 2014.00000000. cited by applicant .
U.S. Appl. No. 14/185,703 Non-Final Office Action dated Dec. 3,
2015. cited by applicant .
U.S. Appl. No. 14/185,703, Requirement for Restriction/Election
dated Sep. 4, 2015. cited by applicant .
U.S. Appl. No. 14/254,715 Final Office Action dated Nov. 30, 2015.
cited by applicant .
U.S. Appl. No. 14/254,715, Advisory Action dated Dec. 9, 2016.
cited by applicant .
U.S. Appl. No. 14/254,715, Final Office Action dated Sep. 19, 2016.
cited by applicant .
U.S. Appl. No. 14/254,715, Non-Final Office Action dated Apr. 21,
2016. cited by applicant .
U.S. Appl. No. 14/254,715, Non-Final Office Action dated Aug. 12,
2015. cited by applicant .
U.S. Appl. No. 14/314,866, Advisory Action dated Aug. 12, 2015.
cited by applicant .
U.S. Appl. No. 14/314,866, Advisory Action dated Aug. 15, 2016.
cited by applicant .
U.S. Appl. No. 14/314,866, Final Office Action dated Apr. 26, 2016.
cited by applicant .
U.S. Appl. No. 14/314,866, Non-Final Office Action dated Sep. 19,
2016. cited by applicant .
U.S. Appl. No. 14/314,866, Non-Final Office Action dated Dec. 26,
2014. cited by applicant .
U.S. Appl. No. 14/314,866, Non-Final Office Action dated Nov. 27,
2015. cited by applicant .
U.S. Appl. No. 14/314,866, Requirement for Restriction/Election
dated Sep. 22, 2014. cited by applicant .
U.S. Appl. No. 14/314,866, Final Office Action dated Jun. 4, 2015.
cited by applicant .
U.S. Appl. No. 14/515,503, Non-Final Office Action dated May 20,
2016. cited by applicant .
U.S. Appl. No. 14/515,503, Notice of Allowance dated Sep. 23, 2016.
cited by applicant .
U.S. Appl. No. 14/515,503, Requirement for Restriction/Election
dated Mar. 4, 2016. cited by applicant .
U.S. Appl. No. 14/578,291, Non-Final Office Action dated Mar. 10,
2015. cited by applicant .
U.S. Appl. No. 14/578,291, Notice of Allowance dated Aug. 26, 2015.
cited by applicant .
U.S. Appl. No. 14/731,914 , Requirement for Restriction/Election
dated Dec. 31, 2015. cited by applicant .
U.S. Appl. No. 14/731,914, Non-Final Office Action dated Jun. 17,
2016. cited by applicant .
U.S. Appl. No. 14/751,807, Requirement for Restriction/Election
dated Aug. 26, 2016. cited by applicant .
U.S. Appl. No. 14/926,773, Non-Final Office Action dated May 6,
2016. cited by applicant .
U.S. Appl. No. 14/926,773, Requirement for Restriction/Election
dated Feb. 16, 2016. cited by applicant .
U.S. Appl. No. 14/928,134, Advisory Action dated Jul. 15, 2016.
cited by applicant .
U.S. Appl. No. 14/928,134, Final Office Action dated Apr. 14, 2016.
cited by applicant .
U.S. Appl. no. 14/928,180, Advisory Action dated Aug. 22, 2016.
cited by applicant .
U.S. Appl. No. 14/928,180, Final Office Action dated Jun. 6, 2016.
cited by applicant .
U.S. Appl. No. 15/242,025, Non-Final Office Action dated Feb. 6,
2017. cited by applicant .
U.S. Appl. No. 13/870,280, Advisory Action dated Jan. 5, 2016.
cited by applicant .
U.S. Appl. No. 13/870,280, Non-Final Office Action dated Mar. 13,
2015. cited by applicant .
U.S. Appl. No. 14/185,703, Final Office Action dated Apr. 20, 2016.
cited by applicant .
U.S. Appl. No. 14/254,715, Requirement for Restriction/Election
dated Jun. 4, 2015. cited by applicant .
U.S. Appl. No. 14/928,180, Non-Final Office Action dated Jan. 5,
2016. cited by applicant .
Ueda et al., "Establishment of Rat Embryonic Stem Cells and Making
of Chimera Rats," PLoS One, vol. 3(7), p. e2800, 2008. cited by
applicant .
Ueda, S., et al., "Establishment of Rat Embryonic Stem Cells and
Making of Chimera Rats," PloS One, 2008, vol. 3(7), pp. 1-9. cited
by applicant .
U.S. Appl. No. 14/928,134 Non-Final Office Action dated Feb. 1,
2016. cited by applicant .
Van Der Oost, "New tool for genome surgery," Science, 2013, vol.
339(6121), pp. 768-770. cited by applicant .
Varlakhanova et al., "Myc Maintains Embryonic Stem Cell
Pluripotency and Self-Renewal," Differentiation, vol. 80(1), pp.
9-19, 2010. cited by applicant .
Wang et al., "One-Step Generation of Mice Carrying Mutations in
Multiple Genes by CRISPR/Cas-Mediated Genome Engineering," Cell,
vol. 153, pp. 910-918 plus supplemental materials, 2013. (published
May 2013). cited by applicant .
Wang et al., "One-Step Generation of Mice Carrying Mutations in
Multiple Genes by CRISPR/Cas-Mediated Genome Engineering," Cell,
vol. 153, pp. 910-918, 2013. (published May 2013). cited by
applicant .
Wang et al., "TALEN-mediated editing of the mouse Y chromosome,"
Nature Biotechnology, vol. 31(6), p. 530-532, 2013 (epub May 12,
2013). cited by applicant .
Wang, H., et al., "One-Step Generation of Mice Carrying Mutations
in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering," Cell,
2013, vol. 153(4), pp. 910-918. cited by applicant .
Wen et al., "Completely ES Cell-Derived Mice Produced by Tetraploid
Complementation Using Inner Cell Mass (ICM) Deficient Blastocysts,"
PLoS One, vol. 9(4), e94730, Apr. 14, 2014. cited by applicant
.
Whitworth et al., "Use of the CRISPR/Cas9 System to Produce
Genetically Engineered Pigs from In Vitro-Derived Oocytes and
Embryos," Biology of Reproduction, vol. 91(3), p. 78, Aug. 6, 2014.
cited by applicant .
Wu, Y., et al., "Correction of a Genetic Disease in Mouse via Use
of CRISPR-Cas9," Cell Stem Cell (2013), vol. 13, pp. 659-662. cited
by applicant .
Xu, T., et al., "Cas9-Based Tools for Targeted Genome Editing and
Transcriptional Control," Applied and Environmental Microbiology
(2014), vol. 80(5), pp. 1544-1552. cited by applicant .
Yamamoto et al., "Derivation of rat embryonic stem cells and
generation of protease-activated receptor-2 knockout rats,"
Transgenic Res., vol. 21, pp. 743-755, 2012. cited by applicant
.
Yamamoto, S., et al., "Derivation of rat embryonic stem cells and
generation of protease-activated receptor-2 knockout rats,"
Transgenic Res., 2012, vol. 21(4), pp. 743-755. cited by applicant
.
Yang et al., "One-step generation of mice carrying reporter and
conditional alleles by CRISPR/Cas-mediated genome engineering,"
Cell, vol. 154(6), pp. 1370-1379, Aug. 29, 2013. cited by applicant
.
Yang et al., "Optimization of scarless human stem cell genome
editing," Nucleic Acids Res., vol. 41(19), pp. 9049-9061, 2013
(epub Jul. 31, 2013). cited by applicant .
Yang et al., "Targeted and genome-wide sequencing reveal single
nucleotide variations impacting specificity of Cas9 in human stem
cells," Nat. Commun., vol. 5, p. 5507, (2014). cited by applicant
.
Yang, S., et al., "Derivation and Genetic Modification of Embryonic
Stem Cells from Disease-Model Inbred Rat Strains," Stem Cells and
Development, 2013, vol. 22(20), Abstract only. cited by applicant
.
Yang, S., et al., Retraction of "Derivation and Genetic
Modification of Embryonic Stem Cells from Disease-Model Inbred Rat
Strains," Stem Cells and Development, 2013, vol. 22(20), 2813.
cited by applicant .
Yen et al., "Somatic mosaicism and allele complexity induced by
CRISPR/Cas9 RNA injections in mouse zygotes," Dev. Biol., vol.
393(1), pp. 3-9, Jun. 28, 2014. cited by applicant .
Yoshimi et al., "Allele-specific genome editing and correction of
disease-associated phenotypes in rats using the CRISPR-Cas
platform," Nature Communications, vol. 5, p. 4240 plus
Supplementary Information, Jun. 26, 2014. cited by applicant .
Yoshimi et al., "ssODN-mediated knock-in with CRISPR-Cas for large
genomic regions in zygotes," Nat. Commun., vol. 7, p. 10431, Jan.
20, 2016. cited by applicant .
Yu et al., "Highly Efficient Genome Modifications Mediated by
CRISPR/Cas9 in Drosophila," Genetics, vol. 195,pp. 289-291 plus
supporting information, Sep. 2013. cited by applicant .
Zetsche et al., "Cpf1 Is a Single RNA-Guided Endonuclease of a
Class 2 CRISPR-Cas System," Cell, vol. 163, pp. 1-13, 2015
(available online Sep. 25, 2015). cited by applicant .
Zhang et al., "Biallelic targeting of expressed genes in mouse
embryonic stem cells using the Cas9 system," Methods, vol. 69(2),
pp. 171-178, Jun. 12, 2014. cited by applicant .
Zhao et al., "Derivation of embryonic stem cells from Brown Norway
rats blastocysts," J. Genet. Genomics, vol. 37, pp. 467-473, 2010.
cited by applicant .
Zhou et al., "Dual sgRNAs facilitate CRISPR/Cas9-mediated mouse
genome targeting," FEBS J., vol. 281(7), pp. 1717-1725, Feb. 26,
2014. cited by applicant .
Zhou, H., et al., "Large chromosomal deletions and heritable small
genetic changes induced by CRISPR/Cas9 in rice," Nucleic Acids
Research (2014), vol. 42(17), pp. 10903-10914. cited by applicant
.
U.S. Appl. No. 15/242,025, Non-Final Office Action dated May 3,
2018. cited by applicant .
Valenzuela, et al., "High-throughput engineering of the mouse
genome coupled with high-resolution expression analysis," Nature
Biotechnology, vol. 21, No. 6, pp. 652-659, (Jun. 2003). cited by
applicant .
Cohen, et al., "A transgenic Alzheimer Rat with Plaques, Tau
Pathology, Behavioral Impairment, Oligomeric A.beta., and Frank
Neuronal Loss," The Journal of Neuroscience, 33(15), 6256-6245,
(Apr. 10, 2013). cited by applicant .
EP Application No. 18187581.6 Extended European Search Report dated
Feb. 18, 2019. cited by applicant .
Kosicki et al., "Repair of double-strand breaks induced by
CRISPR-Cas9 leads to large deletions and complex rearrangements,"
Nat. Biotechnol., 36(8): 765-771, (Jul. 16, 2018). cited by
applicant .
U.S. Appl. No. 14/252,025, Notice of Allowance and Interview
Summary dated Feb. 6, 2019. cited by applicant .
U.S. Appl. No. 15/242,025, Final Office Action dated Nov. 29, 2018.
cited by applicant .
Vechkanov et al., "Fundamentals of Cell Engineering: A Study
Guide", Rostov-on-Don, pp. 15 and 46-47, Full English translation
(2012). cited by applicant .
Verkhovskaya et al, "The action of alkoxy-substituted glycerin on
the morphofunctional properties of a passaged cell culture,"
Cryobiology 1:30-33; Full English translation (1990). cited by
applicant.
|
Primary Examiner: Ton; Thaian N
Attorney, Agent or Firm: Choi; Yongjin Alston & Bird,
LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of U.S. application
Ser. No. 14/254,715, filed Apr. 16, 2014, which claims the benefit
of U.S. Provisional Application No. 61/812,319, filed Apr. 16,
2013, and U.S. Provisional Application No. 61/914,768, filed Dec.
11, 2013, all of which are hereby incorporated herein in their
entirety by reference.
Claims
We claim:
1. A method for targeted modification of a genomic locus of
interest in a population of pluripotent rat cells to produce a
genetically modified rat whose genome comprises a targeted genetic
modification that is transmitted through the germline, comprising:
(a) providing a population of pluripotent rat cells obtained by
culturing isolated rat embryonic stem cells on a feeder cell layer
that is not modified to express leukemia inhibitory factor (LIF)
with a medium comprising N2 supplement, B27 supplement, about 50
U/mL to about 150 U/mL LIF, and a combination of inhibitors
consisting of MEK inhibitor PD0325901 and GSK3 inhibitor CHIR99021,
wherein the pluripotent rat cells: lack expression of c-Myc; form
spherical, free-floating colonies in culture; are diploid; and are
germline competent; (b) obtaining a pluripotent rat cell clone
comprising the targeted genetic modification at the genomic locus
of interest, wherein the obtaining consists of: (i) introducing
into the pluripotent rat cells a large targeting vector (LTVEC)
comprising an insert nucleic acid flanked by a 5' homology arm
homologous to a first nucleic acid sequence at the genomic locus of
interest and a 3' homology arm homologous to a second nucleic acid
sequence at the genomic locus of interest to produce the targeted
genetic modification via homologous recombination, wherein the sum
total of the 5' and the 3' homology arms is at least 10 kb; and
(ii) performing a loss of allele (LOA) assay or a modification of
allele (MOA) assay to identify in a single cloning step a
pluripotent rat cell clone comprising the targeted genetic
modification at the genomic locus of interest and capable of
transmitting the targeted genetic modification through the
germline; (c) introducing the pluripotent rat cell clone into a rat
host embryo; (d) gestating the rat host embryo comprising the
pluripotent rat cell clone in a surrogate mother, wherein the
surrogate mother produces an F0 progeny genetically modified rat
comprising the targeted genetic modification; and (e) breeding the
F0 progeny genetically modified rat with another rat to produce an
F1 progeny genetically modified rat comprising the targeted genetic
modification, wherein the targeted genetic modification is
transmitted through the germline.
2. The method of claim 1, wherein the pluripotent rat cells are rat
embryonic stem (ES) cells.
3. The method of claim 1, wherein the pluripotent rat cells are
derived from a DA strain or an ACI strain.
4. The method of claim 1, wherein the pluripotent rat cells are
characterized by expression of at least one pluripotency marker
selected from the group consisting of Dnmt3L, Eras, Err-beta,
Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4,
Sox15, Sox2, and Utf1.
5. The method of claim 1, wherein the pluripotent rat cells are
characterized by one or more of the following characteristics: (I)
lack of expression of one or more of the pluripotency markers Ecat
1 and Rexo1; (II) lack of expression of one or more of the
mesodermal markers Brachyury and Bmpr2; (III) lack of expression of
one or more of the endodermal markers Gata6, Sox17, and Sox7; and
(IV) lack of expression of one or more of the neural markers Nestin
and Pax6.
6. The method of claim 5, wherein the pluripotent rat cells lack
expression of Ecat1.
7. The method of claim 1, wherein the sum total of the 5' and the
3' homology arms of the LTVEC is from 10 kb to about 150 kb, or
wherein the 5' homology arm ranges from about 5 kb to about 100 kb
or the 3' homology arm ranges from about 5 kb to about 100 kb.
8. The method of claim 1, wherein the LTVEC is from about 20 kb to
about 400 kb.
9. The method of claim 1, wherein the insert nucleic acid is from
about 5 kb to about 400 kb.
10. The method of claim 1, wherein the targeted genetic
modification is biallelic.
11. The method of claim 1, wherein the targeted genetic
modification comprises deletion of an endogenous rat nucleic acid
sequence, wherein the deletion ranges from about 5 kb to about 3
Mb.
12. The method of claim 1, wherein the targeted genetic
modification comprises insertion of an exogenous nucleic acid
sequence ranging from about 5 kb to about 400 kb.
13. The method of claim 1, wherein the targeted genetic
modification comprises: (I) replacement of an endogenous rat
nucleic acid sequence with a homologous or an orthologous nucleic
acid sequence; (II) deletion of an endogenous rat nucleic acid
sequence; (III) insertion of an exogenous nucleic acid sequence
comprising a nucleic acid sequence that is homologous or
orthologous to an endogenous rat nucleic acid sequence; (IV)
insertion of a chimeric nucleic acid sequence comprising a human
nucleic acid sequence and a rat nucleic acid sequence; (V)
insertion of a conditional allele flanked by site-specific
recombinase target sequences; or (VI) insertion of a reporter gene
operably linked to a promoter active in a rat cell.
14. The method of claim 1, wherein the targeted genetic
modification comprises: (I) an insertion of the insert nucleic
acid, wherein the insert nucleic acid comprises a human nucleic
acid sequence; (II) a replacement of a rat nucleic acid sequence at
the genomic locus of interest with a human nucleic acid sequence,
wherein the replaced rat nucleic acid sequence is homologous or
orthologous to the human nucleic acid sequence; (III) a chimeric
nucleic acid sequence comprising a human nucleic acid sequence and
a rat nucleic acid sequence; or (IV) a combination thereof.
15. The method of claim 1, wherein introducing step (b)(i) further
comprises introducing a nucleic acid encoding a nuclease agent that
promotes homologous recombination between the LTVEC and the genomic
locus of interest in the pluripotent rat cells.
16. The method of claim 15, wherein the nuclease agent comprises:
(I) a chimeric protein comprising a zinc finger-based DNA binding
domain fused to a FokI endonuclease; or (II) a chimeric protein
comprising a Transcription Activator-Like Effector Nuclease
(TALEN).
17. The method of claim 15, wherein the nuclease agent comprises a
CRISPR/Cas system.
18. The method of claim 17, wherein the CRISPR/Cas system comprises
a Cas9 nuclease and a guide RNA comprising a fused
crRNA-tracrRNA.
19. The method of claim 18, wherein the guide RNA comprises SEQ ID
NO: 2, 3, 4, 5, 6, 7, or 8.
20. The method of claim 1, wherein the genomic locus of interest
comprises: (i) an Interleukin-2 receptor gamma locus, an ApoE
locus, a Rag1 locus, a Rag2 locus, or a Rag2/Rag1 locus; (ii) an
immunoglobulin locus; or (iii) a T cell receptor locus.
21. The method of claim 1, wherein the concentration of LIF in the
medium is between about 75 U/mL to about 125 U/mL.
22. The method of claim 21, wherein the concentration of LIF in the
medium is between about 90 U/mL to about 110 U/mL.
23. The method of claim 22, wherein the concentration of LIF in the
medium is about 100 U/mL.
24. The method of claim 1, wherein the concentration of the MEK
inhibitor is 0.8 .mu.M to about 1.2 .mu.M, and the concentration of
the GSK3 inhibitor is about 2.5 .mu.M to about 3.5 .mu.M.
25. The method of claim 24, wherein the concentration of the MEK
inhibitor is about 1 .mu.M, and the concentration of the GSK3
inhibitor is about 3 .mu.M.
26. The method of claim 23, wherein the concentration of the MEK
inhibitor is about 1 .mu.M and the concentration of the GSK3
inhibitor is 3 .mu.M.
27. The method of claim 1, wherein the feeder cell layer comprises
mitotically inactivated mouse embryonic fibroblasts, and the medium
is a 2i medium comprising DMEM/F12 basal medium at a concentration
of 1x; neurobasal medium at a concentration of 1x;
penicillin/streptomycin at a concentration of 1%; L-Glutamine at a
concentration of 4 mM; 2-mercaptoethanol at a concentration of 0.1
mM; N2 supplement at a concentration of 1x; B27 supplement at a
concentration 1x; LIF at a concentration of 100U/mL; PD0325901 at a
concentration of 1 .mu.M, and CHIR99021 at a concentration of 3
.mu.M.
28. The method of claim 1, wherein step (b)(i) produces a
population of modified pluripotent rat cells, wherein the modified
pluripotent rat cells retain the ability to form sphere-like
colonies when cultured on the feeder cell layer in vitro such that
at least 50% of the colonies that form when the modified
pluripotent rat cells are plated on the feeder cell layer in vitro
have a sphere-like morphology following modification of the genomic
locus of interest.
29. The method of claim 1, wherein step (b)(i) produces a
population of modified pluripotent rat cells, wherein at least 50%
of the modified pluripotent rat cells remain diploid following
modification of the genomic locus of interest.
30. The method of claim 20, wherein the immunoglobulin locus is an
immunoglobulin heavy chain locus or an immunoglobulin light chain
locus.
31. The method of claim 30, wherein the immunoglobulin light chain
locus is a .lamda. or .kappa. immunoglobulin light chain locus.
32. The method of claim 20, wherein the genomic locus of interest
comprises a T cell receptor alpha locus.
33. The method of claim 1, wherein the insert nucleic acid: (i)
comprises a polynucleotide of interest comprising a genomic nucleic
acid sequence that encodes a human immunoglobulin heavy chain
variable region amino acid sequence; (ii) comprises a
polynucleotide of interest comprising a genomic nucleic acid
sequence that encodes a human immunoglobulin light chain variable
region amino acid sequence; (iii) comprises a polynucleotide of
interest comprising a polynucleotide encoding at least a region of
a T cell receptor; or (iv) comprises a polynucleotide of interest
comprising at least one disease allele.
34. The method of claim 33, wherein the insert nucleic acid
comprises a polynucleotide of interest comprising a polynucleotide
encoding at least a region of a T cell receptor alpha.
35. The method of claim 1, wherein the insert nucleic acid: (i) is
from a human; (ii) comprises a knock-in allele of at least one exon
of an endogenous gene; (iii) comprises a regulatory element; (iv)
comprises a conditional allele; (v) comprises a nucleic acid
flanked by site-specific recombination target sequences; (vi)
comprises a polynucleotide encoding a selection marker; or (vii)
comprises a reporter gene operably linked to a promoter.
36. The method of claim 35, wherein the insert nucleic acid
comprises a polynucleotide encoding a selection marker and/or a
reporter gene flanked by site-specific recombination target
sequences or comprises a self-deleting selection cassette.
37. The method of claim 1, wherein the introducing step is mediated
by electroporation.
38. The method of claim 1, wherein steps (a) and (b) are
sequentially repeated to allow for the targeted integration of at
least two insert nucleic acids into the genomic locus of
interest.
39. The method of claim 14, wherein the size of the insertion is
from about 5 kb to about 500 kb, or wherein the size of the
replacement is from about 5 kb to about 500 kb.
Description
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS
WEB
The official copy of the sequence listing is submitted
electronically via EFS-Web as an ASCII formatted sequence listing
with a file named 491907SEQLIST.TXT, created on Jan. 19, 2017, and
having a size of 15 kilobytes, and is filed concurrently with the
specification. The sequence listing contained in this ASCII
formatted document is part of the specification and is herein
incorporated by reference in its entirety.
FIELD OF INVENTION
Isolated non-human totipotent or pluripotent stem cells, in
particular rat embryonic stem cells, that are capable of sustaining
pluripotency following one or more serial genetic modifications in
vitro, and that are capable of transmitting the targeted genetic
modifications to subsequent generations through germline.
Compositions and methods for modifying a rat genomic locus of
interest via bacterial homologous recombination (BHR) in a
prokaryotic cell. Compositions and methods for genetically
modifying a rat genomic locus of interest using a large targeting
vector (LTVEC) in combination with endonucleases. Compositions and
methods for producing a genetically modified rat comprising one or
more targeted genetic modifications.
BACKGROUND OF THE INVENTION
While rats have been regarded as an important animal model system
that can recapitulate the pathology of various human diseases,
including, but not limited to, cardiovascular (e.g., hypertension),
metabolic (e.g., obesity, diabetes), neurological (e.g., pain
pathologies), and a variety of cancers, the use of rats in modeling
human diseases has been limited as compared to mice, due in part to
unavailability of germline-transmittable pluripotent rat cells,
which can sustain their pluripotency following a series of genetic
modifications in vitro, e.g., one or more serial electroporations,
and due in part to lack of efficient targeting technologies that
allow introduction or deletion of large genomic DNA sequences, or
replacement of large endogenous genomic DNA sequences with
exogenous nucleic acid sequences in pluripotent rat cells.
There is a need in the art for compositions and methods that allow
precise targeted changes in the genome of a rat, which can open or
expand current areas of target discovery and validate therapeutic
agents more quickly and easily.
SUMMARY
Methods are provided for modifying a genomic locus of interest in a
pluripotent cell via targeted genetic modification. Such a method
comprises (a) introducing into the pluripotent cell a large
targeting vector (LTVEC) comprising an insert nucleic acid flanked
with a 5' homology arm and a 3' homology arm; and (b) identifying a
genetically modified pluripotent cell comprising the targeted
genetic modification at the genomic locus of interest, wherein the
targeted genetic modification is capable of being transmitted
through the germline.
In one embodiment, the pluripotent cell is derived from a non-human
animal, including, but not limited to, a rodent, a human, a rat, a
mouse, a hamster, a rabbit, a pig, a bovine, a deer, a sheep, a
goat, a chicken, a cat, a dog, a ferret, a primate (e.g., marmoset,
rhesus monkey), a domesticated mammal or an agricultural mammal, or
any other organism of interest.
In one embodiment, the pluripotent cell is a non-human pluripotent
cell. In one embodiment, the non-human pluripotent cell is a
mammalian pluripotent cell. In one embodiment, the mammalian
pluripotent cell is a rodent pluripotent cell. In one embodiment,
the rodent pluripotent cell is a rat or mouse pluripotent cell. In
one embodiment, the pluripotent cell is a human induced pluripotent
stem (iPS) cell.
In one embodiment, the pluripotent cell is a non-human fertilized
egg at the single cell stage. In one embodiment, the non-human
fertilized egg is a mammalian fertilized egg. In one embodiment,
the mammalian fertilized egg is a rodent fertilized egg at the
single cell stage. In one embodiment, the mammalian fertilized egg
is a rat or mouse fertilized egg at the single cell stage.
In some embodiments, the sum total of the 5' and the 3' homology
arms of the LTVEC is at least 10 kb. In some embodiments, the sum
total of the 5' and the 3' homology arms of the LTVEC is at least
10 kb but less than 100 kb or the sum total of the 5' and the 3'
homology arms of the LTVEC is at least 10 kb but less than 150 kb.
In other embodiments, the size of the sum total of the total of the
5' and 3' homology arms of the LTVEC is about 10 kb to about 150
kb, about 10 kb to about 100 kb, about 10 kb to about 75 kb, about
20 kb to about 150 kb, about 20 kb to about 100 kb, about 20 kb to
about 75 kb, about 30 kb to about 150 kb, about 30 kb to about 100
kb, about 30 kb to about 75 kb, about 40 kb to about 150 kb, about
40 kb to about 100 kb, about 40 kb to about 75 kb, about 50 kb to
about 150 kb, about 50 kb to about 100 kb, or about 50 kb to about
75 kb, about 10 kb to about 30 kb, about 20 kb to about 40 kb,
about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb
to about 100 kb, about 100 kb to about 120 kb, or from about 120 kb
to about 150 kb. In one embodiment, the size of the deletion is the
same or similar to the size of the sum total of the 5' and 3'
homology arms of the LTVEC.
In some such embodiments, the targeted genetic modification is
biallelic.
In some embodiments, the pluripotent cell is a pluripotent rat
cell. In one embodiment, the pluripotent rat cell is a rat
embryonic stem cell. In one embodiment, the pluripotent rat cell is
derived from a DA strain or an ACI strain. In some embodiments, the
pluripotent rat cell is characterized by expression of at least one
pluripotency marker comprising Dnmt3L, Eras, Err-beta, Fbxo15,
Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15,
Sox2, Utf1, or a combination thereof. In some such methods, the
pluripotent rat cell is characterized by one of more of the
following characteristics: (a) lack of expression of one or more
pluripotency markers comprising c-Myc, Ecat1, and/or Rexo1; (b)
lack of expression of mesodermal markers comprising Brachyury
and/or Bmpr2; (c) lack of expression of one or more endodermal
markers comprising Gata6, Sox17 and/or Sox7; or (d) lack of
expression of one or more neural markers comprising Nestin and/or
Pax6. Such methods provide that the sum total of the 5' and the 3'
homology arms of the LTVEC is from about 10 kb to about 30 kb, from
about 20 kb to about 40 kb, from about 40 kb to about 60 kb, from
about 60 kb to about 80 kb, or from about 80 kb to about 100 kb,
from about 100 kb to about 120 kb, from about 120 kb to about 150
kb, or from about 10 kb but less than about 150 kb. In some
embodiments, the sum total of the 5' and the 3' homology arms of
the LTVEC is from about 16 Kb to about 100 Kb. In other
embodiments, the size of the sum total of the total of the 5' and
3' homology arms of the LTVEC is about 10 kb to about 150 kb, about
10 kb to about 100 kb, about 10 kb to about 75 kb, about 20 kb to
about 150 kb, about 20 kb to about 100 kb, about 20 kb to about 75
kb, about 30 kb to about 150 kb, about 30 kb to about 100 kb, about
30 kb to about 75 kb, about 40 kb to about 150 kb, about 40 kb to
about 100 kb, about 40 kb to about 75 kb, about 50 kb to about 150
kb, about 50 kb to about 100 kb, about 50 kb to about 75 kb, about
10 kb to about 30 kb, about 20 kb to about 40 kb, about 40 kb to
about 60 kb, about 60 kb to about 80 kb, about 80 kb to about 100
kb, about 100 kb to about 120 kb, or from about 120 kb to about 150
kb. In one embodiment, the size of the deletion is the same or
similar to the size of the sum total of the 5' and 3' homology arms
of the LTVEC.
The methods further provide that targeted genetic modification (a)
comprises a replacement of an endogenous rat nucleic acid sequence
with a homologous or an orthologous mammalian nucleic acid
sequence; (b) comprises a deletion of an endogenous rat nucleic
acid sequence; (c) comprises a deletion of an endogenous rat
nucleic acid sequence, wherein the deletion ranges from about 5 kb
to about 10 kb, from about 10 kb to about 20 kb, from about 20 kb
to about 40 kb, from about 40 kb to about 60 kb, from about 60 kb
to about 80 kb, from about 80 kb to about 100 kb, from about 100 kb
to about 150 kb, or from about 150 kb to about 200 kb, from about
200 kb to about 300 kb, from about 300 kb to about 400 kb, from
about 400 kb to about 500 kb, from about 500 kb to about 1 Mb, from
about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb, from
about 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb; (d)
comprises an exogenous nucleic acid sequence ranging from about 5
kb to about 10 kb, from about 10 kb to about 20 kb, from about 20
kb to about 40 kb, from about 40 kb to about 60 kb, from about 60
kb to about 80 kb, from about 80 kb to about 100 kb, from about 100
kb to about 150 kb, from about 150 kb to about 200 kb, from about
200 kb to about 250 kb, from about 250 kb to about 300 kb, from
about 300 kb to about 350 kb, or from about 350 kb to about 400 kb;
(e) comprises an exogenous nucleic acid sequence comprising a
homologous or an orthologous nucleic acid sequence; (f) comprises a
chimeric nucleic acid sequence comprising a human and a rat nucleic
acid sequence; (g) ranges from about 5 kb to about 10 kb, from
about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from
about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from
about 80 kb to about 100 kb, from about 100 kb to about 150 kb,
from about 150 kb to about 200 kb, from about 200 kb to about 250
kb, from about 250 kb to about 300 kb, from about 300 kb to about
350 kb, or from about 350 kb to about 400 kb. (h) comprises a
conditional allele flanked with site-specific recombinase target
sequences; or, (i) comprises a reporter gene operably linked to a
promoter active in a rat cell.
Further provided is a method for modifying a genomic locus of
interest in a pluripotent rat cell via targeted genetic
modification, wherein the genomic locus of interest comprises (i) a
first nucleic acid sequence that is complementary to the 5' rat
homology arm; and (ii) a second nucleic acid sequence that is
complementary to the 3' rat homology arm. In some such embodiments,
the first and the second nucleic acid sequence is separated by at
least 5 kb. In some embodiments, the first and the second nucleic
acid sequence is separated by at least 5 kb but less than 3 Mb. In
some such methods, the first and the second nucleic acid sequence
is separated by at least 5 kb but less than 10 kb, at least 10 kb
but less than 20 kb, at least 20 kb but less than 40 kb, at least
40 kb but less than 60 kb, at least 60 kb but less than 80 kb, at
least about 80 kb but less than 100 kb, at least 100 kb but less
than 150 kb, or at least 150 kb but less than 200 kb, at least
about 200 kb but less than about 300 kb, at least about 300 kb but
less than about 400 kb, at least about 400 kb but less than about
500 kb, at least about 500 kb but less than about 1 Mb, at least
about 1 Mb but less than about 1.5 Mb, at least about 1.5 Mb but
less than about 2 Mb, at least about 2 Mb but less than about 2.5
Mb, at least about 2.5 Mb but less than about 3 Mb, at least about
1 Mb but less than about 2 Mb, at least about 2 Mb but less than
about 3 Mb.
In some embodiments, the introducing step further comprises
introducing a second nucleic acid encoding a nuclease agent that
promotes a homologous recombination between the targeting construct
and the genomic locus of interest in the pluripotent rat cell. In
some such embodiments, the nuclease agent comprises (a) a chimeric
protein comprising a zinc finger-based DNA binding domain fused to
a FokI endonuclease; or, (b) a chimeric protein comprising a
Transcription Activator-Like Effector Nuclease (TALEN) fused to a
Fold endonuclease.
In some methods, the introducing step further comprises introducing
into the pluripotent rat cell: (i) a first expression construct
comprising a first promoter operably linked to a first nucleic acid
sequence encoding a Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR)-associated (Cas) protein, (ii) a
second expression construct comprising a second promoter operably
linked to a second nucleic acid sequence encoding a genomic target
sequence operably linked to a guide RNA (gRNA), wherein the genomic
target sequence is immediately flanked on the 3' end by a
Protospacer Adjacent Motif (PAM) sequence. In one embodiment, the
genomic locus of interest comprises the nucleotide sequence of SEQ
ID NO: 1. In one embodiment, the gRNA comprises a third nucleic
acid sequence encoding a Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR) RNA (crRNA) and a trans-activating
CRISPR RNA (tracrRNA). In another embodiment, the genome of the
pluripotent rat cell comprises a target DNA region complementary to
the genomic target sequence. In some such methods, the Cas protein
is Cas9. In some such methods the gRNA comprises (a) the chimeric
RNA of the nucleic acid sequence of SEQ ID NO: 2; or, (b) the
chimeric RNA of the nucleic acid sequence of SEQ ID NO: 3. In some
such methods, the crRNA comprises the sequence set forth in SEQ ID
NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some such methods, the
tracrRNA comprises the sequence set forth in SEQ ID NO: 7 or SEQ ID
NO: 8.
Further provided is a rat genomic locus comprising (i) an insertion
of a homologous or orthologous human nucleic acid sequence; (ii) a
replacement of an endogenous rat nucleic acid sequence with the
homologous or orthologous human nucleic acid sequence; or (iii) a
combination thereof, wherein the rat genomic locus is capable of
being transmitted through the germline. In some such rat genomic
locus, the size of the insertion or replacement is from about 5 kb
to about 400 kb. In some such rat genomic locus, the size of the
insertion or replacement is from about 5 kb to about 10 kb, from
about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from
about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from
about 80 kb to about 100 kb, from about 100 kb to about 150 kb,
from about 150 kb to about 200 kb, from about 200 kb to about 250
kb, from about 250 kb to about 300 kb, from about 300 kb to about
350 kb, from about 350 kb to about 400 kb, from about 400 kb to
about 800 kb, from about 800 kb to 1 Mb, from about 1 Mb to about
1.5 Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb, to about
2.5 Mb, from about 2.5 Mb to about 2.8 Mb, from about 2.8 Mb to
about 3 Mb, at least about 200 kb but less than about 300 kb, at
least about 300 kb but less than about 400 kb, at least about 400
kb but less than about 500 kb, at least about 500 kb but less than
about 1 Mb, at least about 11 Mb but less than about 2 Mb, at least
about 2 Mb but less than about 3 Mb.
Further provided is a method for making a humanized rat,
comprising: (a) targeting a genomic locus of interest in a
pluripotent rat cell with a targeting construct comprising a human
insert nucleic acid to form a genetically modified pluripotent rat
cell; (b) introducing the genetically modified pluripotent rat cell
into a host rat embryo; and (c) gestating the host rat embryo in a
surrogate mother; wherein the surrogate mother produces rat progeny
comprising, a modified genomic locus that comprises: (i) an
insertion of a human nucleic acid sequence; (ii) a replacement of
the rat nucleic acid sequence at the genomic locus of interest with
a homologous or orthologous human nucleic acid sequence; (iii) a
chimeric nucleic acid sequence comprising a human and a rat nucleic
acid sequence; or (iv) a combination thereof, wherein the modified
genomic locus is capable of being transmitted through the
germline.
In some such methods, the targeting construct is a large targeting
vector (LTVEC), and the sum total of the 5' and the 3' homology
arms of the LTVEC is at least 10 kb but less than 100 kb or the sum
total of the 5' and the 3' homology arms of the LTVEC is at least
10 kb but less than 150 kb. In some such methods, the sum total of
the 5' and the 3' homology arms of the targeting construct is from
about 10 kb to about 30 kb, from about 20 kb to 40 kb, from about
40 kb to about 60 kb, from about 60 kb to about 80 kb, from about
80 kb to about 100 kb, from about 100 kb to about 120 kb, or from
about 120 kb to about 150 kb. In some such methods, the human
nucleic acid sequence is at least 5 kb but less than 400 kb. In
some such methods, the human nucleic acid sequence is at least 5 kb
but less than 10 kb, at least 10 kb but less than 20 kb, at least
20 kb but less than 40 kb, at least 40 kb but less than 60 kb, at
least 60 kb but less than 80 kb, at least about 80 kb but less than
100 kb, at least 100 kb but less than 150 kb, at least 150 kb but
less than 200 kb, at least 200 kb but less than 250 kb, at least
250 kb but less than 300 kb, at least 300 kb but less than 350 kb,
or at least 350 kb but less than 400 kb. In other embodiments, the
size of the sum total of the total of the 5' and 3' homology arms
of the LTVEC is about 10 kb to about 150 kb, about 10 kb to about
100 kb, about 10 kb to about 75 kb, about 20 kb to about 150 kb,
about 20 kb to about 100 kb, about 20 kb to about 75 kb, about 30
kb to about 150 kb, about 30 kb to about 100 kb, about 30 kb to
about 75 kb, about 40 kb to about 150 kb, about 40 kb to about 100
kb, about 40 kb to about 75 kb, about 50 kb to about 150 kb, about
50 kb to about 100 kb, about 50 kb to about 75 kb, about 10 kb to
about 30 kb, about 20 kb to about 40 kb, about 40 kb to about 60
kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about
100 kb to about 120 kb, or from about 120 kb to about 150 kb. In
one embodiment, the size of the deletion is the same or similar to
the size of the sum total of the 5' and 3' homology arms of the
LTVEC.
In some methods for making a humanized rat, the pluripotent rat
cell is a rat embryonic stem (ES) cell. In some such methods, the
pluripotent rat cell is derived from a DA strain or an ACI strain.
In some such methods, the pluripotent rat cell is characterized by
expression of at least one pluripotency marker comprises Dnmt3L,
Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor,
Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, and/or a combination
thereof. In some such methods, the pluripotent rat cell is
characterized by one or more of the following features: (a) lack of
expression of one or more pluripotency markers comprising c-Myc,
Ecat1, and/or Rexo1; (b) lack of expression of one or more
mesodermal markers comprising Brachyury and/or Bmpr2; (c) lack of
expression of one or more endodermal markers comprising Gata6,
Sox17, and/or Sox7; or (d) lack of expression of one or more neural
markers comprising Nestin and/or Pax6.
Further provided is a genetically modified rat comprising a
humanized genomic locus, wherein the genetically modified rat
comprises: (i) an insertion of a homologous or orthologous human
nucleic acid sequence; (ii) a replacement of a rat nucleic acid
sequence with a homologous or orthologous human nucleic acid
sequence at an endogenous genomic locus with a homologous or
orthologous human nucleic acid sequence; (iii) a chimeric nucleic
acid sequence comprising a human and a rat nucleic acid sequence;
or, (iv) a combination thereof, wherein the humanized genomic locus
is capable of being transmitted through the germline. In some such
genetically modified rats, the humanized genomic locus comprises a
chimeric nucleic acid sequence comprising a human and a rat nucleic
acid sequence.
Methods for modifying a target genomic locus of a rat via bacterial
homologous recombination (BHR) are also provided and comprise:
introducing into a prokaryotic cell a large targeting vector
(LTVEC) comprising an insert nucleic acid flanked with a 5' rat
homology arm and a 3' rat homology arm, wherein the prokaryotic
cell comprises a rat nucleic acid and is capable of expressing a
recombinase that mediates the BHR at the target locus, and wherein
the sum total of the 5' and 3' homology arms of the LTVEC is at
least 10 kb but less than 100 kb or the sum total of the 5' and the
3' homology arms of the LTVEC is at least 10 kb but less than 150
kb. In other embodiments, the size of the sum total of the total of
the 5' and 3' homology arms of the LTVEC is about 10 kb to about
150 kb, about 10 kb to about 100 kb, about 10 kb to about 75 kb,
about 20 kb to about 150 kb, about 20 kb to about 100 kb, about 20
kb to about 75 kb, about 30 kb to about 150 kb, about 30 kb to
about 100 kb, about 30 kb to about 75 kb, about 40 kb to about 150
kb, about 40 kb to about 100 kb, about 40 kb to about 75 kb, about
50 kb to about 150 kb, about 50 kb to about 100 kb, or about 50 kb
to about 75 kb, about 10 kb to about 30 kb, about 20 kb to about 40
kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about
80 kb to about 100 kb, about 100 kb to about 120 kb, or from about
120 kb to about 150 kb. In one embodiment, the size of the deletion
is the same or similar to the size of the sum total of the 5' and
3' homology arms of the LTVEC.
In some such methods, the target locus of the rat nucleic acid
comprises a first nucleic acid sequence that is complementary to
the 5' homology arm and a second nucleic acid sequence that is
complementary to the 3' homology arm. In some such methods, the
first and the second nucleic acid sequence is separated by at least
5 kb but less than 10 kb, at least 10 kb but less than 20 kb, at
least 20 kb but less than 40 kb, at least 40 kb but less than 60
kb, at least 60 kb but less than 80 kb, at least about 80 kb but
less than 100 kb, at least 100 kb but less than 150 kb, or at least
150 kb but less than 200 kb, at least about 200 kb but less than
about 300 kb, at least about 300 kb but less than about 400 kb, at
least about 400 kb but less than about 500 kb, at least about 500
kb but less than about 1 Mb, at least about 11 Mb but less than
about 2 Mb, at least about 2 Mb but less than about 3 Mb.
In some such methods, introducing the targeting vector into the
prokaryotic cell leads to: (i) a deletion of an endogenous rat
nucleic acid sequence from the target genomic locus; (ii) an
addition of an exogenous nucleic acid sequence at the target
genomic locus; (iii) a replacement of the endogenous rat nucleic
acid sequence with the exogenous nucleic acid sequence at the
target locus; or (iv) a combination thereof. In some such methods,
the insert nucleic acid comprises (a) a polynucleotide that is
homologous or orthologous to the rat nucleic acid sequence at the
target genomic locus; or (b) a conditional allele flanked with
site-specific recombination recognition sequences.
Further provided is a host prokaryotic cell comprising a targeting
vector comprising an insert nucleic acid flanked with a 5' rat
homology arm and a 3' rat homology arm, wherein the insert nucleic
acid ranges from about 5 k to about 400 kb. In some host
prokaryotic cells the size of the insert nucleic acid is from about
5 kb to about 10 kb, from about 10 kb to about 20 kb, from about 20
kb to about 40 kb, from about 40 kb to about 60 kb, from about 60
kb to about 80 kb, from about 80 kb to about 100 kb, from about 100
kb to about 150 kb, from about 150 kb to about 200 kb, from about
200 kb to about 250 kb, from about 250 kb to about 300 kb, from
about 300 kb to about 350 kb, or from 350 kb to about 400 kb. In
some host prokaryotic cells, the prokaryotic cell comprises a
recombinase gene operably linked to a constitutively active
promoter or an inducible promoter.
Methods are also provided for modifying a genomic locus of interest
in a cell via targeted genetic modification comprising introducing
into the cell
(a) a large targeting vector (LTVEC) comprising an insert nucleic
acid flanked with a 5' homology arm and a 3' homology arm, wherein
the sum total of the 5' and 3' homology arms of the LTVEC is at
least 10 kb; and
(b) (i) a first expression construct comprising a first promoter
operably linked to a first nucleic acid sequence encoding a
Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR)-associated (Cas) protein, (ii) a second expression
construct comprising a second promoter operably linked to a second
nucleic acid sequence encoding a genomic target sequence operably
linked to a guide RNA (gRNA); and identifying a genetically
modified pluripotent cell comprising the targeted genetic
modification at the genomic locus of interest.
In one embodiment, the genomic locus of interest comprises the
nucleotide sequence set forth in SEQ ID NO: 1, wherein the gRNA
comprises a third nucleic acid sequence encoding a Clustered
Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA
(crRNA) and a trans-activating CRISPR RNA (tracrRNA), and wherein
the genome of the cell comprises a target DNA region complementary
to the genomic target sequence. In some such methods, the Cas
protein is Cas9. In such methods, the cell can be a pluripotent
cell (such as an embryonic stem cell) or a prokaryotic cell. In one
embodiment, the pluripotent cell is from non-human animal, a
non-human mammal, a rodent, a human, a rat, a mouse, a hamster a
rabbit, a pig, a bovine, a deer, a sheep, a goat, a chicken, a cat,
a dog, a ferret, a primate (e.g., marmoset, rhesus monkey),
domesticated mammal or an agricultural mammal or any other organism
of interest. In another embodiment, the prokaryotic cell is from
bacteria, such as, E. coli.
In other embodiments, the size of the sum total of the total of the
5' and 3' homology arms of the LTVEC is about 10 kb to about 150
kb, about 10 kb to about 100 kb, about 10 kb to about 75 kb, about
20 kb to about 150 kb, about 20 kb to about 100 kb, about 20 kb to
about 75 kb, about 30 kb to about 150 kb, about 30 kb to about 100
kb, about 30 kb to about 75 kb, about 40 kb to about 150 kb, about
40 kb to about 100 kb, about 40 kb to about 75 kb, about 50 kb to
about 150 kb, about 50 kb to about 100 kb, or about 50 kb to about
75 kb, about 10 kb to about 30 kb, about 20 kb to about 40 kb,
about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb
to about 100 kb, about 100 kb to about 120 kb, or from about 120 kb
to about 150 kb. In one embodiment, the size of the deletion is the
same or similar to the size of the sum total of the 5' and 3'
homology arms of the LTVEC.
In one embodiment, the pluripotent cell is a non-human pluripotent
cell. In one embodiment, the non-human pluripotent cell is a
mammalian pluripotent cell. In one embodiment, the mammalian
pluripotent cell is a rodent pluripotent cell. In one embodiment,
the rodent pluripotent cell is a rat or mouse pluripotent cell. In
one embodiment, the pluripotent cell is a human induced pluripotent
stem (iPS) cell.
In one embodiment, the pluripotent cell is a non-human fertilized
egg at the single cell stage. In one embodiment, the non-human
fertilized egg is a mammalian fertilized egg. In one embodiment,
the mammalian fertilized egg is a rodent fertilized egg at the
single cell stage. In one embodiment, the mammalian fertilized egg
is a rat or mouse fertilized egg at the single cell stage.
Further provided is a rat or rat cell comprising a targeted genetic
modification in its genomic locus, wherein the genomic locus is an
Interleukin-2 receptor gamma locus, an ApoE locus, a Rag1 locus, a
Rag2 locus, or a Rag2/Rag1 locus, wherein the targeted genetic
modification comprises: (a) a deletion of an endogenous rat nucleic
acid sequence at the genomic locus; (b) an insertion of a
homologous nucleic acid, an orthologous nucleic acid, or a chimeric
nucleic acid comprising a human and a rat nucleic acid sequence; or
(c) a combination thereof. In such a rat or rat cell, the targeted
genetic modification is transmissible through the germline of the
rat or a rat propagated from the rat cell.
In some such rats or rat cells the deletion of the endogenous rat
nucleic acid at the genomic locus is at least about 10 kb, or the
insertion of the exogenous nucleic acid sequence at the genomic
locus is at least about 5 kb.
Further provided is a rat or rat cell, wherein (a) the targeted
genetic modification at the Interleukin-2 receptor gamma locus
results in a decrease in or absence of Interleukin-2 receptor gamma
protein activity; (b) the targeted genetic modification at the ApoE
locus results in a decrease in or absence of ApoE protein activity;
(c) the targeted genetic modification at the Rag1 locus results in
a decrease in or absence of Rag1 protein activity; (d) the targeted
genetic modification at the Rag2 locus results in a decrease in or
absence of Rag2 protein activity; or, (e) the targeted genetic
modification at the Rag2/Rag1 locus results in a decrease in or
absence of Rag2 protein activity and Rag1 activity.
In some embodiments, the targeted genetic modification of the
Interleukin-2 receptor gamma locus comprises: (a) a deletion of the
entire rat Interleukin-2 receptor gamma coding region or a portion
thereof; (b) a replacement of the entire rat Interleukin-2 receptor
gamma coding region or a portion thereof with a human Interleukin-2
receptor gamma coding region or a portion thereof; (c) a
replacement of an ecto-domain of the rat Interleukin-2 receptor
gamma coding region with the ecto-domain of a human Interleukin-2
receptor gamma; or, (d) at least a 3 kb deletion of the
Interleukin-2 receptor gamma locus. In other such rats or rat cells
the targeted genetic modification of the ApoE locus comprises: (a)
a deletion of the entire ApoE coding region or a portion thereof;
or, (b) at least a 1.8 kb deletion of the ApoE locus comprising the
ApoE coding region.
Further provided is a rat or rat cell, wherein the targeted genetic
modification of the Rag2 locus comprises: (a) a deletion of the
entire Rag2 coding region or a portion thereof; or (b) at least a
5.7 kb deletion of the Rag2 locus comprising the Rag2 coding
region. In some embodiments, the targeted genetic modification of
the Rag2/Rag1 locus comprises: (a) a deletion of the entire Rag2
coding region or a portion thereof and a deletion of the entire
Rag1 coding region or portion thereof; or, (b) a deletion of at
least 16 kb of the Rag2/Rag1 locus comprising the Rag2 coding
region.
Further provided is a rat or rat cell, wherein the targeted genetic
modification comprises an insertion of an expression cassette
comprising a selective marker at the Interleukin-2 receptor gamma
locus, the ApoE locus, the Rag1 locus, the Rag2 locus, or the
Rag2/Rag1 locus. In some such rats or rat cells the expression
cassette comprises a lacZ gene operably linked to the endogenous
promoter at the genomic locus and a human ubiquitin promoter
operably linked to a selective marker.
Further provided is a rat or rat cell, wherein the targeted genetic
modification in the Interleukin-2 receptor gamma locus, the ApoE
locus, the Rag1 locus, the Rag2 locus or the Rag2/Rag1 locus
comprises the insertion of a self-deleting selection cassette. In
some such rats or rat cells, the self-deleting selection cassette
comprises a selective marker gene operably linked to a promoter
active in the rat cell and a recombinase gene operably linked to a
male germ cell-specific promoter, wherein the self-deleting
cassette is flanked by recombination recognition sites recognized
by the recombinase. In some such rats or rat cells, the male germ
cell-specific promoter is a Protamine-1 promoter; the recombinase
gene encodes Cre, and the recombination recognition sites are loxP
sites. In one embodiment, the Protamine-1 promoter is a mouse or a
rat Protamine-1 promoter.
Further provided is a rat or rat cell, wherein the insertion of the
exogenous nucleic acid sequence at the genomic locus comprises a
reporter nucleic acid operably linked to an endogenous
Interleukin-2 receptor gamma promoter, an endogenous ApoE promoter,
an endogenous Rag1 promoter, or an endogenous Rag2 promoter. In
some such rats or rat cells, the reporter nucleic acid encodes a
reporter comprising .beta.-galactosidase, mPlum, mCherry, tdTomato,
mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet,
enhanced yellow fluorescent protein (EYFP), Emerald, enhanced green
fluorescent protein (EGFP), CyPet, cyan fluorescent protein (CFP),
Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a
combination thereof.
Further provided is a rat cell, wherein the rat cell is a
pluripotent rat cell or a rat embryonic stem (ES) cell. In some
such rat cells, the pluripotent rat cell or the rat embryonic stem
(ES) cell (a) is derived from a DA strain or an ACI strain; (b) is
characterized by expression of at least one pluripotency marker
comprisingDnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1,
LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a
combination thereof; or (c) is characterized by one or more of the
following characteristics: (i) lack of expression of one or more
pluripotency markers comprising c-Myc, Ecat1, and Rexo1; (ii) lack
of expression of mesodermal markers comprising Brachyury and Bmpr2;
(iii) lack of expression of one or more endodermal markers
comprising Gata6, Sox17 and Sox7; or (iv) lack of expression of one
or more neural markers comprising Nestin and Pax6.
Further provided is a method for modifying a target genomic locus
in an Interleukin-2 receptor gamma locus, an ApoE locus, a Rag1
locus, a Rag2 locus or a Rag2/Rag1 locus in a pluripotent rat cell,
the method comprising: (a) introducing into the pluripotent rat
cell a targeting vector comprising an insert nucleic acid flanked
with 5' and 3' rat homology arms homologous to the target genomic
locus; and (b) identifying a genetically modified pluripotent rat
cell comprising a targeted genetic modification at the target
genomic locus, wherein the targeted genetic modification is capable
of being transmitted through the germline of a rat propagated from
the pluripotent rat cell. In some such methods, the targeting
vector is a large targeting vector (LTVEC), wherein the sum total
of the 5' and the 3' rat homology arms is at least about 10 kb. In
some embodiments, the sum total of the 5' and the 3' rat homology
arms is at least 10 kb but less than 150 kb. In some embodiments,
the sum total of the 5' and the 3' rat homology arms is at least
about 10 kb but less than about 100 kb. In some embodiments,
introducing the targeting vector into the pluripotent rat cell
leads to: (i) a deletion of an endogenous rat nucleic acid sequence
at the target genomic locus; (ii) an insertion of an exogenous
nucleic acid sequence at the target genomic locus; or (iii) a
combination thereof.
In some embodiments, the deletion of the endogenous rat nucleic
acid at the genomic locus is at least about 10 kb; the deletion of
an endogenous rat nucleic acid sequence at the genomic locus ranges
from about 5 kb to about 10 kb, from about 10 kb to about 20 kb,
from about 20 kb to about 40 kb, from about 40 kb to about 60 kb,
from about 60 kb to about 80 kb, from about 80 kb to about 100 kb,
from about 100 kb to about 150 kb, or from about 150 kb to about
200 kb, from about 200 kb to about 300 kb, from about 300 kb to
about 400 kb, from about 400 kb to about 500 kb, from about 500 kb
to about 1 Mb, from about 1 Mb to about 1.5 Mb, from about 1.5 Mb
to about 2 Mb, from about 2 Mb to about 2.5 Mb, or from about 2.5
Mb to about 3 Mb; the insertion of an exogenous nucleic acid
sequence at the genomic locus is at least about 5 kb; or the
insertion of an exogenous nucleic acid sequence ranges from about 5
kb to about 10 kb, from about 10 kb to about 20 kb, from about 20
kb to about 40 kb, from about 40 kb to about 60 kb, from about 60
kb to about 80 kb, from about 80 kb to about 100 kb, from about 100
kb to about 150 kb, from about 150 kb to about 200 kb, from about
200 kb to about 250 kb, from about 250 kb to about 300 kb, from
about 300 kb to about 350 kb, or from about 350 kb to about 400
kb.
In some embodiments, (a) the targeted genetic modification at the
Interleukin-2 receptor gamma locus results in a decrease in or
absence of Interleukin-2 receptor gamma protein activity; (b) the
targeted genetic modification at the ApoE locus results in a
decrease in or absence of ApoE protein activity; (c) the targeted
genetic modification at the Rag1 locus results in a decrease in or
absence of Rag1 protein activity; (d) the targeted genetic
modification at the Rag2 locus results in a decrease in or absence
of Rag2 protein activity; or, (e) the targeted genetic modification
at the Rag2/Rag1 locus results in a decrease in or absence of Rag2
protein activity and Rag1 protein activity.
In some embodiments, the targeted genetic modification at the
Interleukin-2 receptor gamma locus comprises (a) a deletion of the
entire rat Interleukin-2 receptor gamma coding region or a portion
thereof; (b) a replacement of the entire rat Interleukin-2 receptor
gamma coding region or a portion thereof with a human Interleukin-2
receptor gamma coding region or a portion thereof; (c) a
replacement of an ecto-domain of the rat Interleukin-2 receptor
gamma coding region with the ecto-domain of a human Interleukin-2
receptor gamma; or, (d) at least a 3 kb deletion of the
Interleukin-2 receptor gamma locus comprising the Interleukin-2
receptor gamma coding region.
In some embodiments, the targeted genetic modification at the ApoE
locus comprises: (a) a deletion of the entire ApoE coding region or
a portion thereof; or, (b) at least a 1.8 kb deletion of the ApoE
locus comprising the ApoE coding region.
In some embodiments, the targeted genetic modification at the Rag2
locus comprises: (a) a deletion of the entire Rag2 coding region or
a portion thereof; or, (b) at least a 5.7 kb deletion of the Rag2
locus comprising the Rag2 coding region. In other methods, the
targeted genetic modification of the Rag1/Rag2 locus comprises: (a)
a deletion of the entire Rag2 coding region or a portion thereof
and a deletion of the entire Rag1 coding region or portion thereof;
or, (b) a deletion of at least 16 kb of the Rag2/Rag1 locus
comprising the Rag2 and Rag1 coding regions.
In some such embodiments for modifying a target genomic locus, the
insert nucleic acid comprises an expression cassette comprising a
polynucleotide encoding a selective marker. In some such
embodiments, the expression cassette comprises a lacZ gene operably
linked to an endogenous promoter at the genomic locus and a human
ubiquitin promoter operably linked to a selective marker gene.
In some embodiments, the insert nucleic acid comprises a
self-deleting selection cassette. In some such embodiments, the
self-deleting selection cassette comprises a selective marker
operably linked to a promoter active in the rat pluripotent cell
and a polynucleotide encoding a recombinase operably linked to a
male germ cell-specific promoter, wherein the self-deleting
cassette is flanked by recombination recognition sites recognized
by the recombinase. In some such embodiments, the male germ
cell-specific promoter is a Protamine-1 promoter; or, the
recombinase gene encodes Cre and the recombination recognition
sites are loxP sites. In some embodiments, the Protamine-1 promoter
is a mouse or a rat Protamine-1 promoter.
In other methods, the insertion of the exogenous nucleic acid
sequence at the genomic locus comprises a reporter nucleic acid
sequence operably linked to the endogenous Interleukin-2 receptor
gamma promoter, the endogenous ApoE promoter, the endogenous Rag1
promoter, or the endogenous Rag2 promoter. In some such
embodiments, the reporter nucleic acid sequence encodes a reporter
comprising .beta.-galactosidase, mPlum, mCherry, tdTomato,
mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet,
enhanced yellow fluorescent protein (EYFP), Emerald, enhanced green
fluorescent protein (EGFP), CyPet, cyan fluorescent protein (CFP),
Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a
combination thereof.
In some embodiments for modifying a target genomic locus, the
pluripotent rat cell is a rat embryonic stem (ES) cell. In some
such embodiments, the pluripotent rat cell (a) is derived from a DA
strain or an ACI strain; (b) is characterized by expression of a
pluripotency marker comprising Oct-4, Sox-2, alkaline phosphatase,
or a combination thereof; or, (c) is characterized by one or more
of the following characteristics: (i) lack of expression of one or
more pluripotency markers comprising c-Myc, Ecat1, and Rexo1; (ii)
lack of expression of mesodermal markers comprising Brachyury and
Bmpr2; (iii) lack of expression of one or more endodermal markers
comprising Gata6, Sox17 and Sox7; or (iv) lack of expression of one
or more neural markers comprising Nestin and Pax6.
In some embodiments, the method further comprises identifying the
targeted genetic modification at the target genomic locus, wherein
the identification step employs a quantitative assay for assessing
a modification of allele (MOA) at the target genomic locus.
In some embodiments, the introducing step further comprises
introducing a second nucleic acid encoding a nuclease agent that
promotes a homologous recombination between the targeting vector
and the target genomic locus in the pluripotent rat cell. In some
such embodiments, the nuclease agent comprises a chimeric protein
comprising a zinc finger-based DNA binding domain fused to a FokI
endonuclease. Some such methods result in bi-allelic modification
of the target genomic locus.
In some embodiments, the introducing step of the method further
comprises introducing into the pluripotent rat cell: a first
expression construct comprising a first promoter operably linked to
a first nucleic acid sequence encoding a Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas)
protein, and a second expression construct comprising a second
promoter operably linked to a second nucleic acid sequence encoding
a genomic target sequence operably linked to a guide RNA (gRNA),
wherein the genomic target sequence is immediately flanked on the
3' end by a Protospacer Adjacent Motif (PAM) sequence. In one
embodiment, the genomic target sequence comprises the nucleotide
sequence set forth in SEQ ID NO: 1. In one embodiment the gRNA
comprises a third nucleic acid sequence encoding a Clustered
Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA
(crRNA) and a trans-activating CRISPR RNA (tacrRNA). In some such
embodiments, the Cas protein is Cas9. In some such embodiments, (a)
the gRNA is the chimeric RNA of the nucleic acid sequence set forth
in SEQ ID NO: 2; (b) the gRNA is the chimeric RNA of the nucleic
acid sequence set forth in SEQ ID NO: 3; (c) the crRNA comprises a
sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6; or,
(d) the tracrRNA comprises the sequence set forth in SEQ ID NO: 7
and/or SEQ ID NO: 8.
BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
FIG. 1 depicts rat ESCs, which grow as compact spherical colonies
that routinely detach and float in the dish.
FIGS. 2A through D depict various pluripotency markers expressed by
rat ESCs: FIG. 2A depicts Oct-4 (green); FIG. 2B depicts Sox-2
(red); FIG. 2C depicts DAPI (blue); FIG. 2D depicts an overlay of
pluripotency markers expressed by rESCs.
FIG. 3 depicts that the rat ESCs express light levels of alkaline
phosphatase (a pluripotency marker)(left), and the karyotype for
line DA.2B is 42X,Y (right). Karyotyping was done because rat ESCs
often become tetraploid; lines were thus pre-screened by counting
metaphase chromosome spreads, and lines with mostly normal counts
were then formally karyotyped.
FIGS. 4A-B provide photographs showing the analysis of the
chromosome number of the ACI.G1 rat ES cell line.
FIGS. 5A-B provide photographs showing the analysis of the
chromosome number of the DA.2B rat ES cell line.
FIGS. 6A-B provide photographs showing the analysis of the
chromosome number of the DA.C2 rat ES cell line.
FIG. 7 depicts a closer view of a rat ESC of FIG. 1.
FIG. 8 depicts production of chimeras by blastocyst injection and
transmission of the rat ESC genome through the germline; chimeras
produced by blastocyst injection using parental ACI.G1 rat ESCs;
high percentage chimeras usually have albino snouts.
FIG. 9 depicts F1 agouti pups with albino littermates, sired by
ACI/SD chimera labeled with an asterisk (*) in FIG. 8.
FIG. 10 provides a schematic of the rat ApoE locus and denotes with
grey bars the cutting site for zinc finger nucleases (ZFN1 and
ZFN2). The genomic regions corresponding to the 5' and 3' homology
arms (5 kb and 5.4 kb, respectively) are denoted by the dark grey
boxes. Exon 1 of the ApoE gene is non-coding and is shown as an
open box closest to the 5' homology arm. The three introns of the
ApoE gene are denoted as lines. Exons 2 and 3 comprise coding
regions and are shown as stippled grey boxes. Exon 4 contains both
coding and non-coding sequences as denoted by the stippled grey
shading and the open box.
FIG. 11 provides a summary of the ApoE targeting efficiency when
performed in the presence of zinc finger nucleases (ZFN1 or
ZFN2)
FIGS. 12A-C depict targeting of the rat Rosa 26 locus, which lies
between the Setd5 and Thumpd3 genes as in mouse, with the same
spacing. FIG. 12A shows the structure of the mouse Rosa 26 locus.
Mouse Rosa26 transcripts consist of 2 or 3 exons. FIG. 12B depicts
the structure of the rat Rosa26 locus; the rat locus contains a
second exon 1 (Ex1b) in addition to the homologous exon to mouse
exon1 (Ex1a); no third exon has been identified in rat. FIG. 12C
depicts a targeted rat Rosa26 allele; homology arms of 5 kb each
were cloned by PCR using genomic DNA from DA rESC; the targeted
allele contains a Splicing Acceptor (SA)-lacZ-hUB-neo cassette
replacing a 117 bp deletion in the rat Rosa26 intron.
FIG. 13A depicts a control brain of a 14-week-old wild type rat,
which was stained with X-gal. The control brain showed a low level
of background staining for LacZ (dorsal view).
FIG. 13B depicts LacZ expression in the brain of an rRosa26
heterozygous rat (14-week old). The lacZ reporter was expressed
ubiquitously throughout the brain of the rRosa26 heterozygote.
FIG. 13C depicts a control heart and thymus (inset) of a
14-week-old wild type rat, which were treated with X-gal. The
control heart and thymus showed a low level of background staining
for LacZ.
FIG. 13D depicts LacZ expression in the heart and thymus (inset) of
a 14-week-old rRosa26 heterozygous rat. The lacZ reporter was
expressed ubiquitously throughout the heart and thymus of the
rROSA26 heterozygote.
FIG. 13E depicts a control lung of a 14-week-old wild type rat,
which was treated with X-gal. The control lung showed a low level
of background staining for LacZ.
FIG. 13F depicts LacZ expression in the lung of a 14-week-old
rRosa26 heterozygote rat. The lacZ reporter was expressed
ubiquitously throughout the lung of the rRosa26 heterozygote.
FIGS. 13G and H depict LacZ expression in E12.5 rat embryos. In
contrast to the wild-type control embryo (FIG. 13H), which shows a
low level of background LacZ staining, the rRosa26 heterozygous
embryo exhibited ubiquitous expression of the LacZ reporter
throughout the embryo (FIG. 13G).
FIGS. 13I and J depict LacZ expression in E14.5 rat embryos. In
contrast to the wild-type control embryo (FIG. 13J), which shows a
low level of background LacZ staining, the rRosa26 heterozygous rat
embryo exhibited ubiquitous expression of the LacZ reporter
throughout the embryo (FIG. 13I).
FIG. 14 illustrates a homologous or non-homologous recombination
event that occurs inside a rat ES cell following an electroporation
of a targeting vector comprising a selection cassette (lacZ-neo
cassette).
FIG. 15 illustrates the mechanism by which genome-editing
endonucleases (e.g., ZFNs and TALENs) introduce a double strand
break (DSB) in a target genomic sequence and activate
non-homologous end-joining (NHEJ) in an ES cell.
FIG. 16 illustrates a gene targeting technique that utilizes
ZFN/TALENs to improve the efficiency of homologous recombination of
a targeting vector. DSB represents double strand break.
FIG. 17 provides a summary of the chimera production and germline
transmission of the modified rat ApoE locus. The targeted
modification was assisted by zinc finger nucleases.
FIG. 18 provides a schematic of the IL2r-.gamma. targeting event in
combination with zinc finger nucleases that target ZFN U and ZFN D.
ZFN cut sites are noted in the figure.
FIG. 19 provides the targeting efficiency when targeting
IL2r-.gamma. in combination with the CRISPR/Cas9 system.
FIG. 20 provides a schematic of the rat ApoE locus and a targeting
plasmid. The upper schematic shows the genomic structure of the rat
ApoE locus and the genomic regions corresponding to the 5' and 3'
homology arms (5 kb and 5.4 kb respectively; dark grey boxes). Exon
1 of the ApoE gene is non-coding and is shown as an open box
closest to the 5' homology arm. The three introns of the ApoE gene
are denoted as lines. Exons 2 and 3 comprise coding regions and are
shown as stippled grey boxes. Exon 4 contains both coding and
non-coding sequences as denoted by the stippled grey shading and
the open box. The lower panel shows the targeting plasmid. The 5'
and 3' homology arms (5 kb and 5.4 kb, respectively) are denoted by
the dark grey boxes. The targeting vector comprises a reporter gene
(lacZ) and a self-deleting cassette flanked by loxP sites (open
arrows). The self-deleting cassette comprises a mouse Prm1 promoter
operably linked to the Crei gene and a drug selection cassette
comprising a human ubiquitin promoter operably linked to a neomycin
resistance gene.
FIG. 21 provides a schematic for targeting the ApoE locus in rat ES
cells using zinc-finger nucleases and a targeting vector comprising
a reporter gene (LacZ) and a self-deleting cassette comprising a
mouse Prm1 promoter operably linked to the Crei gene and a drug
selection cassette comprising a human ubiquitin promoter operably
linked to a neomycin resistance gene.
FIG. 22 provides a schematic of the rat ApoE locus and a large
targeting vector (LTVEC). The upper panel shows the genomic
organization of the rat ApoE locus and the genomic regions
corresponding to the 5' and 3' homology arms (45 kb and 23 kb,
respectively; the dark grey boxes). Exon 1 of ApoE is non-coding
and is shown as an open box closet to the 5' homology arm. The
three introns of the ApoE gene are denoted as lines and exons 2 and
3 comprise coding regions and are shown as stippled grey boxes.
Exon 4 contains both coding and non-coding sequences as denoted by
the stippled grey shading and the open box. The lower panel shows
the LTVEC for modifying the rat ApoE locus. The 5' and 3' homology
arms (45 kb and 23 kb, respectively) are denoted by the dark grey
boxes. The LTVEC comprises a reporter gene (lacZ) and a
self-deleting cassette flanked by loxP sites (open arrows), which
comprises a mouse Prm1 promoter operably linked to the Crei gene
and a drug selection cassette comprising a human ubiquitin promoter
operably linked to a neomycin resistance gene.
FIG. 23 provides a schematic of the rat ApoE locus and denotes with
grey bars the cutting sites for zinc finger nucleases (ZFN1 and
ZFN2) used together with the large targeting vector (LTVEC) to
enhance homologous recombination between the targeting vector and
the target cognate chromosomal region.
FIG. 24 depicts the rat IL2r-.gamma. locus that has been disrupted
by a 3.2 kb deletion and the insertion of a reporter gene (eGFP)
and a self-deleting cassette comprising a drug selection cassette
(hUb-neo) and the Crei gene operably linked to a mouse Prm1
promoter.
FIG. 25 provides a summary of the germ-line transmitting,
targetable rat embryonic stem cell lines.
FIG. 26 provides another depiction of the rat IL2r-.gamma. locus
that has been disrupted by a 3.2 kb deletion and the insertion of a
reporter gene (eGFP) and a self-deleting cassette comprising the
Crei gene operably linked to a mouse Prm1 promoter and a drug
selection cassette (hUb-Neo).
FIG. 27 provides a schematic of the rat Rag2 locus and a large
targeting vector (LTVEC) for modifying the rat Rag2 locus. The
upper panel shows the genomic organization of the rat Rag2 locus
and the cognate genomic regions corresponding to the 5' and 3'
homology arms (48 kb and 15 kb, respectively; dark grey boxes).
Rag2 comprises single exon denoted by the stippled grey shading.
The lower panel is the LTVEC. The 5' and 3' homology arms (48 kb
and 15 kb, respectively) are denoted by the dark grey boxes. The
LTVEC comprises a reporter gene (lacZ) and a self-deleting cassette
flanked by loxP sites (open arrows) that contains a rat Prm1
promoter operably linked to the Crei gene and a drug selection
cassette containing a human ubiquitin promoter operably linked to a
neomycin resistance gene.
FIG. 28 provides the genomic structure of the rat Rag1/Rag2 locus
and the genomic regions deleted by either Rag2 targeting (Rag2
deletion) or Rag2/Rag1 double targeting (Rag2/Rag1 deletion).
FIG. 29 provides a schematic of the rat Rag2 and Rag1 loci and a
large targeting vector (LTVEC) used for modifying the loci. The
upper panel shows the genomic organization of the Rag1 and Rag2
loci and the cognate genomic regions corresponding to the 5' and 3'
homology arms (48 kb and 84 kb, respectively; dark grey boxes).
Rag2 and Rag1 each comprise a single exon denoted by the stippled
grey shading. The lower panel is the LTVEC. The 5' and 3' homology
arms (48 kb and 84 kb, respectively) are denoted by the dark grey
boxes. The LTVEC comprises a reporter gene (lacZ) and a
self-deleting cassette flanked by loxP sites (open arrows), which
comprises a rat Prm1 promoter operably linked to the Crei gene and
a drug selection cassette comprising a human ubiquitin promoter
operably linked to a neomycin resistance gene.
FIG. 30 shows that II2rg-/y PBMC do not express mature lymphocyte
markers. GFP-positive lymphocytes were detected in peripheral blood
in 2 of the 3 chimeras.
FIG. 31 provides a schematic of the rat IL-2rg locus and a
targeting plasmid for the full humanization of the rat IL-2rg
locus. The upper panel shows the genomic organization of the rat
IL-2rg locus and the cognate genomic regions corresponding to the
5' and 3' homology arms (4.4 kb and 5.0 kb, respectively; dark grey
boxes). The lower panel is the targeting plasmid. The 5' and 3'
homology arms (4.4 kb and 5.0 kb, respectively) are denoted by the
dark grey boxes. The targeting plasmid comprises the human IL-2rg
genomic region, a reporter gene (GFP) and a self-deleting cassette
flanked by loxP sites (open arrows) that contains a mouse Prm1
promoter operably linked to the Crei gene and a drug selection
cassette containing a human ubiquitin promoter operably linked to a
neomycin resistance gene.
FIG. 32 provides a schematic of the rat IL-2rg locus and a
targeting plasmid for the ecto-domain humanization of the rat
IL-2rg locus. The upper panel shows the genomic organization of the
rat IL-2rg locus and the cognate genomic regions corresponding to
the 5' and 3' homology arms (4.4 kb and 5.0 kb, respectively; dark
grey boxes). The lower panel is the targeting plasmid. The 5' and
3' homology arms (4.4 kb and 5.0 kb, respectively) are denoted by
the dark grey boxes. The targeting plasmid comprises the human
ecto-domain of the IL-2Rg genomic region, a reporter gene (GFP) and
a self-deleting cassette flanked by loxP sites (open arrows) that
contains a mouse Prm1 promoter operably linked to the Crei gene and
a drug selection cassette a human ubiquitin promoter operably
linked to a neomycin resistance gene.
FIG. 33 provides a sequence alignment of the human IL-2rg protein
(SEQ ID NO: 20; NP_000197.1); the rat IL-2rg protein (SEQ ID NO:
21; NP_543165.1); and the chimeric IL-2rg protein (SEQ ID NO: 22)
comprising the human ecto-domain of IL-2rg fused to the remainder
of the rat IL-2rg protein. The junction between the human and rat
IL-2rg is noted by the vertical line.
DETAILED DESCRIPTION OF THE INVENTION
Glossary
The term "embryonic stem cell" or "ES cell" as used herein includes
an embryo-derived totipotent or pluripotent cell that is capable of
contributing to any tissue of the developing embryo upon
introduction into an embryo. The term "pluripotent cell" as used
herein includes an undifferentiated cell that possesses the ability
to develop into more than one differentiated cell types.
The term "homologous nucleic acid" as used herein includes a
nucleic acid sequence that is either identical or substantially
similar to a known reference sequence. In one embodiment, the term
"homologous nucleic acid" is used to characterize a sequence having
amino acid sequence that is at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or even 100% identical to a
known reference sequence.
The term "orthologous nucleic acid" as used herein includes a
nucleic acid sequence from one species that is functionally
equivalent to a known reference sequence in another species.
The term "large targeting vector" or "LTVEC" as used herein
includes large targeting vectors for eukaryotic cells that are
derived from fragments of cloned genomic DNA larger than those
typically used by other approaches intended to perform homologous
gene targeting in eukaryotic cells. Examples of LTVEC, include, but
are not limited to, bacterial homologous chromosome (BAC) and yeast
artificial chromosome (YAC).
The term "modification of allele" (MOA) as used herein includes the
modification of the exact DNA sequence of one allele of a gene(s)
or chromosomal locus (loci) in a genome. Examples of "modification
of allele (MOA)" as described herein includes, but is not limited
to, deletions, substitutions, or insertions of as little as a
single nucleotide or deletions of many kilobases spanning a gene(s)
or chromosomal locus (loci) of interest, as well as any and all
possible modifications between these two extremes.
The term "recombination site" as used herein includes a nucleotide
sequence that is recognized by a site-specific recombinase and that
can serve as a substrate for a recombination event.
"Serial" genetic modifications include two or more modifications
to, e.g., a rat ES cell, conducted independently. For example, a
first modification is made to a rat ES cell genome employing a
suitable first nucleic acid construct. The first modification may
be achieved by electroporation, or any other method known in the
art. Then a second modification is made to the same rat ES cell
genome employing a suitable second nucleic acid construct. The
second modification may be achieved by a second electroporation, or
any other method known in the art. In various embodiments,
following the first and the second genetic modifications of the
same rat ES cell, a third, a fourth, a fifth, a sixth, and so on,
serial genetic modifications (one following another) may be
achieved using, e.g., serial electroporation or any other suitable
method (serially) known in the art.
The term "site-specific recombinase" as used herein includes a
group of enzymes that can facilitate recombination between
"recombination sites" where the two recombination sites are
physically separated within a single nucleic acid molecule or on
separate nucleic acid molecules. Examples of "site-specific
recombinase" include, but are not limited to, Cre, Flp, and Dre
recombinases.
The term "germline" in reference to a nucleic acid sequence
includes a nucleic acid sequence that can be passed to progeny.
The phrase "heavy chain," or "immunoglobulin heavy chain" includes
an immunoglobulin heavy chain sequence, including immunoglobulin
heavy chain constant region sequence, from any organism. Heavy
chain variable domains include three heavy chain CDRs and four FR
regions, unless otherwise specified. Fragments of heavy chains
include CDRs, CDRs and FRs, and combinations thereof. A typical
heavy chain has, following the variable domain (from N-terminal to
C-terminal), a C.sub.H1 domain, a hinge, a C.sub.H2 domain, and a
C.sub.H3 domain. A functional fragment of a heavy chain includes a
fragment that is capable of specifically recognizing an epitope
(e.g., recognizing the epitope with a K.sub.D in the micromolar,
nanomolar, or picomolar range), that is capable of expressing and
secreting from a cell, and that comprises at least one CDR. Heavy
chain variable domains are encoded by variable region nucleotide
sequence, which generally comprises V.sub.H, D.sub.H, and J.sub.H
segments derived from a repertoire of V.sub.H, D.sub.H, and J.sub.H
segments present in the germline. Sequences, locations and
nomenclature for V, D, and J heavy chain segments for various
organisms can be found in IMGT database, which is accessible via
the internet on the world wide web (www) at the URL "imgt.org."
The phrase "light chain" includes an immunoglobulin light chain
sequence from any organism, and unless otherwise specified includes
human kappa (.kappa.) and lambda (.lamda.) light chains and a
VpreB, as well as surrogate light chains. Light chain variable
domains typically include three light chain CDRs and four framework
(FR) regions, unless otherwise specified. Generally, a full-length
light chain includes, from amino terminus to carboxyl terminus, a
variable domain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a
light chain constant region amino acid sequence. Light chain
variable domains are encoded by the light chain variable region
nucleotide sequence, which generally comprises light chain V.sub.L
and light chain J.sub.L gene segments, derived from a repertoire of
light chain V and J gene segments present in the germline.
Sequences, locations and nomenclature for light chain V and J gene
segments for various organisms can be found in IMGT database, which
is accessible via the internet on the world wide web (www) at the
URL "imgt.org." Light chains include those, e.g., that do not
selectively bind either a first or a second epitope selectively
bound by the epitope-binding protein in which they appear. Light
chains also include those that bind and recognize, or assist the
heavy chain with binding and recognizing, one or more epitopes
selectively bound by the epitope-binding protein in which they
appear.
The phrase "operably linked" comprises a relationship wherein the
components operably linked function in their intended manner. In
one instance, a nucleic acid sequence encoding a protein may be
operably linked to regulatory sequences (e.g., promoter, enhancer,
silencer sequence, etc.) so as to retain proper transcriptional
regulation. In one instance, a nucleic acid sequence of an
immunoglobulin variable region (or V(D)J segments) may be operably
linked to a nucleic acid sequence of an immunoglobulin constant
region so as to allow proper recombination between the sequences
into an immunoglobulin heavy or light chain sequence.
1. Target Locus Comprising a Rat Nucleic Acid
Various methods and compositions are provided, which allow for the
integration of at least one insert nucleic acid at a target locus.
As used herein, a "genomic locus of interest" comprises any segment
or region of DNA within the genome that one desires to integrate an
insert nucleic acid. The terms "genomic locus of interest" and
"target genomic locus of interest" can be used interchangeable. The
genomic locus of interest can be native to the cell, or
alternatively can comprise a heterologous or exogenous segment of
DNA that was integrated into the genome of the cell. Such
heterologous or exogenous segments of DNA can include transgenes,
expression cassettes, polynucleotide encoding selection makers, or
heterologous or exogenous regions of genomic DNA. The term "locus"
is a defined herein as a segment of DNA within the genomic DNA.
Genetic modifications as described herein can include one or more
deletions from a locus of interest, additions to a locus of
interest, replacement of a locus of interest, and/or any
combination thereof. The locus of interest can comprise coding
regions or non-coding regulatory regions.
The genomic locus of interest can further comprise any component of
a targeted integration system including, for example, a recognition
site, a selection marker, a previously integrated insert nucleic
acid, polynucleotides encoding nuclease agents, promoters, etc.
Alternatively, the genomic locus of interest can be located within
a yeast artificial chromosome (YAC), bacterial artificial
chromosome (BAC), a human artificial chromosome, or any other
engineered genomic region contained in an appropriate host cell. In
various embodiments, the targeted locus can comprise native,
heterologous, or exogenous nucleic acid sequence from a prokaryote,
a eukaryote, yeast, bacteria, a non-human mammal, a non-human cell,
a rodent, a human, a rat, a mouse, a hamster, a rabbit, a pig, a
bovine, a deer, a sheep, a goat, a chicken, a cat, a dog, a ferret,
a primate (e.g., marmoset, rhesus monkey), domesticated mammal or
an agricultural mammal or any other organism of interest or a
combination thereof.
In specific embodiments, the genomic locus of interest comprises a
target locus of a "rat nucleic acid". Such a region comprises a
nucleic acid from a rat that is integrated within the genome of a
cell.
Non-limiting examples of the target locus include a genomic locus
that encodes a protein expressed in a B cell, a genomic locus that
expresses a polypeptide in an immature B cell, a genomic locus that
expresses a polypeptide in a mature B cell, an immunoglobulin (Ig)
loci, or a T cell receptor loci, including, for example, a T cell
receptor alpha locus. Additional examples of target genomic locus
include an FcER1a locus, a TLR4 locus, a PRLR locus, a Notch4
locus, an Accn2 locus, an Adamts5 locus, a TRPA1 locus, FolH1
locus, an LRP5 locus, an IL2 receptor locus, including, for
example, an IL2 Receptor gamma (IL2Rg) locus, an ApoE locus, a Rag1
locus, a Rag2 locus, a Rag1/Rag2 locus, and an ERBB4 locus. Any
such target locus can be from a rat.
In one embodiment, the target locus encodes a mammalian
immunoglobulin heavy chain variable region amino acid sequence. In
one embodiment, the target locus encodes a rat immunoglobulin heavy
chain variable region amino acid sequence. In one embodiment, the
target locus comprises a genomic DNA sequence comprising an
unrearranged rat, mouse, or human immunoglobulin heavy chain
variable region nucleic acid sequence operably linked to an
immunoglobulin heavy chain constant region nucleic acid sequence.
In one embodiment, the immunoglobulin heavy chain constant region
nucleic acid sequence is a rat, mouse, or human immunoglobulin
heavy chain constant region nucleic acid sequence selected from a
CH1, a hinge, a CH2, a CH3, and a combination thereof. In one
embodiment, the heavy chain constant region nucleic acid sequence
comprises a CH1-hinge-CH2-CH3. In one embodiment, the target locus
comprises a rearranged rat, mouse, or human immunoglobulin heavy
chain variable region nucleic acid sequence operably linked to an
immunoglobulin heavy chain constant region nucleic acid sequence.
In one embodiment, the immunoglobulin heavy chain constant region
nucleic acid sequence is a rat, mouse, or human immunoglobulin
heavy chain constant region nucleic acid sequence selected from a
CH1, a hinge, a CH2, a CH3, and a combination thereof. In one
embodiment, the heavy chain constant region nucleic acid sequence
comprises a CH1-hinge-CH2-CH3.
In one embodiment, the target locus comprises a genomic DNA
sequence that encodes a mammalian immunoglobulin light chain
variable region amino acid sequence. In one embodiment, the genomic
DNA sequence comprises an unrearranged mammalian .lamda. and/or
.kappa. light chain variable region nucleic acid sequence.
In one embodiment, the genomic DNA sequence comprises a rearranged
mammalian .lamda. and/or .kappa. light chain variable region
nucleic acid sequence. In one embodiment, the unrearranged .lamda.
or .kappa. light chain variable region nucleic acid sequence is
operably linked to a mammalian immunoglobulin light chain constant
region nucleic acid sequence selected from a .lamda. light chain
constant region nucleic acid sequence and a .kappa. light chain
constant region nucleic acid sequence. In one embodiment, the
mammalian immunoglobulin light chain constant region nucleic acid
sequence is a rat immunoglobulin light chain constant region
nucleic acid sequence. In one embodiment, the mammalian
immunoglobulin light chain constant region nucleic acid sequence is
a mouse immunoglobulin light chain constant region nucleic acid
sequence. In one embodiment, the mammalian immunoglobulin light
chain constant region nucleic acid sequence is a human
immunoglobulin light chain constant region nucleic acid
sequence.
As used herein, a rat ApoE locus, a rat interleukin-2 receptor
gamma (II-2rg) locus, a rat Rag2 locus, a rat Rag1 locus and/or a
rat Rag2/Rag1 locus comprise the respective regions of the rat
genome in which each of these genes or gene combinations are
located. Modifying any one of the rat ApoE locus, the rat
interleukin-2 receptor gamma locus, the rat Rag2 locus, the rat
Rag1 locus and/or the combined rat Rag2/Rag1 locus can comprise any
desired alteration to the given locus. Non-limiting examples of
modification to the given rat locus are discussed in further detail
herein.
For example, in specific embodiments, one or more of the rat ApoE
locus, the rat interleukin-2 receptor gamma locus, the Rag2 locus,
and/or the Rag2/Rag1 locus is modified such that the activity
and/or level of the encoded ApoE protein or the interleukin-2
receptor gamma protein or the Rag1 protein or the Rag2 protein or a
combination of the Rag1 and Rag2 proteins are decreased. In other
embodiments, the activity of the ApoE protein, the interleukin-2
receptor gamma protein, the Rag1 protein, or the Rag2 protein, or a
combination of the Rag1 and Rag2 proteins is absent.
By "decreased" is intended any decrease in the level or activity of
the gene/protein encoded at the locus of interest. For example, a
decrease in activity can comprise either (1) a statistically
significant decrease in the overall level or activity of a given
protein (i.e., ApoE, interleukin-2 receptor gamma, Rag2, Rag2 or a
combination of Rag1 and Rag2) including, for example, a decreased
level or activity of 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, 120% or greater when compared to an
appropriate control. Methods to assay for a decrease in the
concentration and/or the activity of anyone of ApoE, interleukin-2
receptor gamma, Rag1 and Rag2 are known in the art.
In other embodiments, one or more of the rat ApoE locus, the rat
interleukin-2 receptor gamma locus, the rat Rag2 locus, the rat
Rag1 locus and/or rat Rag2/Rag1 locus comprise a modification such
that the activity and/or level of the encoded ApoE polypeptide, the
interleukin-2 receptor gamma polypeptide, the Rag2 polypeptide, the
Rag1 polypeptide, or both the Rag1 and Rag2 polypeptide is
increased. By "increased" is intended any increase in the level or
activity of the gene/polypeptide encoded at the locus of interest.
For example, an increase in activity can comprise either (1) a
statistically significant increase in the overall level or activity
of a given protein (i.e., ApoE, interleukin-2 receptor gamma, Rag1,
Rag2 or Rag1 and Rag2) including, for example, an increased level
or activity of 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100%, 120% or greater when compared to an appropriate
control. Methods to assay for an increase in the concentration
and/or the activity of anyone of the ApoE, Rag1, Rag2 and
interleukin-2 receptor gamma proteins are known in the art.
The genetic modification to the rat ApoE locus, the rat
interleukin-2 receptor gamma locus, the rat Rag2 locus, the rat
Rag1 locus and/or rat Rag2/Rag1 locus can comprise a deletion of an
endogenous rat nucleic acid sequence at the genomic locus, an
insertion of an exogenous nucleic acid at the genomic locus, or a
combination thereof. The deletion and/or insertion can occur
anywhere within the given locus as discussed elsewhere herein.
Further embodiments provided herein comprise the modification of
one or more of the rat ApoE locus, the rat interleukin-2 receptor
gamma locus, the rat Rag2 locus, the rat Rag1 locus and/or the rat
Rag2/Rag1 locus through the replacement of a portion of the rat
ApoE locus, the interleukin-2 receptor gamma locus, Rag2 locus,
Rag1 locus and/or Rag2/Rag1 locus with the corresponding homologous
or orthologous portion of an ApoE locus, an interleukin-2 receptor
gamma locus, a Rag2 locus, a Rag1 locus and/or a Rag2/Rag1 locus
from another organism.
Still other embodiments, the modification of one or more of the rat
ApoE locus, the rat interleukin-2 receptor gamma locus, Rag2 locus,
Rag1 locus, and/or Rag2/Rag1 locus is carried out through the
replacement of a portion of the rat ApoE locus, the rat
interleukin-2 receptor gamma locus and/or the rat Rag2 locus,
and/or the Rag1 locus and/or Rag2/Rag1 locus with an insert
polynucleotide sharing across its full length least 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to a portion of an ApoE
locus, an interleukin-2 receptor gamma locus, a Rag2 locus, a Rag1
locus and/or a Rag2/Rag1 locus it is replacing.
The given insert polynucleotide and/or the corresponding region of
the rat locus being deleted can be a coding region, an intron, an
exon, an untranslated region, a regulatory region, a promoter, or
an enhancer or any combination thereof or any portion thereof.
Moreover, the given insert polynucleotide and/or the region of the
rat locus being deleted can be of any desired length, including for
example, between 10-100 nucleotides in length, 100-500 nucleotides
in length, 500-1 kb nucleotide in length, 1 Kb to 1.5 kb nucleotide
in length, 1.5 kb to 2 kb nucleotides in length, 2 kb to 2.5 kb
nucleotides in length, 2.5 kb to 3 kb nucleotides in length, 3 kb
to 5 kb nucleotides in length, 5 kb to 8 kb nucleotides in length,
8 kb to 10 kb nucleotides in length or more. In other instances,
the size of the insertion or replacement is from about 5 kb to
about 10 kb, from about 10 kb to about 20 kb, from about 20 kb to
about 40 kb, from about 40 kb to about 60 kb, from about 60 kb to
about 80 kb, from about 80 kb to about 100 kb, from about 100 kb to
about 150 kb, from about 150 kb to about 200 kb, from about 200 kb
to about 250 kb, from about 250 kb to about 300 kb, from about 300
kb to about 350 kb, from about 350 kb to about 400 kb, from about
400 kb to about 800 kb, from about 800 kb to 1 Mb, from about 300
kb to about 400 kb, from about 400 kb to about 500 kb, from about
500 kb to 1 Mb, from about 1 Mb to about 1.5 Mb, from about 1.5 Mb
to about 2 Mb, from about 2 Mb to about 2.5 Mb, from about 2.5 Mb
to about 2.8 Mb, from about 2.8 Mb to about 3 Mb. In other
embodiments, the given insert polynucleotide and/or the region of
the rat locus being deleted is at least 100, 200, 300, 400, 500,
600, 700, 800, or 900 nucleotides or at least 1 kb, 2 kb, 3 kb, 4
kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14
kb, 15 kb, 16 kb or greater.
The given insert polynucleotide can be from any organism,
including, for example, a rodent, a rat, a mouse, a hamster, a
mammal, a non-human mammal, a human, an agricultural animal or a
domestic animal.
As discussed in further detail herein, various methods are provided
to generate targeted modifications of any rat locus of interest,
including for example, targeted modifications in the rat ApoE
locus, the rat interleukin-2 receptor gamma locus, the rat Rag2
locus, the rat Rag1 locus, and/or the rat Rag2/Rag1 locus. Further
provided are genetically modified rats or genetically modified
pluripotent rat cells (e.g., an rat ES cells), which comprise a
deletion, an insertion, a replacement and/or any combination
thereof at the interleukin-2 receptor gamma locus, at the ApoE
locus, at the rat Rag2 locus, at the rat Rag1 locus, and/or at the
rat Rag2/Rag1 locus. Such genetic modifications (including those
that result in an absence, a decrease, an increase or a modulation
in activity of the target locus) and are also capable of being
transmitted through the germline. In specific embodiments, the
genetic modifications result in a knockout of the desired target
locus. Such rats find use in in a variety of experimental systems
as discussed elsewhere herein.
For example, ApoE (Apolipoprotein E) knockouts in rats offer an
animal model to study endothelial function, including, but not
limited to, plaque formation, transcriptional changes (Whole
Transcriptome Shotgun Sequencing (RNA-Seq), and ex vivo function.
Moreover, the larger size of rats facilitate all these assays and
potentially improve the quality of the RNA-Seq data. ApoE is an
important transport molecule and can transport lipids, such as
cholesterol, through the bloodstream. ApoE can also function in the
nervous system, for example, to clear .beta.-amyloid from the
brain. Modifications in ApoE have been implicated in various
conditions, including, for example, atherosclerosis,
hyperlipidemia, and Alzheimer's disease. ApoE knockout animals
display impaired clearing of lipoproteins from the blood and
develop atherosclerosis. Thus, ApoE knockout animals provide a
model to study conditions and/or processes such as, for example,
endothelia function, plaque formation, transcriptional changes
(RNA-Seq), hyperlipidemia, atherosclerosis and Alzheimer's disease.
Assays to measure ApoE activity are known in the art. For example,
a decrease in ApoE activity can be measured by assaying for a
decrease in the ApoE levels in a blood sample obtained from a
subject by immunoassays, such as by ELISA or by Immunoblotting
techniques. Moreover, the large size of rats facilitates all these
assays and improves the quality of the data.
RAG1 (Recombination-Activating Gene 1) and RAG2
(Recombination-Activating Gene 2) are enzymes that are part of a
multi-subunit complex having VDJ recombination activity and play an
important role in the rearrangement and recombination of
immunoglobulin and T-cell receptor genes in lymphocytes. RAG1 and
RAG2 induce a double stranded DNA cleavage to facilitate
recombination and join of segments of the T cell receptor and B
cell receptor (i.e. immunoglobulin) genes. Knockout of RAG1 and/or
RAG2 causes a loss of B cells and T cells in the animal resulting
in severe immunodeficiency. RAG1 and/or RAG2 knockout animals find
use, for example, in studies of xenografts (i.e. human cell
xenografts in rats), cancer, vaccine development, autoimmune
disease, infectious disease and graft versus host disease (GVHD).
Various assays to measure RAG1 and/or RAG2 activity are known in
the art and include, for example, measuring recombination
efficiency or assaying for the presence or absence of B cells
and/or T cells in a subject. Moreover, the large size of rats
facilitates all these assays and potentially improves the quality
of the data.
The IL-2 receptor (IL-2R) is expressed on the surface of certain
immune cells and binds to the cytokine interleukin-2 (IL-2). The
IL-2R is an integral membrane protein comprising at least three
separate subunit chains, including, an alpha chain (IL-2Ra, CD25),
a beta chain (IL-2Rb, CD122) and a gamma chain (IL2-Rg, CD132). The
IL-2 receptor gamma (also referred to as IL2r-.gamma. or IL2Rg)
chain is a common gamma chain that is shared by various cytokine
receptors, including, for example, the receptors for IL-2, IL-4,
IL-7, IL-9, IL-15 and IL-21. IL-2Rg comprises an ectodomain on the
extracellular surface of the cell, which contributes to the binding
of the ligand, a transmembrane domain, and an intracellular domain
which can interact with various molecules to induce intracellular
signal transduction pathways. The Il2rg gene is found on the
X-chromosome in mammals and certain mutations in the gamma chain
gene in humans can cause human X-linked severe combined
immunodeficiency (XSCID) characterized by a profound T-cell defect.
In addition, the gamma chain ecto-domain can be shed off of the
transmembrane receptor and released as a soluble gamma chain
receptor. The soluble gamma chain receptor can be detected in the
blood of a subject and can function to regulate cytokine
signaling.
In some embodiments, the rat IL-2Rg chain is replaced with the
human IL2-Rg chain such that the rat expresses a fully human IL-2Rg
chain. In other instances, it may be useful to replace only the
ectodomain of the rat IL-2Rg chain with the ectodomain of the human
IL-2Rg chain. In such cases, the resulting humanized IL-2Rg chain
expressed in a rat comprises a human ectodomain, with the remainder
of the molecule being from the rat.
The full-length humanization of IL-2Rg is useful because rats
having this modified locus will produce human IL-2Rg. This will
allow for the detection of human IL-2Rg in rats with antibodies
specific to human IL-2Rg. The ecto-humanization (i.e., replacing
the rat ecto-domain of IL-2Rg with the human ecto-domain of IL-2Rg)
will result in an IL-2Rg polypeptide that will bind the human
ligands for IL2-Rg, but because the cytoplasmic domain is still
rat, it ecto-humanized form of IL-2Rg will also interact with the
rat signaling machinery.
2. Modifying a Rat Target Locus
A. Targeting Vectors and Insert Nucleic Acids
i. Insert Nucleic Acid
As used herein, the "insert nucleic acid" comprises a segment of
DNA that one desires to integrate at the target locus. In one
embodiment, the insert nucleic acid comprises one or more
polynucleotides of interest. In other embodiments, the insert
nucleic acid can comprise one or more expression cassettes. A given
expression cassette can comprise a polynucleotide of interest, a
polynucleotide encoding a selection marker and/or a reporter gene
along with the various regulatory components that influence
expression. Non-limiting examples of polynucleotides of interest,
selection markers, and reporter genes that can be included within
the insert nucleic acid are discussed in detail elsewhere
herein.
In specific embodiments, the insert nucleic acid can comprise a
nucleic acid from rat, which can include a segment of genomic DNA,
a cDNA, a regulatory region, or any portion or combination thereof.
In other embodiments, the insert nucleic acid can comprise a
nucleic acid from a non-human mammal, a rodent, a human, a rat, a
mouse, a hamster a rabbit, a pig, a bovine, a deer, a sheep, a
goat, a chicken, a cat, a dog, a ferret, a primate (e.g., marmoset,
rhesus monkey), domesticated mammal or an agricultural mammal or
any other organism of interest. As outlined in further detail
herein, the insert nucleic acid employed in the various methods and
compositions can result in the "humanization" of the a target locus
comprising a rat nucleic acid.
In one embodiment, the insert nucleic acid comprises a knock-in
allele of at least one exon of an endogenous gene. In one
embodiment, the insert nucleic acid comprises a knock-in allele of
the entire endogenous gene (i.e., "gene-swap knock-in").
In one embodiment, the insert nucleic acid comprises a regulatory
element, including for example, a promoter, an enhancer, or a
transcriptional repressor-binding element.
In further embodiments, the insert nucleic acid comprises a
conditional allele. In one embodiment, the conditional allele is a
multifunctional allele, as described in US 2011/0104799, which is
incorporated by reference in its entirety. In specific embodiments,
the conditional allele comprises: (a) an actuating sequence in
sense orientation with respect to transcription of a target gene,
and a drug selection cassette in sense or antisense orientation;
(b) in antisense orientation a nucleotide sequence of interest
(NSI) and a conditional by inversion module (COIN, which utilizes
an exon-splitting intron and an invertible genetrap-like module;
see, for example, US 2011/0104799, which is incorporated by
reference in its entirety); and (c) recombinable units that
recombine upon exposure to a first recombinase to form a
conditional allele that (i) lacks the actuating sequence and the
DSC, and (ii) contains the NSI in sense orientation and the COIN in
antisense orientation.
The insert nucleic acid ranges from about 5 kb to about 10 kb, from
about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from
about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from
about 80 kb to about 100 kb, from about 100 kb to about 150 kb,
from about 150 kb to about 200 kb, from about 200 kb to about 250
kb, from about 250 kb to about 300 kb, from about 300 kb to about
350 kb, or from about 350 kb to about 400 kb.
In one embodiment, the insert nucleic acid comprises a deletion of
a rat genomic DNA sequence ranging from about 1 kb to about 200 kb,
from about 2 kb to about 20 kb, or from about 0.5 kb to about 3 Mb.
In one embodiment, the extent of the deletion of the genomic DNA
sequence is greater than a total length of the 5' homology arm and
the 3' homology arm. In one embodiment, the extent of the deletion
of the genomic DNA sequence ranges from about 5 kb to about 10 kb,
from about 10 kb to about 20 kb, from about 20 kb to about 40 kb,
from about 40 kb to about 60 kb, from about 60 kb to about 80 kb,
from about 80 kb to about 100 kb, from about 100 kb to about 150
kb, from about 150 kb to about 200 kb, from about 20 kb to about 30
kb, from about 30 kb to about 40 kb, from about 40 kb to about 50
kb, from about 50 kb to about 60 kb, from about 60 kb to about 70
kb, from about 70 kb to about 80 kb, from about 80 kb to about 90
kb, from about 90 kb to about 100 kb, from about 100 kb to about
110 kb, from about 110 kb to about 120 kb, from about 120 kb to
about 130 kb, from about 130 kb to about 140 kb, from about 140 kb
to about 150 kb, from about 150 kb to about 160 kb, from about 160
kb to about 170 kb, from about 170 kb to about 180 kb, from about
180 kb to about 190 kb, from about 190 kb to about 200 kb, from
about 200 kb to about 250 kb, from about 250 kb to about 300 kb,
from about 300 kb to about 350 kb, from about 350 kb to about 400
kb, from about 400 kb to about 800 kb, from about 800 kb to 1 Mb,
from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb,
from about 2 Mb, to about 2.5 Mb, from about 2.5 Mb to about 2.8
Mb, from about 2.8 Mb to about 3 Mb, from about 200 kb to about 300
kb, from about 300 kb to about 400 kb, from about 400 kb to about
500 kb, from about 500 kb to about 1 Mb, from about 1 Mb to about
1.5 Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb to about
2.5 Mb, or from about 2.5 Mb to about 3 Mb.
In one embodiment, the insert nucleic acid comprises an insertion
or a replacement of a rat nucleic acid sequence with a homologous
or orthologous human nucleic acid sequence. In one embodiment, the
insert nucleic acid comprises an insertion or replacement of a rat
DNA sequence with a homologous or orthologous human nucleic acid
sequence at an endogenous rat locus that comprises the
corresponding rat DNA sequence.
In one embodiment, the genetic modification is an addition of a
nucleic acid sequence. In one embodiment, the added nucleotide
sequence ranges from 5 kb to 200 kb.
In one embodiment, the insert nucleic acid comprises a genetic
modification in a coding sequence. In one embodiment, the genetic
modification comprises a deletion mutation of a coding sequence. In
one embodiment, the genetic modification comprises a fusion of two
endogenous coding sequences.
In one embodiment, the insert nucleic acid comprises an insertion
or a replacement of a rat nucleic acid sequence with a homologous
or orthologous human nucleic acid sequence. In one embodiment, the
insert nucleic acid comprises an insertion or replacement of a rat
DNA sequence with a homologous or orthologous human nucleic acid
sequence at an endogenous rat locus that comprises the
corresponding rat DNA sequence.
In one embodiment, the genetic modification comprises a deletion of
a non-protein-coding sequence, but does not comprise a deletion of
a protein-coding sequence. In one embodiment, the deletion of the
non-protein-coding sequence comprises a deletion of a regulatory
element. In one embodiment, the genetic modification comprises a
deletion of a promoter. In one embodiment, the genetic modification
comprises an addition of a promoter or a regulatory element. In one
embodiment, the genetic modification comprises a replacement of a
promoter or a regulatory element.
In one embodiment, the nucleic acid sequence of the targeting
vector can comprise a polynucleotide that when integrated into the
genome will produce a genetic modification of a region of the rat
ApoE locus, wherein the genetic modification at the ApoE locus
results in a decrease in ApoE activity, increase in ApoE activity,
or a modulation of ApoE activity. In one embodiment, an ApoE
knockout ("null allele") is generated.
In one embodiment, the nucleic acid sequence of the targeting
vector can comprise a polynucleotide that when integrated into the
genome will produce a genetic modification of a region of the rat
interleukin-2 receptor locus, wherein the genetic modification at
the interleukin-2 receptor locus results in a decrease in
interleukin-2 receptor activity. In one embodiment, an
interleukin-2 receptor knockout ("null allele") is generated.
In further embodiments, the insert nucleic acid results in the
replacement of a portion of the rat ApoE locus, the interleukin-2
receptor gamma locus and/or Rag2 locus, and/or Rag1 locus and/or
Rag2/Rag1 locus with the corresponding homologous or orthologous
portion of an ApoE locus, an interleukin-2 receptor gamma locus, a
Rag2 locus, a Rag1 locus and/or a Rag2/Rag1 locus from another
organism.
Still other embodiments, the insert nucleic acid comprises a
polynucleotide sharing across its full length least 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to a portion of an ApoE
locus, an interleukin-2 receptor gamma locus, a Rag2 locus, a Rag1
locus and/or a Rag2/Rag1 locus it is replacing.
The given insert polynucleotide and the corresponding region of the
rat locus being replaced can be a coding region, an intron, an
exon, an untranslated region, a regulatory region, a promoter, or
an enhancer or any combination thereof. Moreover, the given insert
polynucleotide and/or the region of the rat locus being deleted can
be of any desired length, including for example, between 10-100
nucleotides in length, 100-500 nucleotides in length, 500-1 kb
nucleotide in length, 1 Kb to 1.5 kb nucleotide in length, 1.5 kb
to 2 kb nucleotides in length, 2 kb to 2.5 kb nucleotides in
length, 2.5 kb to 3 kb nucleotides in length, 3 kb to 5 kb
nucleotides in length, 5 kb to 8 kb nucleotides in length, 8 kb to
10 kb nucleotides in length or more. In other instances, the size
of the insertion or replacement is from about 5 kb to about 10 kb,
from about 10 kb to about 20 kb, from about 20 kb to about 40 kb,
from about 40 kb to about 60 kb, from about 60 kb to about 80 kb,
from about 80 kb to about 100 kb, from about 100 kb to about 150
kb, from about 150 kb to about 200 kb, from about 200 kb to about
250 kb, from about 250 kb to about 300 kb, from about 300 kb to
about 350 kb, from about 350 kb to about 400 kb, from about 400 kb
to about 800 kb, from about 800 kb to 1 Mb, from about 1 Mb to
about 1.5 Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb, to
about 2.5 Mb, from about 2.5 Mb to about 2.8 Mb, from about 2.8 Mb
to about 3 Mb. In other embodiments, the given insert
polynucleotide and/or the region of the rat locus being deleted is
at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides
or at least 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb,
10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb or greater.
In one embodiment, the promoter is constitutively active
promoter.
In one embodiment, the promoter is an inducible promoter. In one
embodiment, the inducible promoter is a chemically-regulated
promoter. In one embodiment, the chemically-regulated promoter is
an alcohol-regulated promoter. In one embodiment, the
alcohol-regulated promoter is an alcohol dehydrogenase (alcA) gene
promoter. In one embodiment, the chemically-regulated promoter is a
tetracycline-regulated promoter. In one embodiment, the
tetracycline-regulated promoter is a tetracycline-responsive
promoter. In one embodiment, the tetracycline-regulated promoter is
a tetracycline operator sequence (tetO). In one embodiment, the
tetracycline-regulated promoter is a tet-On promoter. In one
embodiment, the tetracycline-regulated promoter a tet-Off promoter.
In one embodiment, the chemically-regulated promoter is a steroid
regulated promoter. In one embodiment, the steroid regulated
promoter is a promoter of a rat glucocorticoid receptor. In one
embodiment, the steroid regulated promoter is a promoter of an
estrogen receptor. In one embodiment, the steroid-regulated
promoter is a promoter of an ecdysone receptor. In one embodiment,
the chemically-regulated promoter is a metal-regulated promoter. In
one embodiment, the metal-regulated promoter is a metalloprotein
promoter. In one embodiment, the inducible promoter is a
physically-regulated promoter. In one embodiment, the
physically-regulated promoter is a temperature-regulated promoter.
In one embodiment, the temperature-regulated promoter is a heat
shock promoter. In one embodiment, the physically-regulated
promoter is a light-regulated promoter. In one embodiment, the
light-regulated promoter is a light-inducible promoter. In one
embodiment, the light-regulated promoter is a light-repressible
promoter.
In one embodiment, the promoter is a tissue-specific promoter. In
one embodiment, the promoter is a neuron-specific promoter. In one
embodiment, the promoter is a glia-specific promoter. In one
embodiment, the promoter is a muscle cell-specific promoter. In one
embodiment, the promoter is a heart cell-specific promoter. In one
embodiment, the promoter is a kidney cell-specific promoter. In one
embodiment, the promoter is a bone cell-specific promoter. In one
embodiment, the promoter is an endothelial cell-specific promoter.
In one embodiment, the promoter is an immune cell-specific
promoter. In one embodiment, the immune cell promoter is a B cell
promoter. In one embodiment, the immune cell promoter is a T cell
promoter.
In one embodiment, the promoter is a developmentally-regulated
promoter. In one embodiment, the developmentally-regulated promoter
is active only during an embryonic stage of development. In one
embodiment, the developmentally-regulated promoter is active only
in an adult cell.
In some embodiments, the insert nucleic acid comprises a nucleic
acid flanked with site-specific recombination target sequences. It
is recognized the while the entire insert nucleic acid can be
flanked by such site-specific recombination target sequences, any
region or individual polynucleotide of interest within the insert
nucleic acid can also be flanked by such sites. The site-specific
recombinase can be introduced into the cell by any means, including
by introducing the recombinase polypeptide into the cell or by
introducing a polynucleotide encoding the site-specific recombinase
into the host cell. The polynucleotide encoding the site-specific
recombinase can be located within the insert nucleic acid or within
a separate polynucleotide. The site-specific recombinase can be
operably linked to a promoter active in the cell including, for
example, an inducible promoter, a promoter that is endogenous to
the cell, a promoter that is heterologous to the cell, a
cell-specific promoter, a tissue-specific promoter, or a
developmental stage-specific promoter. Site-specific recombination
target sequences, which can flank the insert nucleic acid or any
polynucleotide of interest in the insert nucleic acid can include,
but are not limited to, loxP, lox511, lox2272, lox66, lox71, loxM2,
lox5171, FRT, FRT11, FRT71, attp, att, FRT, rox, and a combination
thereof.
In some embodiments, the site-specific recombination sites flank a
polynucleotide encoding a selection marker and/or a reporter gene
contained within the insert nucleic acid. In such instances
following integration of the insert nucleic acid at the targeted
locus the sequences between the site-specific recombination sites
can be removed.
In one embodiment, the insert nucleic acid comprises a
polynucleotide encoding a selection marker. The selection marker
can be contained in a selection cassette. Such selection markers
include, but are not limited, to neomycin phosphotransferase
(neo.sup.r), hygromycin B phosphotransferase (hyg.sup.r),
puromycin-N-acetyltransferase (puro.sup.r), blasticidin S deaminase
(bsr.sup.r), xanthine/guanine phosphoribosyl transferase (gpt), or
herpes simplex virus thymidine kinase (HSV-k), or a combination
thereof. In one embodiment, the polynucleotide encoding the
selection marker is operably linked to a promoter active in the
cell, rat cell, pluripotent rat cell or the ES rat cell. When
serially stacking polynucleotides of interest into a targeted
locus, the selection marker can comprise a recognition site for a
nuclease agent, as outlined above. In one embodiment, the
polynucleotide encoding the selection marker is flanked with a
site-specific recombination target sequences.
The insert nucleic acid can further comprise a reporter gene
operably linked to a promoter, wherein the reporter gene encodes a
reporter protein selected from the group consisting of or
comprising LacZ, mPlum, mCherry, tdTomato, mStrawberry, J-Red,
DsRed, mOrange, mKO, mCitrine, Venus, YPet, enhanced yellow
fluorescent protein (EYFP), Emerald, enhanced green fluorescent
protein (EGFP), CyPet, cyan fluorescent protein (CFP), Cerulean,
T-Sapphire, luciferase, alkaline phosphatase, and/or a combination
thereof. Such reporter genes can be operably linked to a promoter
active in the cell. Such promoters can be an inducible promoter, a
promoter that is endogenous to the reporter gene or the cell, a
promoter that is heterologous to the reporter gene or to the cell,
a cell-specific promoter, a tissue-specific promoter, or a
developmental stage-specific promoter.
In one embodiment, nucleic acid insert can comprise a mammalian
nucleic acid comprises a genomic locus that encodes a protein
expressed in the nervous system, the skeletal system, the digestive
system, the circulatory system, the muscular system, the
respiratory system, the cardiovascular system, the lymphatic
system, the endocrine system, the urinary system, the reproductive
system, or a combination thereof. In one embodiment, the mammalian
nucleic acid comprises a genomic locus that encodes a protein
expressed in a bone marrow or a bone marrow-derived cell. In one
embodiment, the nucleic acid comprises a genomic locus that encodes
a protein expressed in a spleen cell.
In one embodiment, the mammalian nucleic acid comprises a genomic
locus that encodes a protein expressed in the nervous system, the
skeletal system, the digestive system, the circulatory system, the
muscular system, the respiratory system, the cardiovascular system,
the lymphatic system, the endocrine system, the urinary system, the
reproductive system, or a combination thereof. In one embodiment,
the mammalian nucleic acid comprises a genomic locus that encodes a
protein expressed in a bone marrow or a bone marrow-derived cell.
In one embodiment, the nucleic acid comprises a genomic locus that
encodes a protein expressed in a spleen cell. In one embodiment,
the genomic locus comprises a mouse genomic DNA sequence, a rat
genomic DNA sequence a human genomic DNA sequence, or a combination
thereof. In one embodiment, the genomic locus comprises, in any
order, rat and human genomic DNA sequences. In one embodiment, the
genomic locus comprises, in any order, mouse and human genomic DNA
sequences. In one embodiment, the genomic locus comprises, in any
order, mouse and rat genomic DNA sequences. In one embodiment, the
genomic locus comprises, in any order, rat, mouse, and human
genomic DNA sequences.
In one embodiment, the genomic locus comprises a mouse genomic DNA
sequence, a rat genomic DNA sequence a human genomic DNA sequence,
or a combination thereof. In one embodiment, the genomic locus
comprises, in any order, rat and human genomic DNA sequences. In
one embodiment, the genomic locus comprises, in any order, mouse
and human genomic DNA sequences. In one embodiment, the genomic
locus comprises, in any order, mouse and rat genomic DNA sequences.
In one embodiment, the genomic locus comprises, in any order, rat,
mouse, and human genomic DNA sequences.
In one embodiment, the genetic modification comprises at least one
human disease allele of a human gene. In one embodiment, the human
disease is a neurological disease. In one embodiment, the human
disease is a cardiovascular disease. In one embodiment, the human
disease is a kidney disease. In one embodiment, the human disease
is a muscle disease. In one embodiment, the human disease is a
blood disease. In one embodiment, the human disease is a cancer. In
one embodiment, the human disease is an immune system disease.
In one embodiment, the human disease allele is a dominant allele.
In one embodiment, the human disease allele is a recessive allele.
In one embodiment, the human disease allele comprises a single
nucleotide polymorphism (SNP) allele.
In one embodiment, the genetic modification produces a mutant form
of a protein with an altered binding characteristic, altered
localization, altered expression, and/or altered expression
pattern.
In one embodiment, the insert nucleic acid comprises a selection
cassette. In one embodiment, the selection cassette comprises a
nucleic acid sequence encoding a selective marker, wherein the
nucleic acid sequence is operably linked to a promoter active in
rat ES cells. In one embodiment, the selective marker is selected
from or comprises a hygromycin resistance gene or a neomycin
resistance gene.
In one embodiment, the nucleic acid comprises a genomic locus that
encodes a protein expressed in a B cell. In one embodiment, the
nucleic acid comprises a genomic locus that encodes a protein
expressed in an immature B cell. In one embodiment, the nucleic
acid comprises a genomic locus that encodes a protein expressed in
a mature B cell.
In one embodiment, the insert nucleic acid comprises a regulatory
element. In one embodiment, the regulatory element is a promoter.
In one embodiment, the regulatory element is an enhancer. In one
embodiment, the regulatory element is a transcriptional
repressor-binding element.
In one embodiment, the genetic modification comprises a deletion of
a non-protein-coding sequence, but does not comprise a deletion of
a protein-coding sequence. In one embodiment, the deletion of the
non-protein-coding sequence comprises a deletion of a regulatory
element. In one embodiment, the genetic modification comprises a
deletion of a regulatory element. In one embodiment, the genetic
modification comprises an addition of a promoter or a regulatory
element. In one embodiment, the genetic modification comprises a
replacement of a promoter or a regulatory element.
ii. Expression Cassettes
Provided herein are polynucleotides or nucleic acid molecules
comprising the various components employed in a targeted genomic
integration system provided herein (i.e. any one of or any
combination of nuclease agents, recognition sites, insert nucleic
acids, polynucleotides of interest, targeting vectors, selection
markers, and other components).
The terms "polynucleotide," "polynucleotide sequence," "nucleic
acid sequence," and "nucleic acid fragment" are used
interchangeably herein. These terms encompass nucleotide sequences
and the like. A polynucleotide may be a polymer of RNA or DNA that
is single- or double-stranded, that optionally contains synthetic,
non-natural or altered nucleotide bases. A polynucleotide in the
form of a polymer of DNA may be comprised of one or more segments
of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
Polynucleotides can comprise deoxyribonucleotides and
ribonucleotides include both naturally occurring molecules and
synthetic analogues, and any combination these. The polynucleotides
provided herein also encompass all forms of sequences including,
but not limited to, single-stranded forms, double-stranded forms,
hairpins, stem-and-loop structures, and the like.
Further provided are recombinant polynucleotides comprising the
various components of the targeted genomic integration system. The
terms "recombinant polynucleotide" and "recombinant DNA construct"
are used interchangeably herein. A recombinant construct comprises
an artificial or heterologous combination of nucleic acid
sequences, e.g., regulatory and coding sequences that are not found
together in nature. In other embodiments, a recombinant construct
may comprise regulatory sequences and coding sequences that are
derived from different sources, or regulatory sequences and coding
sequences derived from the same source, but arranged in a manner
different than that found in nature. Such a construct may be used
by itself or may be used in conjunction with a vector. If a vector
is used, then the choice of vector is dependent upon the method
that is used to transform the host cells as is well known to those
skilled in the art. For example, a plasmid vector can be used.
Genetic elements required to successfully transform, select, and
propagate host cells comprising any of the isolated nucleic acid
fragments provided herein are also provided. Screening may be
accomplished by Southern analysis of DNA, Northern analysis of mRNA
expression, immunoblotting analysis of protein expression, or
phenotypic analysis, among others.
In specific embodiments, one or more of the components of the
targeted genomic integration system described herein can be
provided in an expression cassette for expression in a prokaryotic
cell, a eukaryotic cell, a bacterial, a yeast cell, or a mammalian
cell or other organism or cell type of interest. The cassette can
include 5' and 3' regulatory sequences operably linked to a
polynucleotide provided herein. "Operably linked" comprises a
relationship wherein the components operably linked function in
their intended manner. For example, an operable linkage between a
polynucleotide of interest and a regulatory sequence (i.e., a
promoter) is a functional link that allows for expression of the
polynucleotide of interest. Operably linked elements may be
contiguous or non-contiguous. When used to refer to the joining of
two protein coding regions, operably linked means that the coding
regions are in the same reading frame. In another instance, a
nucleic acid sequence encoding a protein may be operably linked to
regulatory sequences (e.g., promoter, enhancer, silencer sequence,
etc.) so as to retain proper transcriptional regulation. In one
instance, a nucleic acid sequence of an immunoglobulin variable
region (or V(D)J segments) may be operably linked to a nucleic acid
sequence of an immunoglobulin constant region so as to allow proper
recombination between the sequences into an immunoglobulin heavy or
light chain sequence.
The cassette may additionally contain at least one additional
polynucleotide of interest to be co-introduced into the organism.
Alternatively, the additional polynucleotide of interest can be
provided on multiple expression cassettes. Such an expression
cassette is provided with a plurality of restriction sites and/or
recombination sites for insertion of a recombinant polynucleotide
to be under the transcriptional regulation of the regulatory
regions. The expression cassette may additionally contain selection
marker genes.
The expression cassette can include in the 5'-3' direction of
transcription, a transcriptional and translational initiation
region (i.e., a promoter), a recombinant polynucleotide provided
herein, and a transcriptional and translational termination region
(i.e., termination region) functional in mammalian cell or a host
cell of interest. The regulatory regions (i.e., promoters,
transcriptional regulatory regions, and translational termination
regions) and/or a polynucleotide provided herein may be
native/analogous to the host cell or to each other. Alternatively,
the regulatory regions and/or a polynucleotide provided herein may
be heterologous to the host cell or to each other. For example, a
promoter operably linked to a heterologous polynucleotide is from a
species different from the species from which the polynucleotide
was derived, or, if from the same/analogous species, one or both
are substantially modified from their original form and/or genomic
locus, or the promoter is not the native promoter for the operably
linked polynucleotide. Alternatively, the regulatory regions and/or
a recombinant polynucleotide provided herein may be entirely
synthetic.
The termination region may be native with the transcriptional
initiation region, may be native with the operably linked
recombinant polynucleotide, may be native with the host cell, or
may be derived from another source (i.e., foreign or heterologous)
to the promoter, the recombinant polynucleotide, the host cell, or
any combination thereof.
In preparing the expression cassette, the various DNA fragments may
be manipulated, so as to provide for the DNA sequences in the
proper orientation. Toward this end, adapters or linkers may be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resubstitutions, e.g., transitions and transversions,
may be involved.
A number of promoters can be used in the expression cassettes
provided herein. The promoters can be selected based on the desired
outcome. It is recognized that different applications can be
enhanced by the use of different promoters in the expression
cassettes to modulate the timing, location and/or level of
expression of the polynucleotide of interest. Such expression
constructs may also contain, if desired, a promoter regulatory
region (e.g., one conferring inducible, constitutive,
environmentally- or developmentally-regulated, or cell- or
tissue-specific/selective expression), a transcription initiation
start site, a ribosome binding site, an RNA processing signal, a
transcription termination site, and/or a polyadenylation
signal.
The expression cassette containing the polynucleotides provided
herein can also comprise a selection marker gene for the selection
of transformed cells. Selectable marker genes are utilized for the
selection of transformed cells or tissues.
Where appropriate, the sequences employed in the methods and
compositions (i.e., the polynucleotide of interest, the nuclease
agent, etc.) may be optimized for increased expression in the cell.
That is, the genes can be synthesized using codons preferred in a
given cell of interest including, for example, mammalian-preferred
codons, human-preferred codons, rodent-preferred codon,
mouse-preferred codons, rat-preferred codons, etc. for improved
expression.
The various methods and compositions provided herein can employ
selection markers. Various selection markers can be used in the
methods and compositions disclosed herein. Such selection markers
can, for example, impart resistance to an antibiotic such as G418,
hygromycin, blasticidin, neomycin, or puromycin. Such selection
markers include neomycin phosphotransferase (neo.sup.r), hygromycin
B phosphotransferase (hyg.sup.r), puromycin-N-acetyltransferase
(puro.sup.r), and blasticidin S deaminase (bsr.sup.r). In still
other embodiments, the selection marker is operably linked to an
inducible promoter and the expression of the selection marker is
toxic to the cell. Non-limiting examples of such selection markers
include xanthine/guanine phosphoribosyl transferase (gpt),
hahypoxanthine-guanine phosphoribosyltransferase (HGPRT) or herpes
simplex virus thymidine kinase (HSV-TK). The polynucleotide
encoding the selection markers are operably linked to a promoter
active in the cell.
iii. Targeting Vectors
Targeting vectors are employed to introduce the insert nucleic acid
into the target locus of the rat nucleic acid. The targeting vector
comprises the insert nucleic acid and further comprises a 5' and a
3' homology arm, which flank the insert nucleic acid. The homology
arms, which flank the insert nucleic acid, correspond to regions
within the target locus of the rat nucleic acid. For ease of
reference, the corresponding cognate genomic regions within the
targeted genomic locus are referred to herein as "target sites".
For example, a targeting vector can comprise a first insert nucleic
acid flanked by a first and a second homology arm complementary to
a first and a second target site As such, the targeting vector
thereby aids in the integration of the insert nucleic acid into the
target locus of the rat nucleic acid through a homologous
recombination event that occurs between the homology arms and the
complementary target sites within the genome of the cell.
In one embodiment, the target locus of the rat nucleic acid
comprises a first nucleic acid sequence that is complementary to
the 5' homology arm and a second nucleic acid sequence that is
complementary to the 3' homology arm. In one embodiment, the first
and the second nucleic acid sequences are separated by at least 5
kb. In another embodiment, the first and the second nucleic acid
sequences are separated by at least 5 kb but less than 200 kb. In
one embodiment, the first and the second nucleic acid sequences are
separated by at least 10 kb. In one embodiment, the first and the
second nucleic acid sequences are separated by at least 20 kb, at
least 30 kb, at least 40 kb, at least 50 kb, at least 60 kb, at
least 70 kb, at least 80 kb, at least 90 kb, at least 100 kb, at
least 110 kb, at least 120 kb, at least 130 kb, at least 140 kb, at
least 150 kb, at least 160 kb, at least 170 kb, at least 180 kb, at
least 190 kb, or at least 200 kb. In still further embodiments, the
first and the second nucleic acid sequence is separated by at least
5 kb but less than 10 kb, at least 5 kb but less than 3 Mb, at
least 10 kb but less than 20 kb, at least 20 kb but less than 40
kb, at least 40 kb but less than 60 kb, at least 60 kb but less
than 80 kb, at least about 80 kb but less than 100 kb, at least 100
kb but less than 150 kb, or at least 150 kb but less than 200 kb,
at least about 200 kb but less than about 300 kb, at least about
300 kb but less than about 400 kb, at least about 400 kb but less
than about 500 kb, at least about 500 kb but less than about 1 Mb,
at least about 1.5 Mb but less than about 2 Mb, at least about 1 Mb
but less than about 1.5 Mb, at least about 2 Mb but less than 2.5
Mb, at least about 2.5 Mb but less than 3 Mb, or at least about 2
Mb but less than about 3 Mb.
A homology arm of the targeting vector can be of any length that is
sufficient to promote a homologous recombination event with a
corresponding target site, including for example, at least 5-10 kb,
5-15 kb, 10-20 kb, 20-30 kb, 30-40 kb, 40-50 kb, 50-60 kb, 60-70
kb, 70-80 kb, 80-90 kb, 90-100 kb, 100-110 kb, 110-120 kb, 120-130
kb, 130-140 kb, 140-150 kb, 150-160 kb, 160-170 kb, 170-180 kb,
180-190 kb, 190-200 kb in length or greater. As outlined in further
detail below, large targeting vectors can employ targeting arms of
greater length. In a specific embodiment, the sum total of the 5'
homology arm and the 3' homology arm is at least 10 kb or the sum
total of the 5' homology arm and the 3' homology arm is at least
about 16 kb to about 100 kb or about 30 kb to about 100 kb. In
other embodiments, the size of the sum total of the total of the 5'
and 3' homology arms of the LTVEC is about 10 kb to about 150 kb,
about 10 kb to about 100 kb, about 10 kb to about 75 kb, about 20
kb to about 150 kb, about 20 kb to about 100 kb, about 20 kb to
about 75 kb, about 30 kb to about 150 kb, about 30 kb to about 100
kb, about 30 kb to about 75 kb, about 40 kb to about 150 kb, about
40 kb to about 100 kb, about 40 kb to about 75 kb, about 50 kb to
about 150 kb, about 50 kb to about 100 kb, or about 50 kb to about
75 kb, about 10 kb to about 30 kb, about 20 kb to about 40 kb,
about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb
to about 100 kb, about 100 kb to about 120 kb, or from about 120 kb
to about 150 kb. In one embodiment, the size of the deletion is the
same or similar to the size of the sum total of the 5' and 3'
homology arms of the LTVEC.
When nuclease agents are employed, the cognate genomic regions
corresponding to the 5' and 3' homology arms of a targeting vector
are "located in sufficient proximity" to nuclease target sites so
as to promote the occurrence of a homologous recombination event
between the cognate genomic regions and the homology arms upon a
nick or double-strand break at the recognition site. For example,
the nuclease target sites can be located anywhere between the
cognate genomic regions corresponding to the 5' and 3' homology
arms. In specific embodiments, the recognition site is immediately
adjacent to at least one or both of the cognate genomic
regions.
As used herein, a homology arm and a target site (i.e., cognate
genomic region) "complement" or are "complementary" to one another
when the two regions share a sufficient level of sequence identity
to one another to act as substrates for a homologous recombination
reaction. By "homology" is meant DNA sequences that are either
identical or share sequence identity to a corresponding or
"complementary" sequence. The sequence identity between a given
target site and the corresponding homology arm found on the
targeting vector can be any degree of sequence identity that allows
for homologous recombination to occur. For example, the amount of
sequence identity shared by the homology arm of the targeting
vector (or a fragment thereof) and the target site (or a fragment
thereof) can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity, such that the
sequences undergo homologous recombination. Moreover, a
complementary region of homology between the homology arm and the
complementary target site can be of any length that is sufficient
to promote homologous recombination at the cleaved recognition
site. For example, a given homology arm and/or complementary target
site can comprise complementary regions of homology that are at
least 5-10 kb, 5-15 kb, 10-20 kb, 20-30 kb, 30-40 kb, 40-50 kb,
50-60 kb, 60-70 kb, 70-80 kb, 80-90 kb, 90-100 kb, 100-110 kb,
110-120 kb, 120-130 kb, 130-140 kb, 140-150 kb, 150-160 kb, 160-170
kb, 170-180 kb, 180-190 kb, 190-200 kb in length or greater (such
as described in the LTVEC vectors described elsewhere herein) such
that the homology arm has sufficient homology to undergo homologous
recombination with the corresponding target sites within the genome
of the cell. For ease of reference the homology arms are referred
to herein as a 5' and a 3' homology arm. This terminology relates
to the relative position of the homology arms to the insert nucleic
acid within the targeting vector.
The homology arms of the targeting vector are therefore designed to
be complementary to a target site with the targeted locus. Thus,
the homology arms can be complementary to a locus that is native to
the cell, or alternatively they can be complementary to a region of
a heterologous or exogenous segment of DNA that was integrated into
the genome of the cell, including, but not limited to, transgenes,
expression cassettes, or heterologous or exogenous regions of
genomic DNA. Alternatively, the homology arms of the targeting
vector can be complementary to a region of a human artificial
chromosome or any other engineered genomic region contained in an
appropriate host cell. Still further, the homology arms of the
targeting vector can be complementary to or be derived from a
region of a BAC library, a cosmid library, or a P1 phage library.
Thus, in specific embodiments, the homology arms of the targeting
vector are complementary to a rat genomic locus that is native,
heterologous or exogenous to a given cell. In further embodiments,
the homology arms are complementary to a rat genomic locus that is
not targetable using a conventional method or can be targeted only
incorrectly or only with significantly low efficiency, in the
absence of a nick or double-strand break induced by a nuclease
agent. In one embodiment, the homology arms are derived from a
synthetic DNA.
In still other embodiments, the 5' and 3' homology arms are
complementary to the same genome as the targeted genome. In one
embodiment, the homology arms are from a related genome, e.g., the
targeted genome is a rat genome of a first strain, and the
targeting arms are from a rat genome of a second strain, wherein
the first strain and the second strain are different. In other
embodiments, the homology arms are from the genome of the same
animal or are from the genome of the same strain, e.g., the
targeted genome is a rat genome of a first strain, and the
targeting arms are from a rat genome from the same rat or from the
same strain.
The targeting vector (such as a large targeting vector) can also
comprise a selection cassette or a reporter gene as discussed
elsewhere herein. The selection cassette can comprise a nucleic
acid sequence encoding a selection marker, wherein the nucleic acid
sequence is operably linked to a promoter. The promoter can be
active in a prokaryotic cell of interest and/or active in a
eukaryotic cell of interest. Such promoters can be an inducible
promoter, a promoter that is endogenous to the reporter gene or the
cell, a promoter that is heterologous to the reporter gene or to
the cell, a cell-specific promoter, a tissue-specific promoter or a
developmental stage-specific promoter. In one embodiment, the
selection marker is selected from or comprises neomycin
phosphotransferase (neo.sup.r), hygromycin B phosphotransferase
(hyg.sup.r), puromycin-N-acetyltransferase (puro.sup.r),
blasticidin S deaminase (bsr.sup.r), xanthine/guanine
phosphoribosyl transferase (gpt), and herpes simplex virus
thymidine kinase (HSV-k), and/or a combination thereof. The
selection marker of the targeting vector can be flanked by the 5'
and 3' homology arms or found either 5' or 3' to the homology
arms.
In one embodiment, the targeting vector (such as a large targeting
vector) comprises a reporter gene operably linked to a promoter,
wherein the reporter gene encodes a reporter protein selected from
the group consisting of or comprises LacZ, mPlum, mCherry,
tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus,
YPet, enhanced yellow fluorescent protein (EYFP), Emerald, enhanced
green fluorescent protein (EGFP), CyPet, cyan fluorescent protein
(CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase,
and/or a combination thereof. Such reporter genes can be operably
linked to a promoter active in the cell. Such promoters can be an
inducible promoter, a promoter that is endogenous to the report
gene or the cell, a promoter that is heterologous to the reporter
gene or to the cell, a cell-specific promoter, a tissue-specific
promoter or a developmental stage-specific promoter.
In one embodiment, combined use of the targeting vector (including,
for example, a large targeting vector) with the nuclease agent
results in an increased targeting efficiency compared to use of the
targeting vector alone. In one embodiment, when the targeting
vector is used in conjunction with the nuclease agent, targeting
efficiency of the targeting vector is increased at least by
two-fold, at least three-fold, or at least 4-fold when compared to
when the targeting vector is used alone.
When employing a targeting vector, the vector design can be such as
to allow for the insertion of a given sequence that is from about 5
kb to about 200 kb as described herein. In one embodiment, the
insertion is from about 5 kb to about 10 kb, from about 10 kb to
about 20 kb, from about 20 kb to about 30 kb, from about 30 kb to
about 40 kb, from about 40 kb to about 50 kb, from about 50 kb to
about 60 kb, from about 60 kb to about 70 kb, from about 80 kb to
about 90 kb, from about 90 kb to about 100 kb, from about 100 kb to
about 110 kb, from about 110 kb to about 120 kb, from about 120 kb
to about 130 kb, from about 130 kb to about 140 kb, from about 140
kb to about 150 kb, from about 150 kb to about 160 kb, from about
160 kb to about 170 kb, from about 170 kb to about 180 kb, from
about 180 kb to about 190 kb, or from about 190 kb to about 200 kb,
from about 5 kb to about 10 kb, from about 10 kb to about 20 kb,
from about 20 kb to about 40 kb, from about 40 kb to about 60 kb,
from about 60 kb to about 80 kb, from about 80 kb to about 100 kb,
from about 100 kb to about 150 kb, from about 150 kb to about 200
kb, from about 200 kb to about 250 kb, from about 250 kb to about
300 kb, from about 300 kb to about 350 kb, or from about 350 kb to
about 400 kb.
When employing a targeting vector, the vector design can be such as
to allow for the replacement of a given sequence that is from about
5 kb to about 200 kb or from about 5 kb to about 3.0 Mb as
described herein. In one embodiment, the replacement is from about
5 kb to about 10 kb, from about 10 kb to about 20 kb, from about 20
kb to about 30 kb, from about 30 kb to about 40 kb, from about 40
kb to about 50 kb, from about 50 kb to about 60 kb, from about 60
kb to about 70 kb, from about 80 kb to about 90 kb, from about 90
kb to about 100 kb, from about 100 kb to about 110 kb, from about
110 kb to about 120 kb, from about 120 kb to about 130 kb, from
about 130 kb to about 140 kb, from about 140 kb to about 150 kb,
from about 150 kb to about 160 kb, from about 160 kb to about 170
kb, from about 170 kb to about 180 kb, from about 180 kb to about
190 kb, from about 190 kb to about 200 kb, from about 5 kb to about
10 kb, from about 10 kb to about 20 kb, from about 20 kb to about
40 kb, from about 40 kb to about 60 kb, from about 60 kb to about
80 kb, from about 80 kb to about 100 kb, from about 100 kb to about
150 kb, or from about 150 kb to about 200 kb, from about 200 kb to
about 300 kb, from about 300 kb to about 400 kb, from about 400 kb
to about 500 kb, from about 500 kb to about 1 Mb, from about 1 Mb
to about 1.5 Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb
to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb.
In one embodiment, the targeting vector comprises a site-specific
recombinase gene. In one embodiment, the site-specific recombinase
gene encodes a Cre recombinase. In one embodiment, the Cre
recombinase gene is Crei, wherein two exons encoding the Cre
recombinase are separated by an intron to prevent its expression in
a prokaryotic cell.
In one embodiment, the Cre recombinase gene further comprises a
nuclear localization signal to facilitate localization of Cre (or
any recombinase or nuclease agent) to the nucleus (e.g., the gene
is an NL-Cre gene). In a specific embodiment, the Cre recombinase
gene further comprises a nuclear localization signal and an intron
(e.g., NL-Crei).
In various embodiments, a suitable promoter for expression of the
nuclease agent (including the Cre or Crei recombinase discussed
above) is selected from or comprises a Prm1, Blimp1, Gata6, Gata4,
Igf2, Lhx2, Lhx5, and/or Pax3. In a specific embodiment, the
promoter is the Gata6 or Gata4 promoter. The various promoters can
be from any organism, including for example, a rodent such as a
mouse or a rat. In another specific embodiment, the promoter is a
Prm1 promoter. In another specific embodiment, the promoter is a
rat Prm1 promoter. In another specific embodiment, the promoter is
a mouse Prm1 promoter. In another specific embodiment, the promoter
is a Blimp1 promoter or a fragment thereof, e.g., a 1 kb or 2 kb
fragment of a Blimp1 promoter. See, for example, U.S. Pat. No.
8,697,851 and U.S. Application Publication 2013-0312129, both of
which are herein incorporated by reference in their entirety.
iv. Large Targeting Vectors
The term "large targeting vector" or "LTVEC" as used herein
comprises large targeting vectors that comprise homology arms that
correspond to and are derived from nucleic acid sequences larger
than those typically used by other approaches intended to perform
homologous targeting in cells and/or comprising insert nucleic
acids comprising nucleic acid sequences larger than those typically
used by other approaches intended to perform homologous
recombination targeting in cells. For example, the LTVEC make
possible the modification of large loci that cannot be accommodated
by traditional plasmid-based targeting vectors because of their
size limitations. In specific embodiments, the homology arms and/or
the insert nucleic acid of the LTVEC comprises genomic sequence of
a eukaryotic cell. The size of the LTVEC is too large to enable
screening of targeting events by conventional assays, e.g.,
southern blotting and long-range (e.g., 1 kb-5 kb) PCR. Examples of
the LTVEC, include, but are not limited to, vectors derived from a
bacterial artificial chromosome (BAC), a human artificial
chromosome or a yeast artificial chromosome (YAC). Non-limiting
examples of LTVECs and methods for making them are described, e.g.,
in U.S. Pat. Nos. 6,586,251, 6,596,541, 7,105,348, and WO
2002/036789 (PCT/US01/45375), and US 2013/0137101, each of which is
herein incorporated by reference.
The LTVEC can be of any length, including, but not limited to, from
about 20 kb to about 400 kb, from about 20 kb to about 30 kb, from
about 30 kb to 40 kb, from about 40 kb to about 50 kb, from about
50 kb to about 75 kb, from about 75 kb to about 100 kb, from about
100 kb to 125 kb, from about 125 kb to about 150 kb, from about 150
kb to about 175 kb, about 175 kb to about 200 kb, from about 200 kb
to about 225 kb, from about 225 kb to about 250 kb, from about 250
kb to about 275 kb or from about 275 kb to about 300 kb, from about
200 kb to about 300 kb, from about 300 kb to about 350 kb, from
about 350 kb to about 400 kb, from about 350 kb to about 550 kb. In
one embodiment, the LTVEC is about 100 kb.
In one embodiment, the LTVEC comprises an insert nucleic acid
ranging from about 5 kb to about 200 kb, from about 5 kb to about
10 kb, from about 10 kb to about 20 kb, from about 20 kb to about
30 kb, from about 0.5 kb to about 30 kb, from about 0.5 kb to about
40 kb, from about 30 kb to about 150 kb, from about 0.5 kb to about
150 kb, from about 30 kb to about 40 kb, from about 40 kb to about
50 kb, from about 60 kb to about 70 kb, from about 80 kb to about
90 kb, from about 90 kb to about 100 kb, from about 100 kb to about
110 kb, from about 120 kb to about 130 kb, from about 130 kb to
about 140 kb, from about 140 kb to about 150 kb, from about 150 kb
to about 160 kb, from about 160 kb to about 170 kb, from about 170
kb to about 180 kb, from about 180 kb to about 190 kb, or from
about 190 kb to about 200 kb, from about 5 kb to about 10 kb, from
about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from
about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from
about 80 kb to about 100 kb, from about 100 kb to about 150 kb,
from about 150 kb to about 200 kb, from about 200 kb to about 250
kb, from about 250 kb to about 300 kb, from about 300 kb to about
350 kb, or from about 350 kb to about 400 kb;
When employing a LTVEC, the vector design can be such as to allow
for the replacement of a given sequence that is from about 5 kb to
about 200 kb or from about 5 kb to about 3 Mb as described herein.
In one embodiment, the replacement is from about 5 kb to about 10
kb, from about 10 kb to about 20 kb, from about 20 kb to about 30
kb, from about 30 kb to about 40 kb, from about 40 kb to about 50
kb, from about 50 kb to about 60 kb, from about 60 kb to about 70
kb, from about 80 kb to about 90 kb, from about 90 kb to about 100
kb, from about 100 kb to about 110 kb, from about 110 kb to about
120 kb, from about 120 kb to about 130 kb, from about 130 kb to
about 140 kb, from about 140 kb to about 150 kb, from about 150 kb
to about 160 kb, from about 160 kb to about 170 kb, from about 170
kb to about 180 kb, from about 180 kb to about 190 kb, from about
190 kb to about 200 kb, from about 5 kb to about 10 kb, from about
10 kb to about 20 kb, from about 20 kb to about 40 kb, from about
40 kb to about 60 kb, from about 60 kb to about 80 kb, from about
80 kb to about 100 kb, from about 100 kb to about 150 kb, or from
about 150 kb to about 200 kb, from about 200 kb to about 300 kb,
from about 300 kb to about 400 kb, from about 400 kb to about 500
kb, from about 500 kb to about 1 Mb, from about 1 Mb to about 1.5
Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb to about 2.5
Mb, or from about 2.5 Mb to about 3 Mb.
In one embodiment, the homology arms of the LTVEC are derived from
a BAC library, a cosmid library, or a P1 phage library. In other
embodiments, the homology arms are derived from the targeted
genomic locus of the cell and in some instances the target genomic
locus, which the LTVEC is designed to target is not targetable
using a conventional method. In still other embodiments, the
homology arms are derived from a synthetic DNA.
In one embodiment, a sum total of the 5' homology arm and the 3'
homology arm in the LTVEC is at least 10 kb. In other embodiments,
the sum total of the 5' and the 3' homology arms of the LTVEC is
from about 10 kb to about 30 kb, from about 20 kb to about 40 kb,
from about 40 kb to about 60 kb, from about 60 kb to about 80 kb,
from about 80 kb to about 100 kb, from 100 kb to about 120 kb, from
about 120 kb to about 140 kb, from about 140 kb to about 160 kb,
from about 160 kb to about 180 kb, from about 180 kb to about 200
kb. In one embodiment the sum total of the 5' and the 3' homology
arms of the LTVEC is from about 30 kb to about 100 kb. In other
embodiments, the size of the sum total of the total of the 5' and
3' homology arms of the LTVEC is about 10 kb to about 150 kb, about
10 kb to about 100 kb, about 10 kb to about 75 kb, about 20 kb to
about 150 kb, about 20 kb to about 100 kb, about 20 kb to about 75
kb, about 30 kb to about 150 kb, about 30 kb to about 100 kb, about
30 kb to about 75 kb, about 40 kb to about 150 kb, about 40 kb to
about 100 kb, about 40 kb to about 75 kb, about 50 kb to about 150
kb, about 50 kb to about 100 kb, or about 50 kb to about 75 kb,
about 10 kb to about 30 kb, about 20 kb to about 40 kb, about 40 kb
to about 60 kb, about 60 kb to about 80 kb, about 80 kb to about
100 kb, about 100 kb to about 120 kb, or from about 120 kb to about
150 kb. In one embodiment, the size of the deletion is the same or
similar to the size of the sum total of the 5' and 3' homology arms
of the LTVEC.
In other embodiments, the 5' homology arm ranges from about 5 kb to
about 100 kb. In one embodiment, the 3' homology arm ranges from
about 5 kb to about 100 kb. In other embodiments, the sum total of
the 5' and 3' homology arms are from about 5 kb to about 10 kb,
from about 10 kb to about 20 kb, from about 20 kb to about 30 kb,
from about 30 kb to about 40 kb, from about 40 kb to about 50 kb,
from about 50 kb to about 60 kb, from about 60 kb to about 70 kb,
from about 70 kb to about 80 kb, from about 80 kb to about 90 kb,
from about 90 kb to about 100 kb, from about 100 kb to about 110
kb, from about 110 kb to about 120 kb, from about 120 kb to about
130 kb, from about 130 kb to about 140 kb, from about 140 kb to
about 150 kb, from about 150 kb to about 160 kb, from about 160 kb
to about 170 kb, from about 170 kb to about 180 kb, from about 180
kb to about 190 kb, from about 190 kb to about 200 kb, or from
about 30 kb to about 100 kb, about 10 kb to about 30 kb, about 20
kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about
80 kb, about 80 kb to about 100 kb, about 100 kb to about 120 kb,
or from about 120 kb to about 150 kb.
In one embodiment, the LTVEC comprises an insert nucleic acid that
is homologous or orthologous to a rat nucleic acid sequence flanked
by the LTVEC homology arms. In one embodiment, the insert nucleic
acid sequence is from a species other than a rat. In one
embodiment, the insert nucleic acid that is homologous or
orthologous to the rat nucleic acid sequence is a mammalian nucleic
acid. In one embodiment, the mammalian nucleic acid is a mouse
nucleic acid. In one embodiment, the mammalian nucleic acid is a
human nucleic acid. In one embodiment, the insert nucleic acid is a
genomic DNA. In one embodiment, the insert is from 5 kb to 200 kb
as described above.
In one embodiment, the LTVEC comprises a selection cassette or a
reporter gene. Various forms of the selection cassette and reporter
gene that can be employed are discussed elsewhere herein.
As described elsewhere herein, the LTVEC can also be used in the
methods provided herein in combination with a nuclease agent that
promotes a homologous recombination between the targeting vector
and the target locus of a rat nucleic acid in a pluripotent rat
cell.
In one embodiment, the large targeting vector (LTVEC) comprises a
site-specific recombinase gene. In one embodiment, the
site-specific recombinase gene encodes a Cre recombinase. In one
embodiment, the Cre recombinase gene is Crei, wherein two exons
encoding the Cre recombinase are separated by an intron to prevent
its expression in a prokaryotic cell. In one embodiment, the Cre
recombinase gene further comprises a nuclear localization signal to
facilitate localization of Cre (or any recombinase or nuclease
agent) to the nucleus (e.g., the gene is an NL-Cre gene). In a
specific embodiment, the Cre recombinase gene further comprises a
nuclear localization signal and an intron (e.g., NL-Crei)
In various embodiments, a suitable promoter for expression of the
nuclease agent (including the Cre or Crei recombinase discussed
above) is selected from or comprises a Prm1, Blimp1, Gata6, Gata4,
Igf2, Lhx2, Lhx5, and/or Pax3. In a specific embodiment, the
promoter is the Gata6 or Gata4 promoter. The various promoters can
be from any organism, including for example, a rodent such as a
mouse or a rat. In another specific embodiment, the promoter is a
Prm1 promoter. In another specific embodiment, the promoter is a
rat Prm1 promoter. In another specific embodiment, the promoter is
a mouse Prm1 promoter. In another specific embodiment, the promoter
is a Blimp1 promoter or a fragment thereof, e.g., a 1 kb or 2 kb
fragment of a Blimp1 promoter. See, for example, U.S. Pat. No.
8,697,851 and U.S. Application Publication 2013-0312129, both of
which are herein incorporated by reference in their entirety.
In one embodiment, the LTVEC comprises an insert nucleic acid that
can produce a deletion, addition, replacement or a combination
thereof of a region of the rat ApoE locus, the IL-2Rg locus, the
Rag2 locus, the Rag1 locus and/or the Rag2/Rag1 locus as discussed
in detail elsewhere herein. In specific embodiments, the genetic
modification at the ApoE locus results in a decrease, an increase
or a modulation in ApoE activity, IL-2Rg activity, Rag2 activity,
Rag1 activity and/or Rag2 and Rag1 activity. In one embodiment, an
ApoE knockout, and IL-2Rg knockout, a Rag2 knockout, a Rag1
knockout, a Rag2/Rag1 knockout is generated. As discussed below,
nuclease agents can be employed with any of the LTVEC targeting
systems to target any genomic locus of interest.
v. Nuclease Agents and Recognition Sites for Nuclease Agents
As outlined in detail above, nuclease agents may be utilized in the
methods and compositions disclosed herein to aid in the
modification of the target locus both in a prokaryotic cell or
within a pluripotent rat cell. Such a nuclease agent may promote
homologous recombination between the targeting vector and the
target locus. In one embodiment, the nuclease agent comprises an
endonuclease agent.
As used herein, the term "recognition site for a nuclease agent"
comprises a DNA sequence at which a nick or double-strand break is
induced by a nuclease agent. The recognition site for a nuclease
agent can be endogenous (or native) to the cell or the recognition
site can be exogenous to the cell. In specific embodiments, the
recognition site is exogenous to the cell and thereby is not
naturally occurring in the genome of the cell. In still further
embodiments, the recognition site is exogenous to the cell and to
the polynucleotides of interest that one desired to be positioned
at the target genomic locus. In further embodiments, the exogenous
or endogenous recognition site is present only once in the genome
of the host cell. In specific embodiments, an endogenous or native
site that occurs only once within the genome is identified. Such a
site can then be used to design nuclease agents that will produce a
nick or double-strand break at the endogenous recognition site.
The length of the recognition site can vary, and includes, for
example, recognition sites that are at least 4, 6, 8, 10, 12, 14,
16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70 or more nucleotides in length. In one embodiment, each
monomer of the nuclease agent recognizes a recognition site of at
least 9 nucleotides. In other embodiments, the recognition site is
from about 9 to about 12 nucleotides in length, from about 12 to
about 15 nucleotides in length, from about 15 to about 18
nucleotides in length, or from about 18 to about 21 nucleotides in
length, and any combination of such subranges (e.g., 9-18
nucleotides). The recognition site could be palindromic, that is,
the sequence on one strand reads the same in the opposite direction
on the complementary strand. It is recognized that a given nuclease
agent can bind the recognition site and cleave that binding site or
alternatively, the nuclease agent can bind to a sequence that is
the different from the recognition site. Moreover, the term
recognition site comprises both the nuclease agent binding site and
the nick/cleavage site irrespective whether the nick/cleavage site
is within or outside the nuclease agent binding site. In another
variation, the cleavage by the nuclease agent can occur at
nucleotide positions immediately opposite each other to produce a
blunt end cut or, in other cases, the incisions can be staggered to
produce single-stranded overhangs, also called "sticky ends", which
can be either 5' overhangs, or 3' overhangs.
Any nuclease agent that induces a nick or double-strand break into
a desired recognition site can be used in the methods and
compositions disclosed herein. A naturally-occurring or native
nuclease agent can be employed so long as the nuclease agent
induces a nick or double-strand break in a desired recognition
site. Alternatively, a modified or engineered nuclease agent can be
employed. An "engineered nuclease agent" comprises a nuclease that
is engineered (modified or derived) from its native form to
specifically recognize and induce a nick or double-strand break in
the desired recognition site. Thus, an engineered nuclease agent
can be derived from a native, naturally-occurring nuclease agent or
it can be artificially created or synthesized. The modification of
the nuclease agent can be as little as one amino acid in a protein
cleavage agent or one nucleotide in a nucleic acid cleavage agent.
In some embodiments, the engineered nuclease induces a nick or
double-strand break in a recognition site, wherein the recognition
site was not a sequence that would have been recognized by a native
(non-engineered or non-modified) nuclease agent. Producing a nick
or double-strand break in a recognition site or other DNA can be
referred to herein as "cutting" or "cleaving" the recognition site
or other DNA.
Active variants and fragments of the exemplified recognition sites
are also provided. Such active variants can comprise at least 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or more sequence identity to the given recognition site,
wherein the active variants retain biological activity and hence
are capable of being recognized and cleaved by a nuclease agent in
a sequence-specific manner. Assays to measure the double-strand
break of a recognition site by a nuclease agent are known in the
art and generally measure the ability of a nuclease to cut the
recognition site.
The recognition site of the nuclease agent can be positioned
anywhere in or near the target locus. The recognition site can be
located within a coding region of a gene, or within regulatory
regions, which influence expression of the gene. Thus, a
recognition site of the nuclease agent can be located in an intron,
an exon, a promoter, an enhancer, a regulatory region, or any
non-protein coding region.
In one embodiment, the nuclease agent is a Transcription
Activator-Like Effector Nuclease (TALEN). TAL effector nucleases
are a class of sequence-specific nucleases that can be used to make
double-strand breaks at specific target sequences in the genome of
a prokaryotic or eukaryotic organism. TAL effector nucleases are
created by fusing a native or engineered transcription
activator-like (TAL) effector, or functional part thereof, to the
catalytic domain of an endonuclease, such as, for example, FokI.
The unique, modular TAL effector DNA binding domain allows for the
design of proteins with potentially any given DNA recognition
specificity. Thus, the DNA binding domains of the TAL effector
nucleases can be engineered to recognize specific DNA target sites
and thus, used to make double-strand breaks at desired target
sequences. See, WO 2010/079430; Morbitzer et al. (2010) PNAS
10.1073/pnas.1013133107; Scholze & Boch (2010) Virulence
1:428-432; Christian et al. Genetics (2010) 186:757-761; Li et al.
(2010) Nuc. Acids Res. (2010) doi:10.1093/nar/gkq704; and Miller et
al. (2011) Nature Biotechnology 29:143-148; all of which are herein
incorporated by reference.
Examples of suitable TAL nucleases, and methods for preparing
suitable TAL nucleases, are disclosed, e.g., in US Patent
Application No. 2011/0239315 A1, 2011/0269234 A1, 2011/0145940 A1,
2003/0232410 A1, 2005/0208489 A1, 2005/0026157 A1, 2005/0064474 A1,
2006/0188987 A1, and 2006/0063231 A1 (each hereby incorporated by
reference). In various embodiments, TAL effector nucleases are
engineered that cut in or near a target nucleic acid sequence in,
e.g., a genomic locus of interest, wherein the target nucleic acid
sequence is at or near a sequence to be modified by a targeting
vector. The TAL nucleases suitable for use with the various methods
and compositions provided herein include those that are
specifically designed to bind at or near target nucleic acid
sequences to be modified by targeting vectors as described
herein.
In one embodiment, each monomer of the TALEN comprises 12-25 TAL
repeats, wherein each TAL repeat binds a 1 bp subsite. In one
embodiment, the nuclease agent is a chimeric protein comprising a
TAL repeat-based DNA binding domain operably linked to an
independent nuclease. In one embodiment, the independent nuclease
is a Fold endonuclease. In one embodiment, the nuclease agent
comprises a first TAL-repeat-based DNA binding domain and a second
TAL-repeat-based DNA binding domain, wherein each of the first and
the second TAL-repeat-based DNA binding domain is operably linked
to a FokI nuclease, wherein the first and the second
TAL-repeat-based DNA binding domain recognize two contiguous target
DNA sequences in each strand of the target DNA sequence separated
by about 6 bp to about 40 bp cleavage site, and wherein the FokI
nucleases dimerize and make a double strand break at a target
sequence.
In one embodiment, the nuclease agent comprises a first
TAL-repeat-based DNA binding domain and a second TAL-repeat-based
DNA binding domain, wherein each of the first and the second
TAL-repeat-based DNA binding domain is operably linked to a FokI
nuclease, wherein the first and the second TAL-repeat-based DNA
binding domain recognize two contiguous target DNA sequences in
each strand of the target DNA sequence separated by a 5 bp or 6 bp
cleavage site, and wherein the FokI nucleases dimerize and make a
double strand break.
The nuclease agent employed in the various methods and compositions
disclosed herein can further comprise a zinc-finger nuclease (ZFN).
In one embodiment, each monomer of the ZFN comprises 3 or more zinc
finger-based DNA binding domains, wherein each zinc finger-based
DNA binding domain binds to a 3 bp subsite. In other embodiments,
the ZFN is a chimeric protein comprising a zinc finger-based DNA
binding domain operably linked to an independent nuclease. In one
embodiment, the independent endonuclease is a FokI endonuclease. In
one embodiment, the nuclease agent comprises a first ZFN and a
second ZFN, wherein each of the first ZFN and the second ZFN is
operably linked to a FokI nuclease, wherein the first and the
second ZFN recognize two contiguous target DNA sequences in each
strand of the target DNA sequence separated by about 6 bp to about
40 bp cleavage site or about a 5 bp to about 6 bp cleavage site,
and wherein the FokI nucleases dimerize and make a double strand
break. See, for example, US20060246567; US20080182332;
US20020081614; US20030021776; WO/2002/057308A2; US20130123484;
US20100291048; and, WO/2011/017293A2, each of which is herein
incorporated by reference.
In one embodiment of the methods provided herein, the nuclease
agent comprises (a) a chimeric protein comprising a zinc
finger-based DNA binding domain fused to a FokI endonuclease; or,
(b) a chimeric protein comprising a Transcription Activator-Like
Effector Nuclease (TALEN) fused to a FokI endonuclease.
In still another embodiment, the nuclease agent is a meganuclease.
Meganucleases have been classified into four families based on
conserved sequence motifs, the families are the LAGLIDADG (SEQ ID
NO: 16), GIY-YIG, H-N-H, and His-Cys box families. These motifs
participate in the coordination of metal ions and hydrolysis of
phosphodiester bonds. HEases are notable for their long recognition
sites, and for tolerating some sequence polymorphisms in their DNA
substrates. Meganuclease domains, structure and function are known,
see for example, Guhan and Muniyappa (2003) Grit Rev Biochem Mol
Biol 38:199-248; Lucas et al., (2001) Nucleic Acids Res 29:960-9;
Jurica and Stoddard, (1999) Cell Mol Life Sci 55:1304-26; Stoddard,
(2006) Q Rev Biophys 38:49-95; and Moure et al., (2002) Nat Struct
Biol 9:764. In some examples a naturally occurring variant, and/or
engineered derivative meganuclease is used. Methods for modifying
the kinetics, cofactor interactions, expression, optimal
conditions, and/or recognition site specificity, and screening for
activity are known, see for example, Epinat et al., (2003) Nucleic
Acids Res 31:2952-62; Chevalier et al., (2002) Mol Cell 10:895-905;
Gimble et al., (2003) Mol Biol 334:993-1008; Seligman et al.,
(2002) Nucleic Acids Res 30:3870-9; Sussman et al., (2004) J Mol
Biol 342:31-41; Rosen et al., (2006) Nucleic Acids Res 34:4791-800;
Chames et al., (2005) Nucleic Acids Res 33:e178; Smith et al.,
(2006) Nucleic Acids Res 34:e149; Gruen et al., (2002) Nucleic
Acids Res 30:e29; Chen and Zhao, (2005) Nucleic Acids Res 33:e154;
WO2005105989; WO2003078619; WO2006097854; WO2006097853;
WO2006097784; and WO2004031346.
Any meganuclease can be used herein, including, but not limited to,
I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII,
I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP,
I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI,
F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII,
I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI,
I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I-NgrIP,
I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI,
I-PcuVI, I-PgrIP, I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP,
I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP,
I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP,
I-TdeIP, I-TevI, I-TevII, I-TevIII, I-UarAP, I-UarHGPAIP,
I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP PI-MtuHIIP,
PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP,
PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, PI-TliII, or any
active variants or fragments thereof.
In one embodiment, the meganuclease recognizes double-stranded DNA
sequences of 12 to 40 base pairs. In one embodiment, the
meganuclease recognizes one perfectly matched target sequence in
the genome. In one embodiment, the meganuclease is a homing
nuclease. In one embodiment, the homing nuclease is a LAGLIDADG
(SEQ ID NO: 16) family of homing nuclease. In one embodiment, the
LAGLIDADG (SEQ ID NO: 16) family of homing nuclease is selected
from I-SceI, I-CreI, and I-DmoI.
Nuclease agents can further comprise restriction endonucleases,
which include Type I, Type II, Type III, and Type IV endonucleases.
Type I and Type III restriction endonucleases recognize specific
recognition sites, but typically cleave at a variable position from
the nuclease binding site, which can be hundreds of base pairs away
from the cleavage site (recognition site). In Type II systems the
restriction activity is independent of any methylase activity, and
cleavage typically occurs at specific sites within or near to the
binding site. Most Type II enzymes cut palindromic sequences,
however Type IIa enzymes recognize non-palindromic recognition
sites and cleave outside of the recognition site, Type IIb enzymes
cut sequences twice with both sites outside of the recognition
site, and Type IIs enzymes recognize an asymmetric recognition site
and cleave on one side and at a defined distance of about 1-20
nucleotides from the recognition site. Type IV restriction enzymes
target methylated DNA. Restriction enzymes are further described
and classified, for example in the REBASE database (webpage at
rebase.neb.com; Roberts et al., (2003) Nucleic Acids Res
31:418-20), Roberts et al., (2003) Nucleic Acids Res 31:1805-12,
and Belfort et al., (2002) in Mobile DNA II, pp. 761-783, Eds.
Craigie et al., (ASM Press, Washington, D.C.).
The nuclease agent employed in the various methods and compositions
can also comprise a CRISPR/Cas system. Such systems can employ, for
example, a Cas9 nuclease, which in some instances, is
codon-optimized for the desired cell type in which it is to be
expressed. The system further employs a fused crRNA-tracrRNA
construct that functions with the codon-optimized Cas9. This single
RNA is often referred to as a guide RNA or gRNA. Within a gRNA, the
crRNA portion is identified as the `target sequence` for the given
recognition site and the tracrRNA is often referred to as the
`scaffold`. Briefly, a short DNA fragment containing the target
sequence is inserted into a guide RNA expression plasmid. The gRNA
expression plasmid comprises the target sequence (in some
embodiments around 20 nucleotides), a form of the tracrRNA sequence
(the scaffold) as well as a suitable promoter that is active in the
cell and necessary elements for proper processing in eukaryotic
cells. Many of the systems rely on custom, complementary oligos
that are annealed to form a double stranded DNA and then cloned
into the gRNA expression plasmid. The gRNA expression cassette and
the Cas9 expression cassette is then introduced into the cell. See,
for example, Mali P et al. (2013) Science 2013 Feb. 15;
339(6121):823-6; Jinek M et al. Science 2012 Aug. 17;
337(6096):816-21; Hwang W Y et al. Nat Biotechnol 2013 March;
31(3):227-9; Jiang W et al. Nat Biotechnol 2013 March; 31(3):233-9;
and, Cong L et al. Science 2013 Feb. 15; 339(6121):819-23, each of
which is herein incorporated by reference.
In one embodiment, the method for modifying a genomic locus of
interest in a pluripotent rat cell further comprises introducing
into the pluripotent rat cell: (a) a first expression construct
comprising a first promoter operably linked to a first nucleic acid
sequence encoding a Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR)-associated (Cas) protein; (b) a second
expression construct comprising a second promoter operably linked
to a genomic target sequence linked to a guide RNA (gRNA), wherein
the genomic target sequence is flanked on the 3'end by a
Protospacer Adjacent Motif (PAM) sequence. In one embodiment, the
genomic target sequence comprises the nucleotide sequence of
GNNNNNNNNNNNNNNNNNNNNGG (GN.sub.1-20 GG; SEQ ID NO: 1). In one
embodiment, the genomic target sequence comprises SEQ ID NO:23,
wherein N is between 1 and 20 nucleotides in length. In another
embodiment, the genomic target sequence comprises between 14 and 20
nucleotides in length of SEQ ID NO:1.
In one embodiment, the gRNA comprises a third nucleic acid sequence
encoding a Clustered Regularly Interspaced Short Palindromic
Repeats (CRISPR) RNA (crRNA) and a trans-activating CRISPR RNA
(tracrRNA). In specific embodiments, the Cas protein is Cas9.
In some embodiments, the gRNA comprises (a) the chimeric RNA of the
nucleic acid sequence
5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU-
CGGUGCUUUU-3' (SEQ ID NO: 2); or, (b) the chimeric RNA of the
nucleic acid sequence
5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG-3' (SEQ ID NO:
3).
In another embodiment, the crRNA comprises
5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAU-3' (SEQ ID NO: 4);
5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG (SEQ ID NO: 5); or
5'-GAGUCCGAGCAGAAGAAGAAGUUUUA-3' (SEQ ID NO: 6).
In yet other embodiments, the tracrRNA comprises,
5'-AAGGCUAGUCCG-3' (SEQ ID NO: 7) or
5'-AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3' (SEQ ID
NO: 8).
In one embodiment, the Cas protein is a type I Cas protein. In one
embodiment, the Cas protein is a type II Cas protein. In one
embodiment, the type II Cas protein is Cas9. In one embodiment, the
first nucleic acid sequence encodes a human codon-optimized Cas
protein.
In one embodiment, the first nucleic acid comprises a mutation that
disrupts at least one amino acid residue of nuclease active sites
in the Cas protein, wherein the mutant Cas protein generates a
break in only one strand of the target DNA region, and wherein the
mutation diminishes nonhomologous recombination in the target DNA
region.
In one embodiment, the first nucleic acid that encodes the Cas
protein further comprises a nuclear localization signal (NLS). In
one embodiment, the nuclear localization signal is a SV40 nuclear
localization signal.
In one embodiment, the second promoter that drives the expression
of the genomic target sequence and the guide RNA (gRNA) is an RNA
polymerase III promoter. In one embodiment, the RNA polymerase III
promoter is a human U6 promoter. In one embodiment, the RNA
polymerase III promoter is a rat U6 polymerase III promoter. In one
embodiment, the RNA polymerase III promoter is a mouse U6
polymerase III promoter.
In one embodiment, the nucleic acid sequences encoding crRNA and
the tracrRNA are linked via a synthetic loop, wherein, upon
expression, the crRNA and the tracrRNA forms a crRNA:tracrRNA
duplex.
In one embodiment, the first expression construct and the second
expression construct are expressed from a same plasmid.
In one embodiment, the first and the second expression constructs
are introduced together with the LTVEC. In one embodiment, the
first and the second expression constructs are introduced
separately from the LTVEC over a period of time.
In one embodiment, the method comprises introducing a plurality of
the second construct and a plurality of the LTVEC for multiplex
editing of distinct target loci as described herein.
Active variants and fragments of nuclease agents (i.e. an
engineered nuclease agent) are also provided. Such active variants
can comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the
native nuclease agent, wherein the active variants retain the
ability to cut at a desired recognition site and hence retain nick
or double-strand-break-inducing activity. For example, any of the
nuclease agents described herein can be modified from a native
endonuclease sequence and designed to recognize and induce a nick
or double-strand break at a recognition site that was not
recognized by the native nuclease agent. Thus in some embodiments,
the engineered nuclease has a specificity to induce a nick or
double-strand break at a recognition site that is different from
the corresponding native nuclease agent recognition site. Assays
for nick or double-strand-break-inducing activity are known and
generally measure the overall activity and specificity of the
endonuclease on DNA substrates containing the recognition site.
The nuclease agent may be introduced into the cell by any means
known in the art. The polypeptide encoding the nuclease agent may
be directly introduced into the cell. Alternatively, a
polynucleotide encoding the nuclease agent can be introduced into
the cell. When a polynucleotide encoding the nuclease agent is
introduced into the cell, the nuclease agent can be transiently,
conditionally or constitutively expressed within the cell. Thus,
the polynucleotide encoding the nuclease agent can be contained in
an expression cassette and be operably linked to a conditional
promoter, an inducible promoter, a constitutive promoter, or a
tissue-specific promoter. Such promoters of interest are discussed
in further detail elsewhere herein. Alternatively, the nuclease
agent is introduced into the cell as an mRNA encoding or comprising
a nuclease agent.
In one embodiment, the crRNA and the tracrRNA are expressed as
separate RNA transcripts.
In specific embodiments, the polynucleotide encoding the nuclease
agent is stably integrated in the genome of the cell and operably
linked to a promoter active in the cell. In other embodiments, the
polynucleotide encoding the nuclease agent is in the same targeting
vector comprising the insert nucleic acid, while in other instances
the polynucleotide encoding the nuclease agent is in a vector or a
plasmid that is separate from the targeting vector comprising the
insert nucleic acid.
When the nuclease agent is provided to the cell through the
introduction of a polynucleotide encoding the nuclease agent, such
a polynucleotide encoding a nuclease agent can be modified to
substitute codons having a higher frequency of usage in the cell of
interest, as compared to the naturally occurring polynucleotide
sequence encoding the nuclease agent. For example the
polynucleotide encoding the nuclease agent can be modified to
substitute codons having a higher frequency of usage in a given
prokaryotic or eukaryotic cell of interest, including a bacterial
cell, a yeast cell, a human cell, a non-human cell, a mammalian
cell, a rodent cell, a mouse cell, a rat cell or any other host
cell of interest, as compared to the naturally occurring
polynucleotide sequence.
In one embodiment, the endonuclease agent is introduced together
with the LTVEC. In one embodiment, the endonuclease agent is
introduced separately from the LTVEC over a period of time. In one
embodiment, the endonuclease agent is introduced prior to the
introduction of the LTVEC. In one embodiment, the endonuclease
agent is introduced into the rat ES cell following introduction of
the LTVEC.
In one embodiment, the endonuclease agent is an expression
construct comprising a nucleic acid sequence encoding an
endonuclease, wherein the nucleic acid sequence is operably linked
to a promoter. In one embodiment, the promoter is a constitutively
active promoter. In one embodiment, the promoter is an inducible
promoter. In one embodiment, the promoter is active in the
pluripotent rat cell. In one embodiment, the endonuclease agent is
an mRNA encoding an endonuclease.
B. Methods for Integrating a Polynucleotide of Interest into a
Target Locus
Methods for modifying a target locus of interest are provided. In
one embodiment, a target locus in a pluripotent rat cell is
targeted for genetic modification. Such a method comprises: (a)
introducing into the pluripotent rat cell a targeting vector
comprising an insert nucleic acid flanked with a 5' rat homology
arm and a 3' rat homology arm; and (b) identifying a genetically
modified pluripotent rat cell comprising the targeted genetic
modification at the target locus, wherein the targeted genetic
modification is capable of being transmitted through the germline.
In specific embodiments, the sum total of the 5' homology arm and
the 3' homology arm is at east 10 kb and/or a large targeting
vector is employed.
In other embodiments, the size of the sum total of the total of the
5' and 3' homology arms of the LTVEC is about 10 kb to about 150
kb, about 10 kb to about 100 kb, about 10 kb to about 75 kb, about
20 kb to about 150 kb, about 20 kb to about 100 kb, about 20 kb to
about 75 kb, about 30 kb to about 150 kb, about 30 kb to about 100
kb, about 30 kb to about 75 kb, about 40 kb to about 150 kb, about
40 kb to about 100 kb, about 40 kb to about 75 kb, about 50 kb to
about 150 kb, about 50 kb to about 100 kb, or about 50 kb to about
75 kb, about 10 kb to about 30 kb, about 20 kb to about 40 kb,
about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb
to about 100 kb, about 100 kb to about 120 kb, or from about 120 kb
to about 150 kb. In one embodiment, the size of the deletion is the
same or similar to the size of the sum total of the 5' and 3'
homology arms of the LTVEC.
The pluripotent rat cell can be a rat embryonic stem cell. In a
specific embodiment, (a) the rat ES cell is derived from a DA
strain or an ACI strain; or, (b) the rat ES cell is characterized
by expression of a pluripotency marker comprising Oct-4, Sox-2,
alkaline phosphatase, or a combination thereof. In other instances,
the rat embryonic stem cell employed comprises a rat ES cell as
described in U.S. patent application Ser. No. 14/185,103, filed on
Feb. 20, 2014, herein incorporated by reference in its
entirety.
As described elsewhere herein, the insert nucleic acid can be any
nucleic acid sequence. In non-limiting embodiments, (a) the insert
nucleic acid comprises a replacement of an endogenous rat nucleic
acid sequence with a homologous or a orthologous mammalian nucleic
acid sequence; (b) the insert nucleic acid comprises a deletion of
an endogenous rat nucleic acid sequence; (c) the insert nucleic
acid comprises a deletion of an endogenous rat nucleic acid
sequence, wherein the deletion ranges from 5 kb to 200 kb or from 5
kb to 3 Mb (as discussed in detail elsewhere herein); (d) the
insert nucleic acid comprises an addition of an exogenous nucleic
acid sequence (including for example an exogenous nucleic acid
sequence ranging from about 5 kb to about 10 kb, from about 10 kb
to about 20 kb, from about 20 kb to about 40 kb, from about 40 kb
to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb
to about 100 kb, from about 100 kb to about 150 kb, from about 150
kb to about 200 kb, from about 200 kb to about 250 kb, from about
250 kb to about 300 kb, from about 300 kb to about 350 kb, or from
about 350 kb to about 400 kb); (e) the insert nucleic acid
comprises an exogenous nucleic acid sequence comprising a
homologous or an orthologous nucleic acid sequence; (f) the
homologous or the orthologous nucleic acid sequence of (a) wherein
the nucleic acid sequence is a human nucleic acid sequence; (g) the
insert nucleic acid comprises the homologous or the orthologous
nucleic acid sequence of (a) wherein the nucleic acid sequence is a
chimeric nucleic acid sequence comprising a human and a rat nucleic
acid sequence; (h) the insert nucleic acid comprises the exogenous
nucleic acid sequence of (e), wherein the insert nucleic acid
ranges from about 5 kb to about 200 kb; (i) the insert nucleic acid
comprises a conditional allele flanked with site-specific
recombinase target sequences; (j) the insert nucleic acid comprises
a reporter gene operably linked to a promoter; (k) the insert
nucleic acid comprises one or more unrearranged human
immunoglobulin heavy chain V.sub.H gene segments, one or more
unrearranged human immunoglobulin heavy chain D gene segments, and
one or more unrearranged human immunoglobulin heavy chain J.sub.H
gene segments, which are operably linked to a rodent heavy chain
constant region nucleic acid sequence; (l) the insert nucleic acid
comprises a rearranged human immunoglobulin heavy chain variable
region nucleic acid sequence operably linked to a rodent heavy
chain constant region nucleic acid sequence; (m) the insert nucleic
acid comprises one or more unrearranged human immunoglobulin
V.sub..kappa. or V.sub..lamda. gene segments and one or more
unrearranged human immunoglobulin J.sub..kappa. or J.sub..lamda.
gene segments, which are operably linked to a mammalian
immunoglobulin .lamda. or .kappa. light chain light chain constant
region nucleic acid sequence; (n) the insert nucleic acid comprises
a rearranged human immunoglobulin .lamda. or .kappa. light chain
variable region nucleic acid sequence operably linked to a
mammalian immunoglobulin .lamda. or .kappa. light chain light chain
constant region nucleic acid sequence; (o) the mammalian heavy
chain constant region nucleic acid sequence of (k) and/or (l)
comprises a rat constant region nucleic acid sequence, a human
constant region nucleic acid sequence, or a combination thereof;
or, (p) the mammalian immunoglobulin .lamda. or .kappa. light chain
constant region nucleic acid of (m) and/or (n) comprises a rat
constant region nucleic acid sequence, a human constant region
nucleic acid sequence, or a combination thereof.
In one embodiment, the insert nucleic acid comprises one or more
functional human V.sub.H gene segments comprising V.sub.H1-2,
V.sub.H1-3, V.sub.H1-8, V.sub.H1-18, V.sub.H1-24, V.sub.H1-45,
V.sub.H1-46, V.sub.H1-58, V.sub.H1-69, V.sub.H2-5, V.sub.H2-26,
V.sub.H2-70, V.sub.H3-7, V.sub.H3-9, V.sub.H3-11, V.sub.H3-13,
V.sub.H3-15, V.sub.H3-16, V.sub.H3-20, V.sub.H3-21, V.sub.H3-23,
V.sub.H3-30, V.sub.H3-30-3, V.sub.H3-30-5, V.sub.H3-33,
V.sub.H3-35, V.sub.H3-38, V.sub.H3-43, V.sub.H3-48, V.sub.H3-49,
V.sub.H3-53, V.sub.H3-64, V.sub.H3-66, V.sub.H3-72, V.sub.H3-73,
V.sub.H3-74, V.sub.H4-4, V.sub.H4-28, V.sub.H4-30-1, V.sub.H4-30-2,
V.sub.H4-30-4, V.sub.H4-31, V.sub.H4-34, V.sub.H4-39, V.sub.H4-59,
V.sub.H4-61, V.sub.H5-51, V.sub.H6-1, V.sub.H7-4-1, V.sub.H7-81, or
a combination thereof.
In one embodiment, the insert nucleic acid comprises one or more
functional human D gene segments comprising D1-1, D1-7, D1-14,
D1-20, D1-26, D2-2, D2-8, D2-15, D2-21, D3-3, D3-9, D3-10, D3-16,
D3-22, D4-4, D4-11, D4-17, D4-23, D5-12, D5-5, D5-18, D5-24, D6-6,
D6-13, D6-19, D6-25, D7-27, or a combination thereof.
In one embodiment, the insert nucleic acid comprises one or more
functional J.sub.H gene segments comprising J.sub.H1, J.sub.H2,
J.sub.H3, J.sub.H4, J.sub.H5, J.sub.H6, or a combination thereof.
In one embodiment, the insert nucleic acid comprises one or more
human V.sub..kappa.gene segments comprising V.kappa.4-1,
V.kappa.5-2, V.kappa. 7-3, V.kappa. 2-4, V.kappa.1-5, V.kappa.1-6,
V.kappa.3-7, V.kappa.1-8, V.kappa.1-9, V.kappa.2-10, V.kappa.3-11,
V.kappa.1-12, V.kappa.1-13, V.kappa.2-14, V.kappa.3-15,
V.kappa.1-16, V.kappa.1-17, V.kappa.2-18, V.kappa.2-19,
V.kappa.3-20, V.kappa.6-21, V.kappa.1-22, V.kappa.1-23,
V.kappa.2-24, V.kappa.3-25, V.kappa.2-26, V.kappa.1-27,
V.kappa.2-28, V.kappa.2-29, V.kappa.2-30, V.kappa.3-31,
V.kappa.1-32, V.kappa.1-33, V.kappa.3-34, V.kappa.1-35,
V.kappa.2-36, V.kappa.1-37, V.kappa.2-38, V.kappa.1-39,
V.kappa.2-40, or a combination thereof.
In one embodiment, the insert nucleic acid comprises one or more
human V.lamda. gene segments comprising V.lamda.3-1, V.lamda.4-3,
V.lamda.2-8, V.lamda.3-9, V.lamda.3-10, V.lamda.2-11, V.lamda.3-12,
V.lamda.2-14, V.lamda.3-16, V.lamda.2-18, V.lamda.3-19,
V.lamda.3-21, V.lamda.3-22, V.lamda.2-23, V.lamda.3-25,
V.lamda.3-27, or a combination thereof.
In one embodiment, the insert nucleic acid comprises one or more
human J.kappa. gene segments comprising J.kappa.1, J.kappa.2,
J.kappa.3, J.kappa.4, J.kappa.5, or a combination thereof.
In specific embodiments, upon modification of the target locus in a
pluripotent rat cell, the genetic modification is transmitted
through the germline.
In one embodiment, the insert nucleic acid sequence comprises a
polynucleotide that when integrated into the genome will produce a
genetic modification of a region of the rat ApoE locus, wherein the
genetic modification at the ApoE locus results in a decrease in
ApoE activity, an increase in ApoE activity or a modulation of ApoE
activity. In one embodiment, an ApoE knockout is generated.
In one embodiment, the insert nucleic acid sequence comprises a
polynucleotide that when integrated into the genome will produce a
genetic modification of a region of the rat interleukin-2 receptor
gamma locus, wherein the genetic modification at the interleukin-2
receptor gamma locus results in a decrease in interleukin-2
receptor activity, an increase in interleukin-2 receptor gamma
activity, or a modulation of interleukin-2 receptor activity. In
one embodiment, an interleukin-2 receptor knockout is
generated.
In still another embodiment, the insert nucleic acid sequence
comprises a polynucleotide that when integrated into the genome
will produce a genetic modification of a region of the rat Rag1
locus, the rat Rag2 locus and/or the rat Rag2/Rag1 locus, wherein
the genetic modification at the rat Rag1, Rag2 and/or Rag2/Rag1
locus results in a decrease in in Rag1, Rag2 or Rag1 and Rag2
protein activity, an increase in Rag1, Rag2 or Rag1 and Rag2
protein activity, or a modulation in Rag1, Rag2 or Rag 1 and Rag2
protein activity. In one embodiment, a Rag1, Rag2 or Rag2/Rag1
knockout is generated.
In further embodiments, the insert nucleic acid results in the
replacement of a portion of the rat ApoE locus, the interleukin-2
receptor gamma locus and/or Rag2 locus, and/or Rag1 locus and/or
Rag2/Rag1 locus with the corresponding orthologous portion of an
ApoE locus, an interleukin-2 receptor gamma locus, a Rag2 locus, a
Rag1 locus and/or a Rag2/Rag1 locus from another organism.
Still other embodiments, the insert nucleic acid comprises a
polynucleotide sharing across its full length least 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to a portion of an ApoE
locus, an interleukin-2 receptor gamma locus, a Rag2 locus, a Rag1
locus and/or a Rag2/Rag1 locus it is replacing.
The given insert polynucleotide and the corresponding region of the
rat locus being replaced can be a coding region, an intron, an
exon, an untranslated region, a regulatory region, a promoter, or
an enhancer or any combination thereof. Moreover, the given insert
polynucleotide and/or the region of the rat locus being replaced
can be of any desired length, including for example, between 10-100
nucleotides in length, 100-500 nucleotides in length, 500-1 kb
nucleotide in length, 1 Kb to 1.5 kb nucleotide in length, 1.5 kb
to 2 kb nucleotides in length, 2 kb to 2.5 kb nucleotides in
length, 2.5 kb to 3 kb nucleotides in length, 3 kb to 5 kb
nucleotides in length, 5 kb to 8 kb nucleotides in length, 8 kb to
10 kb nucleotides in length or more. In other instances, the size
of the insertion or replacement is from about 5 kb to about 10 kb,
from about 10 kb to about 20 kb, from about 20 kb to about 40 kb,
from about 40 kb to about 60 kb, from about 60 kb to about 80 kb,
from about 80 kb to about 100 kb, from about 100 kb to about 150
kb, from about 150 kb to about 200 kb, from about 200 kb to about
250 kb, from about 250 kb to about 300 kb, from about 300 kb to
about 350 kb, from about 350 kb to about 400 kb, from about 400 kb
to about 800 kb, from about 800 kb to 1 Mb, from about 1 Mb to
about 1.5 Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb, to
about 2.5 Mb, from about 2.5 Mb to about 2.8 Mb, from about 2.8 Mb
to about 3 Mb. In other embodiments, the given insert
polynucleotide and/or the region of the rat locus being replaced is
at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides
or at least 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb,
10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb or greater.
i. Methods for Modifying a Target Locus of a Rat Nucleic Acid Via
Bacterial Homologous Recombination (BHR)
Methods and compositions are provided for modifying a target locus
of a rat nucleic acid via bacterial homologous recombination (BHR)
in a prokaryotic cell. Such methods find use in utilizing bacterial
homologous recombination in a prokaryotic cell to genetically
modify a target locus of a rat nucleic acid in order to create a
targeting vector. Such a targeting vector comprising the
genetically modified target locus can be introduced into a
eukaryotic cell, for example, a pluripotent rat cell. "Homologous
recombination" includes the exchange of DNA fragments between two
DNA molecules at cross-over sites within regions of homology. Thus,
"bacterial homologous recombination" or "B FIR" includes homologous
recombination that occurs in bacteria.
Methods for modifying a target locus of a rat nucleic acid via
bacterial homologous recombination (BHR) are provided that comprise
introducing into a prokaryotic cell a targeting vector comprising
an insert nucleic acid flanked with a 5' rat homology arm and a 3'
rat homology arm, wherein the prokaryotic cell comprises a rat
nucleic acid and is capable of expressing a recombinase that
mediates the BUR at the target locus. Such targeting vectors can
include any of the large targeting vectors described herein.
In one embodiment, the method comprises introducing into a
prokaryotic cell: (i) a first construct comprising a rat nucleic
acid having a DNA sequence of interest; (ii) a second targeting
construct comprising an insert nucleic acid flanked with a rat 5'
homology arm and a rat 3' homology arm, and (iii) a third construct
encoding a recombinase that mediates bacterial homologous
recombination. In one embodiment, the first, the second, and the
third construct are introduced into the prokaryotic cell separately
over a period of time. In one embodiment, the prokaryotic cell
comprises a nucleic acid that encodes the recombinase, and the
method does not require introduction of the third construct. In one
embodiment, the recombinase is expressed under the control of an
inducible promoter.
In one embodiment the first construct comprising the rat nucleic
acid is derived from a bacterial artificial chromosome (BAC) or
yeast artificial chromosome (YAC).
A prokaryotic cell comprising the insert nucleic acid at the target
genomic locus can be selected. This method can be serially repeated
as disclosed herein to allow the introduction of multiple insert
nucleic acids at the targeted rat locus in the prokaryotic cell.
Once the target rat nucleic acid locus is "built" within the
prokaryotic cell, a targeting vector comprising the modified rat
target locus can be isolated from the prokaryotic cell and
introduced into a target genomic locus within a mammalian cell
(i.e., a rat cell, a pluripotent rat cell, or a rat embryonic stem
cell).
Preferred rat cells for receiving targeting vectors are described
in U.S. application Ser. No. 14/185,703, filed Feb. 20, 2014, the
contents of which are summarized herein. These rat cells are
pluripotent rat cells capable of sustaining their pluripotency
following one or more targeted genetic modifications in vitro, and
are capable of transmitting the targeted genetic modifications
through the germline.
Electroporated pluripotent rat cells are plated at a high density
for the selection of drug-resistant cells comprising the targeting
vector. The drug selection process removes the majority of the
plated cells (.about.99%), leaving behind individual colonies, each
of which is a clone derived from a single cell. Of the remaining
cells, most cells (.about.80-100%) contain the targeting vector
(comprising a drug selection cassette) integrated at a random
location in the genome. Therefore, the colonies are picked
individually and genotyped to identify rat ES cells harboring the
targeting vector at the correct genomic location (e.g., using the
modification of allele assay described below).
A high-throughput quantitative assay, namely, modification of
allele (MOA) assay, can be used for genotyping. Such an assay
allows a large-scale screening of a modified allele(s) in a
parental chromosome following a genetic modification. The MOA assay
can be carried out via various analytical techniques, including,
but not limited to, a quantitative PCR, e.g., a real-time PCR
(qPCR). For example, the real-time PCR comprises a first primer set
that recognizes the target locus and a second primer set that
recognizes a non-targeted reference locus. In addition, the primer
set comprises a fluorescent probe that recognizes the amplified
sequence. In one embodiment, the quantitative assay is carried out
via Invader Probes.RTM.. In one embodiment, the quantitative assay
is carried out via MMP Assays.RTM.. In one embodiment, the
quantitative assay is carried out via TaqMan.RTM. Molecular Beacon.
In one embodiment, the quantitative assay is carried out via
Eclipse.TM. probe technology. (See, for example, US2005/0144655,
which is incorporated by reference herein in its entirety).
The selected pluripotent rat cell or the rat ES cells comprising
the targeted genetic modification can then be introduced into a
host rat embryo, for example, a pre-morula stage or blastocyst
stage rat embryo, and implanted in the uterus of a surrogate mother
to generate a founder rat (F0 rat). Subsequently, the founder rat
can be bred to a wild-type rat to create F1 progeny heterozygous
for the genetic modification. Mating of the heterozygous F1 rat can
produce progeny homozygous for the genetic modification. Mating of
the heterozygous F1 rat can produce progeny homozygous for the
genetic modification. In some embodiments, various genetic
modifications of the target loci described herein can be carried
out using a large targeting vector (LTVEC) as described in detail
elsewhere herein. For example, an LTVEC can be derived from
Bacterial Artificial Chromosome (BAC) DNA using VELOCIGENE.RTM.
genetic engineering technology (see, e.g., U.S. Pat. No. 6,586,251
and Valenzuela, D. M. et al. (2003), High-throughput engineering of
the mouse genome coupled with high-resolution expression analysis,
Nature Biotechnology 21(6): 652-659, which is incorporated herein
by reference in their entireties).
Use of bacterial homologous recombination (BHR) to generate a large
targeting vector (LTVEC) circumvents the limitations of plasmids in
accommodating a large genomic DNA fragment and consequent low
efficiency of introducing a targeted modification into an
endogenous locus in pluripotent rat cells. One or more targeted
genetic modifications can be performed in generating a LTVEC. An
exemplary LTVEC produced in the prokaryotic cell can comprises an
insert nucleic acid that carries a rat genomic sequence with one or
more genetic modifications or an exogenous nucleic acid (e.g., a
homolog or ortholog of a rat nucleic acid), which is flanked by rat
homologous arms complementary to specific genomic regions.
Host prokaryotic cells comprising the various targeting vectors
described herein are also provided. Such prokaryotic cells include,
but are not limited to, bacteria such as E. coli. In one
embodiment, a host prokaryotic cell comprises a targeting vector
comprising an insert nucleic acid flanked with a 5' rat homology
arm and a 3' rat homology arm, wherein the insert nucleic acid
ranges from about 5 kb to about 200 kb.
The host prokaryotic cell can further comprise a nucleic acid that
encodes a recombinase polypeptide or the nucleic acid that encodes
the recombinase polypeptide is operably linked to an inducible
promoter.
Further provided are various methods and compositions, which employ
the LTVEC as described herein in combination with a prokaryotic
cell in order to produce targeted genetic modifications. Such
compositions and methods are discussed elsewhere herein.
Methods for modifying a target locus of a nucleic acid via
bacterial homologous recombination (BHR) are provided that comprise
introducing into a prokaryotic cell a targeting vector comprising
an insert nucleic acid flanked with a 5' homology arm and a 3'
homology arm, wherein the prokaryotic cell comprises nucleic acids
corresponding to the 5' and 3' homology arms and the prokaryotic
cell is capable of expressing a recombinase that mediates the BHR
at the target locus. Such targeting vectors can include any of the
large targeting vectors described herein. Such methods can employ a
LTVEC as discussed in detail herein and further employ the
CRISPR/Cas system as discussed elsewhere herein.
ii. Methods for Modifying a Target Locus of Interest in a
Pluripotent Rat Cell
Further provided is a method for modifying a target locus of
interest in a pluripotent rat cell via targeted genetic
modification, comprising (a) introducing into the pluripotent rat
cell a targeting vector comprising an insert nucleic acid flanked
with a 5' rat homology arm and a 3' rat homology arm, wherein the
sum total of the 5' homology arm and the 3' homology arm is at
least 10 kb; and (b) identifying a genetically modified pluripotent
rat cell comprising the targeted genetic modification at the target
locus of interest. In one embodiment, the sum total of the 5'
homology arm and the 3' homology arm is at least about 16 kb to
about 30 kb. En specific embodiments, the targeted genetic
modification is capable of being transmitted through the germline.
Such targeting vectors can include any of the large targeting
vectors described herein.
In one aspect, a method for modifying a genomic locus of interest
in a pluripotent rat cell via targeted genetic modification is
provided, comprising: (a) providing a pluripotent rat cell that is
able to sustain its pluripotency following at least one targeted
genetic modification of its genome and is able to transmit the
targeted modification to a germline of an F1 generation; (b)
introducing a large targeting vector (LTVEC) into the pluripotent
rat cell, wherein the LTVEC comprises an insert nucleic acid
flanked with a 5' homology arm and a 3' homology arm, wherein the
5' homology arm and the 3' homology arm comprise a rat genomic DNA
fragment; and (c) identifying a genetically modified pluripotent
rat cell comprising the targeted genetic modification.
Various methods can be used to identify cells having the insert
nucleic acid integrated at the target locus of interest. Insertion
of the insert nucleic acid at the target locus of interest results
in a "modification of allele". The term "modification of allele"
and methods for the detection of the modified allele are discussed
in further detail elsewhere herein.
In one aspect, a method for modifying a genomic locus of interest
in a pluripotent rat cell via endonuclease-mediated gene targeting
is provided, the method comprising: (a) providing an isolated
pluripotent rat cell that is able to transmit the genetically
modified genome to a germline of an F1 generation; (b) introducing
into the pluripotent rat cell an endonuclease agent; wherein the
endonuclease agent makes a nick or a double strand break at a
target DNA sequence located in the genomic locus of interest, and
wherein the nick or the double strand break at the target DNA
sequence in the rat ES cell induces: (i) non-homologous end joining
(NHEJ)-mediated DNA repair of the nick or the double strand break,
wherein the NHEJ-mediated DNA repair generates a mutant allele
comprising an insertion or a deletion of a nucleic acid sequence at
the target DNA sequence; or (ii) homologous recombination-mediated
DNA repair that results in restoration of a wild-type nucleic acid
sequence; and (c) identifying the modified genomic locus of
interest.
In one aspect, a method for modifying a genomic locus of interest
in an isolated rat embryonic stem cell (ES) via a nuclease agent is
provided, comprising: (a) providing an isolated rat ES cell that is
able to transmit the targeted genetic modification to a germline of
an F1 generation; (b) introducing into the rat ES cell: (i) a large
targeting vector (LTVEC) comprising an insert nucleic acid flanked
with a rat 5' homology arm and a rat 3' homology arm, wherein the
insert is a nucleic acid sequence that is at least 5 kb; and (ii)
an endonuclease agent, wherein the endonuclease agent makes a nick
or a double strand break at a target DNA sequence located in the
genomic locus of interest, and wherein the target sequence is not
present in the insert nucleic acid; and (c) identifying the
targeted genetic modification in the rat embryonic stem (ES)
cell.
In one aspect, a method for modifying a genomic locus of interest
in a pluripotent rat cell via RNA-guided genome engineering is
provided, the method comprising: (a) providing a pluripotent rat
cell that is able to transmit the genetically modified genome to a
germline of an F1 generation; (b) introducing into the pluripotent
rat cell: (i) a first expression construct comprising a first
promoter operably linked to a first nucleic acid sequence encoding
a Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR)-associated (Cas) protein, (ii) a second expression
construct comprising a second promoter operably linked to a genomic
target sequence linked to a guide RNA (gRNA), wherein the genomic
target sequence is flanked on the 3'end by a Protospacer Adjacent
Motif (PAM) sequence. In one embodiment, the genomic target
sequence comprises the nucleotide sequence of
GNNNNNNNNNNNNNNNNNNNNGG (GN.sub.1-20GG; SEQ ID NO: 1). In one
embodiment, the genomic target sequence comprises SEQ ID NO:1,
wherein N is between 14 and 20 nucleotides in length. In one
embodiment, the gRNA comprises a third nucleic acid sequence
encoding a Clustered Regularly Interspaced Short Palindromic
Repeats (CRISPR) RNA (crRNA) and a fourth nucleic acid sequence
encoding a trans-activating CRISPR RNA (tracrRNA). In one
embodiment, upon expression, the Cas protein forms a CRISPR-Cas
complex comprising the crRNA and the tracrRNA, and the CRISPR-Cas
complex makes a nick or a double strand break at a target DNA
sequence located in the genomic locus of interest, and wherein the
nick or the double strand break at the target DNA sequence in the
pluripotent rat cell induces: (i) non-homologous end joining
(NHEJ)-mediated DNA repair of the nick or the double strand break
created by the CRISPR-Cas complex, wherein the NHEJ generates a
mutant allele comprising an insertion or a deletion of a nucleic
acid sequence at the target DNA sequence; or (ii) homologous
recombination-mediated DNA repair that results in restoration of a
wild-type nucleic acid sequence; and (c) identifying the modified
the genomic locus of interest.
In one embodiment, the pluripotent rat cell is an induced rat
pluripotent stem cell (iPS). In one embodiment, the pluripotent rat
cell is a developmentally restricted progenitor cell.
The presence of a nick or a double-strand break in the recognition
site within the selection marker, in various embodiments, increases
the efficiency and/or frequency of recombination between a
targeting vector (such as a LTVEC) and the targeted locus of
interest. In one embodiment, the recombination is homologous
recombination. In another embodiment, the recombination is an
insertion by non-homologous end joining. In various embodiments, in
the presence of the nick or double strand break, targeting
efficiency of a targeting vector (such as a LTVEC) at the target
genomic locus is at least about 2-fold higher, at least about
3-fold higher, at least about 4-fold higher than in the absence of
the nick or double-strand break (using, e.g., the same targeting
vector and the same homology arms and corresponding target sites at
the genomic locus of interest but in the absence of an added
nuclease agent that makes the nick or double strand break).
In one embodiment, the targeted genetic modification at the target
locus is biallelic. By "biallelic" is meant that both alleles of a
gene comprise the targeted genetic modification. In certain
embodiments, the combined use of a targeting vector (including, for
example, an LTVEC) with a nuclease agent results in biallelic
targeted genetic modification of the genomic locus of interest in a
cell as compared to use of the targeting vector alone. When the
targeting vector is used in conjunction with a nuclease agent,
biallelic targeting efficiency is increased at least by two-fold,
at least three-fold, at least 4-fold or more as compared to when
the targeting vector is used alone. In further embodiments, the
biallelic targeting efficiency is at least 0.2%, 0.3%, 0.4%, 0.5%,
0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4% or 5% or higher.
Compositions are provided which comprise a genetically modified rat
having a targeted genetic modification in the interleukin-2
receptor gamma locus or in the ApoE locus. The various methods and
compositions provided herein allows for these modified loci to be
transmitted through the germline.
In specific embodiments, a genetically modified rat or a
genetically modified pluripotent rat cell comprises a genomic locus
having a targeted genetic modification in the interleukin-2 gamma
receptor locus or having a targeted genetic modification in the
ApoE locus, wherein the interleukin-2 gamma receptor genomic locus
or the ApoE locus comprise: (i) a deletion of at least a portion of
the interleukin-2 gamma receptor locus or at least a portion of the
ApoE locus; (ii) an insertion of a heterologous nucleic acid
sequence into the ApoE locus or into the interleukin-2 gamma
receptor locus; or (iii) a combination thereof, wherein the
genetically modified genomic locus is capable of being transmitted
through the germline.
Methods are further provided that allow for such genetically
modified rats and for such genetically modified pluripotent rat
cells to be made. Such methods include a method for modifying an
ApoE genomic locus or a interleukin-2 gamma receptor locus in a
pluripotent rat cell via targeted genetic modification. The method
comprises (a) introducing into the pluripotent rat cell a targeting
vector comprising an insert nucleic acid flanked with a 5' rat
homology arm to the ApoE locus and a 3' rat homology arm to the
ApoE locus, (b) identifying a genetically modified pluripotent rat
cell comprising the targeted genetic modification at the ApoE
genomic locus of interest, wherein the targeted genetic
modification is capable of being transmitted through germline.
Additional methods include (a) introducing into the pluripotent rat
cell a targeting vector comprising an insert nucleic acid flanked
with a 5' rat homology arm to the interleukin-2 receptor gamma
locus and a 3' rat homology arm to the interleukin-2 receptor gamma
locus, (b) identifying a genetically modified pluripotent rat cell
comprising the targeted genetic modification at the interleukin-2
receptor gamma locus, wherein the targeted genetic modification is
capable of being transmitted through germline.
iii. Methods of Integrating Multiple Polynucleotides of Interest at
the Targeted Locus
The various methods and compositions provided herein allow for the
targeted integration of multiple polynucleotides of interest with a
given target locus. The various methods set forth above can be
sequentially repeated to allow for the targeted integration of any
number of insert nucleic acids into a given targeted locus. Thus,
the various methods provide for the insertion of at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or
more insert nucleic acids into the target locus. In particular
embodiments, such sequential stacking methods allow for the
reconstruction of large genomic regions from a mammalian cell
(i.e., a human, a non-human, a rodent, a mouse, a monkey, a rat, a
hamster, a domesticated mammal or an agricultural animal) into a
targeted locus. In such instances, the transfer and reconstruction
of genomic regions that include both coding and non-coding regions
allow for the complexity of a given region to be preserved by
retaining, at least in part, the coding regions, the non-coding
regions and the copy number variations found within the native
genomic region. Thus, the various methods provide, for example,
methods to generate "heterologous" or "exogenous" genomic regions
within any mammalian cell or animal of interest, particularly
within a prokaryotic host cell or within a pluripotent rat cell or
a rat ES cell. In one non-limiting example, a "humanized" genomic
region within a non-human animal (i.e., within a rat) is
generated.
3. A Humanized Genomic Locus
Provided herein are various methods and compositions comprising a
humanized rat locus. As used herein, by "humanized" genomic locus
is meant a region of a non-human genome comprising at least one
human nucleic acid sequence. A "humanized rat locus" comprises a
region of rat DNA that has a human DNA sequence inserted therein.
The human DNA sequence can be a naturally occurring human DNA
sequence or it can be modified from its native form. In specific
embodiments, the human DNA shares at least 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a native human
sequence. If a human sequence is not a native human sequence it at
least has greater sequence identity to a native human sequence than
it does to an orthologous rat sequence. Moreover, the human DNA
sequence can comprise a cDNA, a region of human genomic DNA, a
non-coding regulatory region, or any portion of a coding, genomic,
or regulatory region of the human DNA. The human DNA sequence
inserted into the rat locus can comprise any of the insert
polynucleotides as described elsewhere herein. In specific
embodiments, the human DNA sequence is orthologous to the rat
target locus, while in other instances, the human DNA sequence is
homologous to the rat target locus.
In one embodiment, the targeted genetic modification is an
insertion or a replacement of an endogenous rat nucleic acid
sequence with a homologous or orthologous human nucleic acid
sequence. In one embodiment, the targeted genetic modification
comprises an insertion or replacement of an endogenous rat nucleic
acid sequence with a homologous or orthologous human nucleic acid
sequence at an endogenous rat locus that comprises the
corresponding rat nucleic acid sequence.
Methods for making a humanized rat locus (or a rat or rat cell
comprising the humanized rat locus) comprise introducing into the
target locus comprising a rat nucleic acid a human nucleic acid
sequence. In one embodiment, a method of making a humanized rat is
provided. Such a method comprises (a) modifying a genome of a
pluripotent rat cell with a targeting vector comprising an insert
nucleic acid that comprises a human nucleic acid sequence to form a
donor cell; (b) introducing the donor cell into a host rat embryo;
and (c) gestating the host rat embryo in a surrogate mother;
wherein the surrogate mother produces a rat progeny that comprises
the human nucleic acid sequence. In specific embodiments, the
humanized rat locus is capable of being transmitted through the
germline. In a further embodiment, the targeting vector comprises a
large targeting vector (LTVEC) and the insert nucleic acid that
comprises a human nucleic acid sequence is at least 5 kb.
In other methods, the humanized rat locus is made by modifying a
target locus of a rat nucleic acid via bacterial homologous
recombination (BHR). The method comprises introducing into a
prokaryotic cell a targeting vector comprising an insert nucleic
acid flanked with a 5' rat homology arm and a 3' rat homology arm,
wherein the insert nucleic acid comprises a human nucleic acid
sequence, and wherein the prokaryotic cell comprises a rat nucleic
acid and is capable of expressing a recombinase that mediates the
BHR at the target locus.
The humanized rat genomic locus can comprise (a) an insertion of a
homologous or orthologous human nucleic acid sequence; (b) a
replacement of an endogenous rat nucleic acid sequence with a
homologous or orthologous human nucleic acid sequence; or (c) a
combination thereof. In specific embodiments, the humanized rat
genomic locus is capable of being transmitted through the germline.
In still other embodiments, the human orthologous sequence replaces
the corresponding sequence found in the rat.
Any human nucleic acid sequence can be used in the methods and
compositions provided herein. Non-limiting examples of human
nucleic acid sequences that can be used in the methods and
compositions are discussed in detail elsewhere herein.
The human nucleic acid sequence for insertion into the rat locus of
interest can be any size. In one embodiment, the human nucleic acid
sequence can be from about 500 nucleotides to about 200 kb, from
about 500 nucleotides to about 5 kb, from about 5 kb to about 200
kb, from about 5 kb to about 10 kb, from about 10 kb to about 20
kb, from about 20 kb to about 30 kb, from about 30 kb to about 40
kb, from about 40 kb to about 50 kb, from about 60 kb to about 70
kb, from about 80 kb to about 90 kb, from about 90 kb to about 100
kb, from about 100 kb to about 110 kb, from about 120 kb to about
130 kb, from about 130 kb to about 140 kb, from about 140 kb to
about 150 kb, from about 150 kb to about 160 kb, from about 160 kb
to about 170 kb, from about 170 kb to about 180 kb, from about 180
kb to about 190 kb, or from about 190 kb to about 200 kb. In a
specific embodiment, the human nucleic acid sequence is at least 5
kb.
In one embodiment, a rat genomic locus is provided wherein the
homologous or orthologous human nucleic acid sequence comprises (a)
one or more unrearranged human immunoglobulin heavy chain V.sub.H
gene segments, one or more unrearranged human immunoglobulin heavy
chain D gene segments, and one or more unrearranged human
immunoglobulin heavy chain J.sub.H gene segments, which are
operably linked to a mammalian heavy chain constant region nucleic
acid sequence; (b) a rearranged human immunoglobulin heavy chain
variable region nucleic acid sequence operably linked to a
mammalian immunoglobulin heavy chain constant region nucleic acid
sequence; (c) one or more unrearranged human immunoglobulin
V.sub..kappa. or V.sub..lamda. gene segments and one or more
unrearranged human immunoglobulin J.sub..kappa. or J.sub..lamda.
gene segments, which are operably linked to a mammalian,
immunoglobulin .lamda. or .kappa. light chain light chain constant
region nucleic acid sequence; or, (d) a rearranged human
immunoglobulin .lamda. or .kappa. light chain variable region
nucleic acid sequence operably linked to a mammalian immunoglobulin
.lamda. or .kappa. light chain light chain constant region nucleic
acid sequence.
In another embodiment, a rat genomic locus is provided wherein (a)
the mammalian immunoglobulin heavy chain constant region nucleic
acid sequence is a rat constant region nucleic acid sequence, a
human constant region nucleic acid sequence, or a combination
thereof; or, (b) the mammalian immunoglobulin .lamda. or .kappa.
light chain light chain constant region nucleic acid sequence is a
rat constant region nucleic acid sequence, a human constant region
nucleic acid sequence, or a combination thereof.
In a specific embodiment, a rat genomic locus is provided wherein
the immunoglobulin heavy chain constant region nucleic acid
sequence is selected from or comprises a CH1, a hinge, a CH2, a
CH3, and/or a combination thereof.
In one embodiment, the rat genomic locus comprises one or more
functional human V.sub.H gene segments comprising V.sub.H1-2,
V.sub.H1-3, V.sub.H1-8, V.sub.H1-18, V.sub.H1-24, V.sub.H1-45,
V.sub.H1-46, V.sub.H1-58, V.sub.H1-69, V.sub.H2-5, V.sub.H2-26,
V.sub.H2-70, V.sub.H3-7, V.sub.H3-9, V.sub.H3-11, V.sub.H3-13,
V.sub.H3-15, V.sub.H3-16, V.sub.H3-20, V.sub.H3-21, V.sub.H3-23,
V.sub.H3-30, V.sub.H3-30-3, V.sub.H3-30-5, V.sub.H3-33,
V.sub.H3-35, V.sub.H3-38, V.sub.H3-43, V.sub.H3-48, V.sub.H3-49,
V.sub.H3-53, V.sub.H3-64, V.sub.H3-66, V.sub.H3-72, V.sub.H3-73,
V.sub.H3-74, V.sub.H4-4, V.sub.H4-28, V.sub.H4-30-1, V.sub.H4-30-2,
V.sub.H4-304, V.sub.H4-31, V.sub.H4-34, V.sub.H4-39, V.sub.H4-59,
V.sub.H4-61, V.sub.H5-51, V.sub.H6-1, V.sub.H7-4-1, V.sub.H7-81, or
a combination thereof.
In one embodiment, the rat genomic locus comprises one or more
functional human D gene segments comprising D1-1, D1-7, D1-14,
D1-20, D1-26, D2-2, D2-8, D2-15, D2-21, D3-3, D3-9, D3-10, D3-16,
D3-22, D4-4, D4-11, D4-17, D4-23, D5-12, D5-5, D5-18, D5-24, D6-6,
D6-13, D6-19, D6-25, D7-27, or a combination thereof.
In one embodiment, the rat genomic locus comprises one or more
functional J.sub.H gene segments comprising J.sub.H1, J.sub.H2,
J.sub.H3, J.sub.H4, J.sub.HS, J.sub.H6, and/or a combination
thereof. In one embodiment, the insert nucleic acid comprises one
or more human V.kappa. gene segments comprises V.kappa.4-1,
V.kappa.5-2, V.kappa. 7-3, V.kappa. 2-4, V.kappa.1-5, V.kappa.1-6,
V.kappa.3-7, V.kappa.1-8, V.kappa.1-9, V.kappa.2-10, V.kappa.3-11,
V.kappa.1-12, V.kappa.1-13, V.kappa.2-14, V.kappa.3-15,
V.kappa.1-16, V.kappa.1-17, V.kappa.2-18, V.kappa.2-19,
V.kappa.3-20, V.kappa.6-21, V.kappa.1-22, V.kappa.1-23,
V.kappa.2-24, V.kappa.3-25, V.kappa.2-26, V.kappa.1-27,
V.kappa.2-28, V.kappa.2-29, V.kappa.2-30, V.kappa.3-31,
V.kappa.1-32, V.kappa.1-33, V.kappa.3-34, V.kappa.1-35,
V.kappa.2-36, V.kappa.1-37, V.kappa.2-38, V.kappa.1-39,
V.kappa.2-40, or a combination thereof.
In one embodiment, the rat genomic locus comprises one or more
human V.lamda. gene segments comprising V.lamda.3-1, V.lamda.4-3,
V.lamda.2-8, V.lamda.3-9, V.lamda.3-10, V.lamda.2-11, V.lamda.3-12,
V.lamda.2-14, V.lamda.3-16, V.lamda.2-18, V.lamda.3-19,
V.lamda.3-21, V.lamda.3-22, V.lamda.2-23, V.lamda.3-25,
V.lamda.3-27, or a combination thereof.
In one embodiment, the rat genomic locus comprises one or more
human J.kappa. gene segments comprising J.kappa.1, J.kappa.2,
J.kappa.3, J.kappa.4, J.kappa.5, or a combination thereof.
In yet another embodiment, the rat genomic locus comprises a
humanized genomic locus comprising a human interleukin-2 receptor
(IL2R) nucleic acid sequence or a variant or a fragment thereof is
provided. In specific embodiments, the IL2R nucleic acid sequence
comprises an interleukin-2 receptor alpha, an interleukin-2
receptor beta, or an interleukin-2 receptor gamma nucleic acid
sequence or variants or fragments thereof.
In further embodiments, a rat genomic locus comprises a humanized
genomic locus comprising of a portion of the human ApoE locus, the
human interleukin-2 receptor gamma locus, the human Rag2 locus, the
human Rag1 locus and/or the human Rag2/Rag1 locus replacing the
corresponding homologous or orthologous portion of the rat ApoE
locus, the rat interleukin-2 receptor gamma locus, the rat Rag2
locus, the rat Rag1 locus and/or the rat Rag2/Rag1 locus. In one
embodiment, the rat ecto-domain of IL-2Rg is replaced with the
ecto-domain of human IL-2Rg, with the remainder of the molecule
being from the rat.
In another embodiment, a genetically modified rat comprising a
humanized genomic locus is provided. Such genetically modified rats
comprise (a) an insertion of a homologous or orthologous human
nucleic acid sequence; (b) a replacement of rat nucleic acid
sequence with a homologous or orthologous human nucleic acid
sequence at an endogenous genomic locus; or (c) a combination
thereof, wherein the humanized genomic locus is capable of being
transmitted through the germline.
Genetically modified rats comprising any of the various humanized
genomic loci provided herein and described above are also
provided.
4. Polynucleotides of Interest
Any polynucleotide of interest may be contained in the various
insert nucleic acids and thereby integrated at the target locus.
The methods disclosed herein, provide for at least 1, 2, 3, 4, 5, 6
or more polynucleotides of interest to be integrated into the
targeted genomic locus.
The polynucleotide of interest within the insert nucleic acid when
integrated at the target genomic locus can introduce one or more
genetic modifications into the cell. The genetic modification can
comprise a deletion of an endogenous nucleic acid sequence and/or
the addition of an exogenous or heterologous or orthologous
polynucleotide into the target genomic locus. In one embodiment,
the genetic modification comprises a replacement of an endogenous
nucleic acid sequence with an exogenous polynucleotide of interest
at the target genomic locus. Thus, methods provided herein allow
for the generation of a genetic modification comprising a knockout,
a deletion, an insertion, a replacement ("knock-in"), a point
mutation, a domain swap, an exon swap, an intron swap, a regulatory
sequence swap, a gene swap, or a combination thereof. Such
modifications may occur upon integration of the first, second,
third, fourth, fifth, six, seventh, or any subsequent insert
nucleic acids into the target genomic locus.
The polynucleotide of interest within the insert nucleic acid
and/or integrated at the target locus can comprise a sequence that
is native to the cell it is introduced into; the polynucleotide of
interest can be heterologous to the cell it is introduced to; the
polynucleotide of interest can be exogenous to the cell it is
introduced into; the polynucleotide of interest can be orthologous
to the cell it is introduced into; or the polynucleotide of
interest can be from a different species than the cell it is
introduced into. As used herein "native" in reference to a sequence
inserted at the target locus is a sequence that is native to the
cell having the target locus or native to the cell from which the
target locus was derived (i.e., from a rat). As used herein,
"heterologous" in reference to a sequence includes a sequence that
originates from a foreign species, or, if from the same species, is
substantially different or modified from its native form in
composition and/or genomic locus by deliberate human intervention.
As used herein, "exogenous" in reference to a sequence is a
sequence that originates from a foreign species. The polynucleotide
of interest can be from any organism of interest including, but not
limited to, non-human, a rodent, a hamster, a mouse, a rat, a
human, a monkey, an agricultural mammal or a non-agricultural
mammal. The polynucleotide of interest can further comprise a
coding region, a non-coding region, a regulatory region, or a
genomic DNA. Thus, the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, and/or
any of the subsequent insert nucleic acids can comprise such
sequences.
In one embodiment, the polynucleotide of interest within the insert
nucleic acid and/or integrated at the target locus is native to a
mouse nucleic acid sequence, a human nucleic acid, a non-human
nucleic acid, a rodent nucleic acid, a rat nucleic acid, a hamster
nucleic acid, a monkey nucleic acid, an agricultural mammal nucleic
acid, or a non-agricultural mammal nucleic acid. In still further
embodiments, the polynucleotide of interest integrated at the
target locus is a fragment of a genomic nucleic acid. In one
embodiment, the genomic nucleic acid is a mouse genomic nucleic
acid, a human genomic nucleic acid, a non-human nucleic acid, a
rodent nucleic acid, a rat nucleic acid, a hamster nucleic acid, a
monkey nucleic acid, an agricultural mammal nucleic acid or a
non-agricultural mammal nucleic acid or a combination thereof.
In one embodiment, the polynucleotide of interest can range from
about 500 nucleotides to about 200 kb as described above. The
polynucleotide of interest can be from about 500 nucleotides to
about 5 kb, from about 5 kb to about 200 kb, from about 5 kb to
about 10 kb, from about 10 kb to about 20 kb, from about 20 kb to
about 30 kb, from about 30 kb to about 40 kb, from about 40 kb to
about 50 kb, from about 60 kb to about 70 kb, from about 80 kb to
about 90 kb, from about 90 kb to about 100 kb, from about 100 kb to
about 110 kb, from about 120 kb to about 130 kb, from about 130 kb
to about 140 kb, from about 140 kb to about 150 kb, from about 150
kb to about 160 kb, from about 160 kb to about 170 kb, from about
170 kb to about 180 kb, from about 180 kb to about 190 kb, or from
about 190 kb to about 200 kb, from about 5 kb to about 10 kb, from
about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from
about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from
about 80 kb to about 100 kb, from about 100 kb to about 150 kb,
from about 150 kb to about 200 kb, from about 200 kb to about 250
kb, from about 250 kb to about 300 kb, from about 300 kb to about
350 kb, or from about 350 kb to about 400 kb.
The polynucleotide of interest within the insert nucleic acid
and/or inserted at the target genomic locus can encode a
polypeptide, can encode an miRNA, or it can comprise any regulatory
regions or non-coding regions of interest including, for example, a
regulatory sequence, a promoter sequence, an enhancer sequence, a
transcriptional repressor-binding sequence, or a deletion of a
non-protein-coding sequence, but does not comprise a deletion of a
protein-coding sequence. In addition, the polynucleotide of
interest within the insert nucleic acid and/or inserted at the
target genomic locus can encode a protein expressed in the nervous
system, the skeletal system, the digestive system, the circulatory
system, the muscular system, the respiratory system, the
cardiovascular system, the lymphatic system, the endocrine system,
the urinary system, the reproductive system, or a combination
thereof. In one embodiment, the polynucleotide of interest within
the insert nucleic acid and/or inserted at the target genomic locus
encodes a protein expressed in a bone marrow or a bone
marrow-derived cell. In one embodiment, the polynucleotide of
interest within the insert nucleic acid and/or integrated at the
target locus encodes a protein expressed in a spleen cell. In still
further embodiments, the polynucleotide of interest within the
insert nucleic acid and/or inserted at the target locus encodes a
protein expressed in a B cell, encodes a protein expressed in an
immature B cell or encodes a protein expressed in a mature B
cell.
The polynucleotide of interest within the insert polynucleotide can
comprise a portion of an ApoE locus, an IL-2-Rg locus, a Rag locus,
a Rag2 locus and/or a Rag2/Rag1 locus. Such portions of these given
loci are discussed elsewhere herein, as are the various homologous
and orthologous regions from any organism of interest that can be
employed.
In one embodiment, polynucleotide of interest within the insert
nucleic acid and/or inserted at the target locus comprises a
genomic nucleic acid sequence that encodes an immunoglobulin heavy
chain variable region amino acid sequence. The phrase "heavy
chain," or "immunoglobulin heavy chain" are described elsewhere
herein.
In one embodiment, the polynucleotide of interest within the insert
nucleic acid and/or integrated at the target locus comprises a
genomic nucleic acid sequence that encodes a human immunoglobulin
heavy chain variable region amino acid sequence.
In one embodiment, the genomic nucleic acid sequence comprises one
or more unrearranged human immunoglobulin heavy chain. V.sub.H gene
segments, one or more unrearranged human immunoglobulin heavy chain
D gene segments, and one or more unrearranged human immunoglobulin
heavy chain J.sub.H gene segments, which are operably linked to a
mammalian heavy chain constant region nucleic acid sequence. In one
embodiment, the genomic nucleic acid sequence comprises a
rearranged human immunoglobulin heavy chain variable region nucleic
acid sequence operably linked to a mammalian heavy chain constant
region nucleic acid sequence. In one embodiment, the genomic
nucleic acid sequence comprises one or more unrearranged human
immunoglobulin V.sub..kappa. or V.sub..lamda. gene segments and one
or more unrearranged human immunoglobulin J.sub..kappa. or
J.sub..lamda. gene segments, which are operably linked to a
mammalian immunoglobulin .lamda. or .kappa. light chain light chain
constant region nucleic acid sequence. In one embodiment, the
genomic nucleic acid sequence comprises a rearranged human
immunoglobulin .lamda. or .kappa. light chain variable region
nucleic acid sequence operably linked to a mammalian immunoglobulin
.lamda. or .kappa. light chain light chain constant region nucleic
acid sequence. In one embodiment, the heavy chain constant region
nucleic acid sequence comprises a rat constant region nucleic acid
sequence, a human constant region nucleic acid sequence, or a
combination thereof. In one embodiment, the immunoglobulin .lamda.
or .kappa. light chain constant region nucleic acid comprises a rat
constant region nucleic acid sequence, a human constant region
nucleic acid sequence, or a combination thereof.
In one embodiment, the immunoglobulin heavy chain constant region
nucleic acid sequence is selected from or comprises a CH1, a hinge,
a CH2, a CH3, and/or a combination thereof. In one embodiment, the
heavy chain constant region nucleic acid sequence comprises a
CH1-hinge-CH2-CH3.
In one embodiment, the polynucleotide of interest within the insert
nucleic acid and/or integrated at the target locus comprises a
genomic nucleic acid sequence that encodes an immunoglobulin light
chain variable region amino acid sequence. The phrase "light chain"
includes an immunoglobulin light chain sequence from any organism,
and is described elsewhere herein.
In one embodiment, the polynucleotide of interest within the insert
nucleic acid and/or integrated at the target genomic locus
comprises a genomic nucleic acid sequence that encodes a human
immunoglobulin light chain variable region amino acid sequence.
In one embodiment, the genomic nucleic acid sequence comprises one
or more unrearranged human immunoglobulin V.sub..kappa. or
V.sub..lamda. gene segments and one or more unrearranged human
immunoglobulin J.sub..kappa. or J.sub..lamda. gene segments, which
are operably linked to a rodent immunoglobulin .lamda. or .kappa.
light chain light chain constant region nucleic acid sequence. In
one embodiment, the genomic nucleic acid sequence comprises a
rearranged human immunoglobulin .lamda. or .kappa. light chain
variable region nucleic acid sequence operably linked to a rodent
immunoglobulin .lamda. or .kappa. light chain light chain constant
region nucleic acid sequence. In one embodiment, the light chain
constant region nucleic acid sequence comprises a rat constant
region nucleic acid sequence, a human constant region nucleic acid
sequence, or a combination thereof. In one embodiment, the
immunoglobulin .lamda. or .kappa. light chain constant region
nucleic acid comprises a rat constant region nucleic acid sequence,
a human constant region nucleic acid sequence, or a combination
thereof
The polynucleotide of interest within the insert nucleic acid
and/or integrated at the target locus can encode an extracellular
protein or a ligand for a receptor. In specific embodiments, the
encoded ligand is a cytokine. Cytokines of interest includes a
chemokine selected from or comprising CCL, CXCL, CX3CL, and/or XCL.
The cytokine can also comprise a tumor necrosis factor (TNF). In
still other embodiments, the cytokine is an interleukin (IL). In
one embodiment, the interleukin is selected from or comprises IL-1,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,
IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,
IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and/or IL-36. In one
embodiment, the interleukin is IL-2. In specific embodiments, such
polynucleotides of interest within the insert nucleic acid and/or
integrated at the target genomic locus are from a human and, in
more specific embodiments, can comprise human genomic sequence.
The polynucleotide of interest within the insert nucleic acid
and/or integrated at the target genomic locus can encode
Apolipoprotein E (ApoE).
The polynucleotide of interest within the insert nucleic acid
and/or integrated at the target locus can encode a cytoplasmic
protein or a membrane protein. In one embodiment, the membrane
protein is a receptor, such as, a cytokine receptor, an interleukin
receptor, an interleukin 2 receptor-alpha, an interleukin-2
receptor beta, an interleukin-2 receptor gamma or receptor tyrosine
kinase. In other instances, the polynucleotide of interest within
the insert nucleic acid and/or integrated at the target locus can
comprise an orthologous or homologous region of the target
locus.
The polynucleotide of interest within the insert nucleic acid
and/or integrated at the target locus can comprise a polynucleotide
encoding at least a region of a T cell receptor, including the T
cell receptor alpha. In specific methods each of the insert nucleic
acids comprise a genomic region of the T cell receptor locus (i.e.
the T cell receptor alpha locus) such that upon completion of the
serial integration, a portion or the entirety of the genomic T cell
receptor locus has been integrated at the target locus. Such insert
nucleic acids can comprise at least one or more of a variable
segment or a joining segment of a T cell receptor locus (i.e. of
the T cell receptor alpha locus). In still further embodiments, the
polynucleotide of interest encoding the region of the T cell
receptor can be from, for example, a mammal, a nonhuman mammal,
rodent, mouse, rat, a human, a monkey, an agricultural mammal or a
domestic mammal polynucleotide encoding a mutant protein.
In other embodiments, the polynucleotide of interest integrated at
the target locus encodes a nuclear protein. In one embodiment, the
nuclear protein is a nuclear receptor. In specific embodiments,
such polynucleotides of interest within the insert nucleic acid
and/or integrated at the target locus are from a human and, in more
specific embodiments, can comprise human genomic sequence.
The polynucleotide of interest within the insert nucleic acid
and/or integrated at the target genomic locus can comprise a
genetic modification in a coding sequence. Such genetic
modifications include, but are not limited to, a deletion mutation
of a coding sequence or the fusion of two coding sequences.
The polynucleotide of interest within the insert nucleic acid
and/or integrated at the target locus can comprise a polynucleotide
encoding a mutant protein, including, for example, a human mutant
protein. In one embodiment, the mutant protein is characterized by
an altered binding characteristic, altered localization, altered
expression, and/or altered expression pattern. In one embodiment,
the polynucleotide of interest within the insert nucleic acid
and/or integrated at the target locus comprises at least one
disease allele, including for example, an allele of a neurological
disease, an allele of a cardiovascular disease, an allele of a
kidney disease, an allele of a muscle disease, an allele of a blood
disease, an allele of a cancer-causing gene, or an allele of an
immune system disease. In such instances, the disease allele can be
a dominant allele or the disease allele is a recessive allele.
Moreover, the disease allele can comprises a single nucleotide
polymorphism (SNP) allele. The polynucleotide of interest encoding
the mutant protein can be from any organism, including, but not
limited to, a mammal, a non-human mammal, rodent, mouse, rat, a
human, a monkey, an agricultural mammal or a domestic mammal
polynucleotide encoding a mutant protein.
In one embodiment, the genetic modification produces a mutant form
of a protein with an altered binding characteristic, altered
localization, altered expression, and/or altered expression
pattern.
In one embodiment, the genetic modification produces a deletion,
addition, replacement or a combination thereof of a region of the
rat ApoE locus, wherein the genetic modification at the ApoE locus
results in a decrease in ApoE activity. In one embodiment, an ApoE
knockout is generated.
In one embodiment, the genetic modification produces a deletion,
addition, replacement or a combination thereof of a region of the
rat Rag1 locus, wherein the genetic modification at the Rag1 locus
results in a decrease in Rag1 activity. In one embodiment, a Rag1
knockout is generated. In one embodiment, the genetic modification
produces a deletion, addition, replacement or a combination thereof
of a region of the rat Rag2 locus, wherein the genetic modification
at the Rag2 locus results in a decrease in Rag2 activity. In one
embodiment, a Rag2 knockout is generated. In one embodiment, the
genetic modification produces a deletion, addition, replacement or
a combination thereof of a region of the rat Rag1/Rag2 locus,
wherein the genetic modification at the Rag1/Rag2 locus results in
a decrease in Rag1 activity and a decrease in Rag2 activity. In one
embodiment, a Rag1/Rag2 knockout is generated.
In one embodiment, the genetic modification produces a deletion,
addition, replacement or a combination thereof of a region of the
rat interleukin-2 receptor gamma locus, wherein the genetic
modification at the interleukin-2 receptor gamma locus results in a
decrease in interleukin-2 receptor gamma. In one embodiment, a
interleukin-2 receptor gamma knockout is generated.
As discussed elsewhere herein, further embodiments provided herein
comprises one or more of the rat ApoE locus, the rat interleukin-2
receptor gamma locus, the Rag2 locus, the Rag1 locus and/or the
Rag2/Rag1 locus is modified through the replacement of a portion of
the rat ApoE locus, the interleukin-2 receptor gamma locus, the
Rag2 locus, the Rag1 locus and/or Rag2/Rag1 locus with the
corresponding orthologous portion of an ApoE locus, an
interleukin-2 receptor gamma locus, a Rag2 locus, a Rag1 locus
and/or a Rag2/Rag1 locus from another organism.
In one embodiment, multiple genetic modifications are generated. In
one embodiment, a genetic modification produces a deletion,
addition, replacement or a combination thereof of a region of the
rat interleukin-2 receptor gamma locus, wherein the genetic
modification at the interleukin-2 receptor gamma locus results in a
decrease in interleukin-2 receptor gamma and a second genetic
modification produces a deletion, addition, replacement or a
combination thereof of a region of the rat Rag2 locus, wherein the
genetic modification at the Rag2 locus results in a decrease in
Rag2 activity. In one embodiment, an interleukin-2 receptor
gamma/Rag2 knockout is generated. Such a rat has a SCID
phenotype.
In one embodiment, the mammalian nucleic acid comprises a genomic
locus that encodes a protein expressed in the nervous system, the
skeletal system, the digestive system, the circulatory system, the
muscular system, the respiratory system, the cardiovascular system,
the lymphatic system, the endocrine system, the urinary system, the
reproductive system, or a combination thereof. In one embodiment,
the mammalian nucleic acid comprises a genomic locus that encodes a
protein expressed in a bone marrow or a bone marrow-derived cell.
In one embodiment, the nucleic acid comprises a genomic locus that
encodes a protein expressed in a spleen cell. In one embodiment,
the genomic locus comprises a mouse genomic DNA sequence, a rat
genomic DNA sequence a human genomic DNA sequence, or a combination
thereof. In one embodiment, the genomic locus comprises, in any
order, rat and human genomic DNA sequences. In one embodiment, the
genomic locus comprises, in any order, mouse and human genomic DNA
sequences. In one embodiment, the genomic locus comprises, in any
order, mouse and rat genomic DNA sequences. In one embodiment, the
genomic locus comprises, in any order, rat, mouse, and human
genomic DNA sequences.
In one embodiment, the insert nucleic acid comprises a genetic
modification in a coding sequence of a gene. In one embodiment, the
genetic modification comprises a deletion mutation in the coding
sequence. In one embodiment, the genetic modification comprises a
fusion of two endogenous coding sequences.
In one embodiment, the genetic modification comprises a deletion of
a non-protein-coding sequence, but does not comprise a deletion of
a protein-coding sequence. In one embodiment, the deletion of the
non-protein-coding sequence comprises a deletion of a regulatory
element. In one embodiment, the genetic modification comprises an
addition of a promoter. In one embodiment, the genetic modification
comprises a replacement of a promoter or regulatory element. In one
embodiment, the regulatory element is an enhancer. In one
embodiment, the regulatory element is a transcriptional
repressor-binding element.
In one embodiment, the genetic modification comprises placement of
a human nucleic acid sequence encoding a mutant human protein. In
one embodiment, the genetic modification comprises at least one
human disease allele of a human gene. In one embodiment, the human
disease is a neurological disease. In one embodiment, the human
disease is a cardiovascular disease. In one embodiment, the human
disease is a kidney disease. In one embodiment, the human disease
is a muscle disease. In one embodiment, the human disease is a
blood disease. In one embodiment, the human disease is a cancer. In
one embodiment, the human disease is an immune system disease. In
one embodiment, the human disease allele is a dominant allele. In
one embodiment, the human disease allele is a recessive allele. In
one embodiment, the human disease allele comprises a single
nucleotide polymorphism (SNP) allele.
The polynucleotide of interest within the insert nucleic acid
and/or integrated at the target locus can also comprise a
regulatory sequence, including for example, a promoter sequence, an
enhancer sequence, or a transcriptional repressor-binding sequence.
In specific embodiments, the polynucleotide of interest within the
insert nucleic acid and/or integrated at the target genomic locus
comprises a polynucleotide having a deletion of a
non-protein-coding sequence, but does not comprise a deletion of a
protein-coding sequence. In one embodiment, the deletion of the
non-protein-coding sequence comprises a deletion of a regulatory
sequence. In another embodiment, the deletion of the regulatory
element comprises a deletion of a promoter sequence. In one
embodiment, the deletion of the regulatory element comprises a
deletion of an enhancer sequence. Such a polynucleotide of interest
can be from any organism, including, but not limited to, a mammal,
a non-human mammal, rodent, mouse, rat, a human, a monkey, an
agricultural mammal or a domestic mammal polynucleotide encoding a
mutant protein.
5. Methods of Introducing Sequences and Generation of Transgenic
Animals
As outlined above, methods and compositions are provided herein to
allow for the targeted integration of one or more polynucleotides
of interest into a target locus. Such systems employ a variety of
components and for ease of reference, herein the term "targeted
integration system" generically comprises all the components
required for an integration event (i.e. in non-limiting examples,
the various nuclease agents, recognition sites, insert DNA
polynucleotides, targeting vectors, target genomic locus, and/or
polynucleotides of interest).
The methods provided herein comprise introducing into a cell one or
more polynucleotides or polypeptide constructs comprising the
various components of the targeted genomic integration system.
"Introducing" means presenting to the cell the sequence
(polypeptide or polynucleotide) in such a manner that the sequence
gains access to the interior of the cell. The methods provided
herein do not depend on a particular method for introducing any
component of the targeted genomic integration system into the cell,
only that the polynucleotide gains access to the interior of a
least one cell. Methods for introducing polynucleotides into
various cell types are known in the art and include, but are not
limited to, stable transfection methods, transient transfection
methods, and virus-mediated methods.
In some embodiments, the cells employed in the methods and
compositions have a DNA construct stably incorporated into their
genome. "Stably incorporated" or "stably introduced" means the
introduction of a polynucleotide into the cell such that the
nucleotide sequence integrates into the genome of the cell and is
capable of being inherited by progeny thereof. Any protocol may be
used for the stable incorporation of the DNA constructs or the
various components of the targeted genomic integration system.
Transfection protocols as well as protocols for introducing
polypeptides or polynucleotide sequences into cells may vary.
Non-limiting transfection methods include chemical-based
transfection methods include the use of liposomes; nanoparticles;
calcium phosphate (Graham et al. (1973). Virology 52 (2): 456-67,
Bacchetti et al. (1977) Proc Nati Acad Sci USA 74 (4): 1590-A and,
Kriegler, M (1991). Transfer and Expression: A Laboratory Manual.
New York: W. H. Freeman and Company. pp. 96-97); dendrimers; or
cationic polymers such as DEAE-dextran or polyethylenimine. Non
chemical methods include electroporation; Sono-poration; and
optical transfection. Particle-based transfection include the use
of a gene gun, magnet assisted transfection (Bertram, J. (2006)
Current Pharmaceutical Biotechnology 7, 277-28). Viral methods can
also be used for transfection.
In one embodiment, the introducing one or more of the
polynucleotides into a cell is mediated by electroporation, by
intracytoplasmic injection, by a viral infection, by an adenovirus,
by lentivirus, by retrovirus, by transfection, by lipid-mediated
transfection or is mediated via Nucleofection.TM..
In one embodiment, introduction one or more of the polynucleotides
into a cell further comprises: introducing an expression construct
comprising a nucleic acid sequence of interest operably linked to a
promoter. In one embodiment, the promoter is a
constitutively-active promoter. In one embodiment, the promoter is
an inducible promoter. In one embodiment, the promoter is active in
the rat embryonic stem cell.
In one embodiment, the expression construct is introduced together
with the LTVEC. In one embodiment, the expression construct is
introduced separately from the LTVEC over a period of time.
In one embodiment, the introduction of the one or more
polynucleotides into the cell can be performed multiple times over
a period of time. In one embodiment, the introduction of the one or
more polynucleotides into the cell are performed at least two times
over a period of time, at least three times over a period of time,
at least four times over a period of time, at least five times over
a period of time, at least six times over a period of time, at
least seven times over a period of time, at least eight times over
a period of time, at least nine times over a period of times, at
least ten times over a period of time, at least eleven times, at
least twelve times over a period of time, at least thirteen times
over a period of time, at least fourteen times over a period of
time, at least fifteen times over a period of time, at least
sixteen times over a period of time, at least seventeen times over
a period of time, at least eighteen times over a period of time, at
least nineteen times over a period of time, or at least twenty
times over a period of time.
In one embodiment, the nuclease agent is introduced into the cell
simultaneously with the targeting vector or the large targeting
vector (LTVEC). Alternatively, the nuclease agent is introduced
separately from the targeting vector or the LTVEC over a period of
time. In one embodiment, the nuclease agent is introduced prior to
the introduction of the targeting vector or the LTVEC, while in
other embodiments, the nuclease agent is introduced following
introduction of the targeting vector or the LTVEC.
In one embodiment, screening step comprises a quantitative assay
for assessing modification of allele (MOA) of a parental
chromosome. In one embodiment, the quantitative assay is carried
out via a quantitative PCR. In one embodiment, the quantitative PCR
is a real-time PCR (qPCR). In one embodiment, the real-time PCR
comprises a first primer set that recognizes the target locus and a
second primer set that recognizes a non-targeted reference locus.
In one embodiment, the primer set comprises a fluorescent probe
that recognizes the amplified sequence. In one embodiment, the
quantitative assay is carried out via fluorescence-mediated in situ
hybridization (FISH). In one embodiment, the quantitative assay is
carried out via comparative genomic hybridization. In one
embodiment, the quantitative assay is carried out via isothermic
DNA amplification. In one embodiment, the quantitative assay is
carried out via isothermic DNA amplification. In one embodiment,
the quantitative assay is carried out via quantitative
hybridization to an immobilized probe(s). In one embodiment, the
quantitative assay is carried out via Invader Probes.RTM.. In one
embodiment, the quantitative assay is carried out via MMP
Assays.RTM.. In one embodiment, the quantitative assay is carried
out via TaqMan.RTM. Molecular Beacon. In one embodiment, the
quantitative assay is carried out via Eclipse.TM. probe technology.
(See, for example, US2005/0144655, which is incorporated by
reference herein in its entirety).
Further provided is a method for making a humanized rat,
comprising: (a) modifying a genome of a pluripotent rat cell with a
targeting vector comprising an insert nucleic acid that comprises a
human nucleic acid sequence to form a donor cell; (b) introducing
the donor cell into a host rat embryo; and (c) gestating the host
rat embryo in a surrogate mother; wherein the surrogate mother
produces a rat progeny that comprises the human nucleic acid
sequence. In one embodiment, the donor cell is introduced into a
host rat embryo that is at the blastocyst stage or at a pre-morula
stage (i.e., a 4 cell stage or an 8 cell stage). Moreover, step (a)
can also be performed with a large targeting vector (LTVEC) and/or
a human nucleic acid sequence at least 5 Kb in length. In still
further embodiments, the genetic modification is capable of being
transmitted through the germline.
Genetically modified rats can be generated employing the various
methods disclosed herein. Such methods comprise (1) integrating one
or more polynucleotide of interest at the target locus of a
pluripotent rat cell to generate a genetically modified pluripotent
rat cell comprising the insert nucleic acid in the targeted genomic
locus employing the methods disclosed herein; (2) selecting the
genetically modified pluripotent rat cell having the one or more
polynucleotides of interest at the target genomic locus; (3)
introducing the genetically modified pluripotent rat cell into a
rat host embryo; and (4) implanting the host rat embryo comprising
the genetically modified pluripotent rat cell into a surrogate
mother. A progeny from the genetically modified pluripotent rat
cell is generated. In one embodiment, the donor cell is introduced
into a rat host embryo at the blastocyst stage or at the pre-morula
stage (i.e., the 4 cell stage or the 8 cell stage). Progeny that
are capable of transmitting the genetic modification though the
germline are generated. The pluripotent rat cell can be a rat ES
cell as discussed elsewhere herein.
Nuclear transfer techniques can also be used to generate the
genetically modified rats. Briefly, methods for nuclear transfer
include the steps of: (1) enucleating an oocyte; (2) isolating a
donor cell or nucleus to be combined with the enucleated oocyte;
(3) inserting the cell or nucleus into the enucleated oocyte to
form a reconstituted cell; (4) implanting the reconstituted cell
into the womb of an animal to form an embryo; and (5) allowing the
embryo to develop. In such methods oocytes are generally retrieved
from deceased animals, although they may be isolated also from
either oviducts and/or ovaries of live animals. Oocytes can be
matured in a variety of medium known to those of ordinary skill in
the art prior to enucleation. Enucleation of the oocyte can be
performed in a number of manners well known to those of ordinary
skill in the art. Insertion of the donor cell or nucleus into the
enucleated oocyte to form a reconstituted cell is usually by
microinjection of a donor cell under the zona pellucida prior to
fusion. Fusion may be induced by application of a DC electrical
pulse across the contact/fusion plane (electrofusion), by exposure
of the cells to fusion-promoting chemicals, such as polyethylene
glycol, or by way of an inactivated virus, such as the Sendai
virus. A reconstituted cell is typically activated by electrical
and/or non-electrical means before, during, and/or after fusion of
the nuclear donor and recipient oocyte. Activation methods include
electric pulses, chemically induced shock, penetration by sperm,
increasing levels of divalent cations in the oocyte, and reducing
phosphorylation of cellular proteins (as by way of kinase
inhibitors) in the oocyte. The activated reconstituted cells, or
embryos, are typically cultured in medium well known to those of
ordinary skill in the art and then transferred to the womb of an
animal. See, for example, US20080092249, WO/1999/005266A2,
US20040177390, WO/2008/017234A1, and U.S. Pat. No. 7,612,250, each
of which is herein incorporated by reference.
In one aspect, a method for making a genetically modified rat is
provided, comprising modifying a genomic locus of interest in a
pluripotent rat cell employing endonuclease-mediated gene targeting
to introduce a modification at a rat genomic locus of interest to
form a modified pluripotent rat cell, maintaining the modified
pluripotent rat cell under conditions sufficient to maintain
pluripotency, employing the modified pluripotent rat cell as a
donor cell in a rat host embryo, and gestating the host embryo
comprising the modified pluripotent rat cell in a surrogate mother,
wherein the host embryo is gestated by the surrogate mother and a
genetically modified rat progeny is born.
In one embodiment, the target sequence is located in an intron. In
one embodiment, the target sequence is located in an exon. In one
embodiment, the target sequence is located in a promoter. In one
embodiment, the target sequence is located in a promoter regulatory
region. In one embodiment, the target sequence is located in an
enhancer region.
In one embodiment, introducing step is performed multiple times
over a period of time using a plurality of endonucleases that
recognize distinct target sequences. In one embodiment, step is
performed at least two times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least three times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least four times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least five times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least six times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least seven times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least eight times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least nine times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least ten times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least eleven times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least twelve times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least thirteen times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least fourteen times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least fifteen times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least sixteen times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least seventeen times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least eighteen times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, at least nineteen times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences, or at least twenty times over a period of time using a
plurality of endonucleases that recognize distinct target
sequences.
In one embodiment, introducing step is mediated by electroporation,
by intracytoplasmic injection, by an adenovirus, by lentivirus, by
retrovirus, by transfection, by lipid-mediated transfection or is
mediated via Nucleofection.TM.
In one embodiment, the method further comprises introducing an
exogenous nucleic acid into the genetically modified pluripotent
rat cell. In one embodiment, the exogenous nucleic acid is a
transgene. In one embodiment, the exogenous nucleic acid is
introduced into an endogenous locus. In one embodiment, the
exogenous nucleic acid is introduced ectopically (e.g., at a locus
different from its endogenous locus).
In one aspect, a method for making a genetically modified rat is
provided, comprising modifying a genomic locus of interest in a
pluripotent rat cell employing RNA-guided genome engineering to
introduce a modification at a rat genomic locus of interest to form
a modified pluripotent rat cell, maintaining the modified
pluripotent rat cell under conditions sufficient to maintain
pluripotency, employing the modified pluripotent rat cell as a
donor cell in a rat host embryo, and gestating the host embryo
comprising the modified pluripotent rat cell in a surrogate mother,
wherein the host embryo is gestated by the surrogate mother and a
genetically modified rat progeny is born.
In one embodiment, the method has a targeting rate ranging from
about 2% to about 80%.
In one embodiment, the method comprises co-introducing a plurality
of the second expression construct comprising distinct genomic
target sequences for multiplex editing of distinct genomic loci. In
on embodiment, the method comprises introducing a plurality of the
second expression construct comprising distinct genomic target
sequences for multiplex editing of distinct genomic loci over a
period of time.
In one embodiment, introducing step is performed multiple times
over a period of time. In one embodiment, introducing step (b) is
performed at least two times over a period of time, at least three
times over a period of time, at least four times over a period of
time, at least five times over a period of time, at least six times
over a period of time, at least seven times over a period of time,
at least eight times over a period of time, at least nine times
over a period of time, at least ten times over a period of time, at
least eleven times over a period of time, at least twelve times
over a period of time, at least thirteen times over a period of
time, at least fourteen times over a period of time, at least
fifteen times over a period of time, at least sixteen times over a
period of time, at least seventeen times over a period of time, at
least eighteen times over a period of time, at least nineteen times
over a period of time, at least twenty times over a period of
time.
In one embodiment, the first expression construct and the second
expression construct are expressed from a same plasmid.
In one embodiment, introducing step is mediated by electroporation,
by intracytoplasmic injection, by an adenovirus, by lentivirus, by
retrovirus, by transfection, by lipid-mediated transfection or is
mediated via Nucleofection.TM..
In one embodiment, the method further comprises introducing an
exogenous nucleic acid into the pluripotent rat cell comprising the
mutant allele.
In one embodiment, the exogenous nucleic acid is a transgene. In
one embodiment, the exogenous nucleic acid is introduced into an
endogenous locus. In one embodiment, the exogenous nucleic acid is
placed ectopically (e.g., at a locus different from its endogenous
locus).
In one embodiment, the method further comprises introducing an
exogenous nucleic acid into the genetically modified pluripotent
rat cell. In one embodiment, the exogenous nucleic acid is a
transgene. In one embodiment, the exogenous nucleic acid is
introduced into an endogenous locus. In one embodiment, the
exogenous nucleic acid is introduced ectopically (e.g., at a locus
different from its endogenous locus).
In one aspect, a method for making a humanized rat is provided,
comprising modifying a genome of a pluripotent rat cell with an
LTVEC comprising an insert that comprises a human sequence of at
least 5 kb, and employing the pluripotent rat cell as a donor cell,
introducing the donor cell into a host embryo, and gestating the
host embryo in a surrogate mother, wherein the surrogate mother
births a rat progeny that comprises the humanization.
Other methods for making a genetically modified rat comprising in
its germline one or more genetic modifications as described herein
is provided, comprising: (a) modifying a targeted rat locus
contained in a prokaryotic cell employing the various methods
described herein; (b) selecting a modified prokaryotic cell
comprising the genetic modification at the targeted rat locus; (c)
isolating the genetically modified targeting vector from the genome
of the modified prokaryotic cell; (d) introducing the genetically
modified targeting vector into a pluripotent rat cell to generate a
genetically modified pluripotent cell comprising the insert nucleic
acid at the targeted genomic locus; (e) selecting the genetically
modified rat pluripotent cell; (f) introducing the genetically
modified pluripotent rat cell into a host rat embryo at a
pre-morula stage; and (g) implanting the host rat embryo comprising
the genetically modified pluripotent rat cell into a surrogate
mother to generate an F0 generation derived from the genetically
modified pluripotent rat cell. In such methods the targeting vector
can comprise a large targeting vector. The pluripotent rat cell can
be a rat ES cell. In further methods, the isolating step (c)
further comprises (c1) linearizing the genetically modified
targeting vector (i.e., the genetically modified. LTVEC). In still
further embodiments, the introducing step (d) further comprises
(d1) introducing a nuclease agent as described herein into the
pluripotent rat cell. In one embodiment, selecting steps (b) and/or
(e) are carried out by applying a selectable agent as described
herein to the prokaryotic cell or the pluripotent rat cell. In one
embodiment, selecting steps (b) and/or (e) are carried out via a
modification of allele (MOA) assay as described herein.
Further methods for modifying a target genomic locus of a mammalian
cell via bacterial homologous recombination (BHR) in a prokaryotic
cell are provided and comprise: (a) providing a prokaryotic cell
comprising a target locus comprising a rat nucleic acid, (b)
introducing into the prokaryotic cell a targeting vector comprising
an insert nucleic acid flanked with a 5' rat homology arm and a 3'
rat homology arm, wherein the insert nucleic acid comprises a
mammalian region (including, for example, a DNA insert from a
human), and (c) selecting a targeted prokaryotic cell comprising
the insert nucleic acid at the target rat locus, wherein the
prokaryotic cell is capable of expressing a recombinase that
mediates the BHR. Step (a1) can comprise providing a prokaryotic
cell comprising a target locus comprising a rat nucleic acid
comprising a first polynucleotide comprising a first recognition
site for a first nuclease agent, and step (b1) can further comprise
expressing in the prokaryotic cell a nuclease agent that makes a
nick or double-strand break at or near the first recognition site.
Steps (a)-(c) can be serially repeated as disclosed herein to allow
the introduction of multiple insert nucleic acids at the targeted
rat locus in the prokaryotic cell. Once the targeted genomic locus
is "built" with the prokaryotic cell, a targeting vector comprising
the modified target rat locus can be isolated from the prokaryotic
cell and introduced into a target genomic locus within a
pluripotent rat cell. Pluripotent rat cells (i.e., rat ES cells)
comprising the modified genomic locus can then be made into
genetically modified rats.
In some embodiments, various genetic modifications of the target
genomic loci described herein can be carried out by a series of
homologous recombination reactions (BHR) in bacterial cells using
an LTVEC derived from Bacterial Artificial Chromosome (BAC) DNA
using VELOCIGENE.RTM. genetic engineering technology (see, e.g.,
U.S. Pat. No. 6,586,251 and Valenzuela, D. M. et al. (2003),
High-throughput engineering of the mouse genome coupled with
high-resolution expression analysis, Nature Biotechnology 21(6):
652-659, which is incorporated herein by reference in their
entireties).
In some embodiments, targeted rat ES cells comprising various
genetic modifications as described herein are used as insert ES
cells and introduced into a pre-morula stage embryo from a
corresponding organism, e.g., an 8-cell stage mouse embryo, via the
VELOCIMOUSE.RTM. method (see, e.g., U.S. Pat. Nos. 7,576,259,
7,659,442, 7,294,754, and US 2008-0078000 A1, all of which are
incorporated by reference herein in their entireties). The rat
embryo comprising the genetically modified rat ES cells is
incubated until the blastocyst stage and then implanted into a
surrogate mother to produce an F0. Rats bearing the genetically
modified genomic locus can be identified via modification of allele
(MOA) assay as described herein. The resulting F0 generation rat
derived from the genetically modified ES rat cells is crossed to a
wild-type rat to obtain F1 generation offspring. Following
genotyping with specific primers and/or probes, F1 rats that are
heterozygous for the genetically modified genomic locus are crossed
to each other to produce rats that are homozygous for the
genetically modified genomic locus. Alternatively, an F0 female rat
and an F0 male rat each having the genetic modification can be
crossed to obtain an F1 rat homozygous for the genetic
modification.
In one aspect, a genetically modified rat genome is provided,
comprising a targeted modification of an endogenous rat nucleic
acid sequence with a homologous or orthologous non-rat nucleic acid
sequence.
In one embodiment, the homologous or orthologous non-rat nucleic
acid sequence is of a length from about 5 kb to about 200 kb. In
one embodiment, the homologous or orthologous non-rat nucleic acid
sequence ranges from about 5 kb to about 10 kb. In one embodiment,
the homologous or orthologous non-rat nucleic acid sequence ranges
from about 10 kb to about 20 kb. In one embodiment, the homologous
or orthologous non-rat nucleic acid sequence ranges from about 20
kb to about 30 kb. In one embodiment, the homologous or orthologous
non-rat nucleic acid sequence ranges from about 30 kb to about 40
kb. In one embodiment, the homologous or orthologous non-rat
nucleic acid sequence ranges from about 40 kb to about 50 kb. In
one embodiment, the homologous or orthologous non-rat nucleic acid
sequence ranges from about 50 kb to about 60 kb. In one embodiment,
the homologous or orthologous non-rat nucleic acid sequence ranges
from about 60 kb to about 70 kb. In one embodiment, the homologous
or orthologous non-rat nucleic acid sequence ranges from about 70
kb to about 80 kb. In one embodiment, the homologous or orthologous
non-rat nucleic acid sequence ranges from about 80 kb to about 90
kb. In one embodiment, the homologous or orthologous non-rat
nucleic acid sequence ranges from about 90 kb to about 100 kb. In
one embodiment, the homologous or orthologous non-rat nucleic acid
sequence ranges from about 100 kb to about 110 kb. In one
embodiment, the homologous or orthologous non-rat nucleic acid
sequence ranges from about 110 kb to about 120 kb. In one
embodiment, the homologous or orthologous non-rat nucleic acid
sequence ranges from about 120 kb to about 130 kb. In one
embodiment, the homologous or orthologous non-rat nucleic acid
sequence ranges from about 140 kb to about 150 kb. In one
embodiment, the homologous or orthologous non-rat nucleic acid
sequence ranges from about 150 kb to about 160 kb. In one
embodiment, the homologous or orthologous non-rat nucleic acid
sequence ranges from about 160 kb to about 170 kb. In one
embodiment, the homologous or orthologous non-rat nucleic acid
sequence ranges from about 170 kb to about 180 kb. In one
embodiment, the homologous or orthologous non-rat nucleic acid
sequence ranges from about 180 kb to about 190 kb. In one
embodiment, the homologous or orthologous non-rat nucleic acid
sequence ranges from about 190 kb to about 200 kb. Various
polynucleotides of interest that can be employed in the insert
nucleic acid are described elsewhere herein.
6. Cells
The various methods and compositions described herein employ a
genomic locus targeting system in a cell. In one embodiment, the
cell is a pluripotent cell. In one embodiment, the pluripotent cell
is a non-human pluripotent cell. In one embodiment, the non-human
pluripotent cell is a mammalian pluripotent cell. In one
embodiment, the pluripotent cell is a human induced pluripotent
stem (iPS) cell.
In one embodiment, the pluripotent cell is a pluripotent rat cell.
In one embodiment, the pluripotent rat cell is a rat embryonic stem
(ES) cell. In one embodiment, the pluripotent rat cell is an
induced pluripotent stem (iPS) cell or is a developmentally
restricted progenitor cell. In other embodiments, the pluripotent
rat cell is able to sustain its pluripotency following at least one
targeted genetic modification of its genome and is able to transmit
the targeted modification to a germline of an F1 generation.
In one embodiment, the pluripotent cell is a non-human fertilized
egg at the single cell stage. In one embodiment, the non-human
fertilized egg is a mammalian fertilized egg. In one embodiment,
the mammalian fertilized egg is a rodent fertilized egg at the
single cell stage. In one embodiment, the mammalian fertilized egg
is a rat or mouse fertilized egg at the single cell stage.
The various cells employed in the method and compositions disclosed
herein can also comprise prokaryotic cells, such as a bacterial
cell, including E. coli. In specific embodiments, the prokaryotic
cell is a recombination-competent strain of E. coli. In one
embodiment, the prokaryotic cell comprises a nucleic acid that
encodes the recombinase, while in other instances, the prokaryotic
cell does not comprise the nucleic acid that encodes the
recombinase, and the nucleic acid encoding the recombinase is
introduced into the prokaryotic cell. In one embodiment, the
nucleic acid encoding the recombinase comprises a DNA or an mRNA.
In some embodiments, the nucleic acid encoding the recombinase is
pABG. In one embodiment, the recombinase is expressed under the
control of an inducible promoter. In one embodiment, expression of
the recombinase is controlled by arabinose.
A. Rat Embryonic Stem (ES) Cells
As outlined in detail above, the various compositions and methods
provided herein can employ embryonic stem (ES) cells from rat. In
one embodiment, the pluripotent rat cell is a rat ES cell. In one
embodiment, the rat ES cell is derived from a rat strain is a
Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer
strain, F344, F6, and Dark Agouti or ACI. In one embodiment, the
rat strain is a mix of two or more of a strain selected from the
group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6,
and Dark Agouti. In one embodiment, the rat ES cell is derived from
an inbred strain. In one embodiment, the rat ES cell is derived
from a strain selected from a DA strain and an ACI strain. In a
specific embodiment, the rat ES cell is derived from an ACI strain.
In one embodiment, the rat ES cell is derived from a rat
blastocyst.
In other embodiments, the rat ES cell is characterized by
expression of at least one pluripotency marker. In specific
embodiments, the rat ES cell is characterized by expression of a
pluripotency marker comprising Oct-4, Sox-2, alkaline phosphatase,
or a combination thereof. In one embodiment, the rat ES cell is a
male (XY) rat ES cell or a female (XX) rat ES cell.
In one embodiment, following the one to 15 serial genetic
modifications, the genetically modified rat ES cells upon exposure
to differentiation medium are capable of differentiation into a
plurality of cell types.
In one embodiment, following the one to 15 serial genetic
modifications, the genetically modified rat ES cells are capable of
being maintained in an undifferentiated state in culture. In one
embodiment, the genetically modified and cultured rat ES cells in
the undifferentiated state, when employed as donor cells in a rat
host embryo, populate the embryo and form a blastocyst comprising
the one to fifteen genetic modifications. In one embodiment, the
blastocyst, when implanted into a surrogate mother under conditions
suitable for gestation, develops into an F0 rat progeny that
comprises the one to 15 genetic modifications.
In one aspect, an isolated rat ES cell is provided that is capable
of sustaining pluripotency following one or more genetic
modifications in vitro, and that is capable of transmitting a
genetically modified genome to a germline of an F1 generation.
In one embodiment, the rat ES cell maintains its pluripotency to
develop into a plurality of cell types following the one or more
serial genetic modifications in vitro (e.g., two, three, four,
five, or six or more serial genetic modifications). In one
embodiment, the genetic modification is mediated by an
electroporation, by intracytoplasmic injection, by a viral
infection, by an adenovirus, by lentivirus, by retrovirus, by
transfection, by lipid-mediated transfection, or by
Nucleofaction.TM..
In one embodiment, the rat ES cell maintains its pluripotency to
develop into a plurality of cell types following a single round of
electroporation with an exogenous nucleic acid. In one embodiment,
the rat ES cell maintains its pluripotency to develop into a
plurality of cell types following a 2.sup.nd, 3.sup.rd, 4.sup.th,
5.sup.th, 6.sup.th, 7.sup.th, 8.sup.th, 9.sup.th, 10.sup.th,
11.sup.th, 12.sup.th, 13.sup.th, 14.sup.th, 15.sup.th round of
electroporation with an exogenous nucleic acid.
In other embodiments, the rat ES cells employed are those described
in U.S. application Ser. No. 14/185,703, filed Feb. 20, 2014 and
herein incorporated by reference in its entirety.
The pluripotent rat cell employed in the various methods and
compositions disclosed herein can be characterized by expression of
at least one pluripotency marker comprising Dnmt3L, Eras, Err-beta,
Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4,
Sox15, Sox2, Utf1, and/or a combination thereof. In other
instances, the pluripotent rat cell employed in the various methods
and compositions disclosed herein is characterized by one or more
of the following features: (a) lack of expression of one or more
pluripotency markers comprising c-Myc, Ecat1, and/or Rexo1; (b)
lack of expression of one or more mesodermal markers comprising
Brachyury and/or Bmpr2; (c) lack of expression of one or more
endodermal markers comprising Gata6, Sox17, and/or Sox7; or (d)
lack of expression of one or more neural markers comprising Nestin
and/or Pax6. As used herein, "lack of expression" as it relates to
expression of a pluripotency marker means that the expression of
the pluripotency marker is at or below the experimental background
as determined for each individual experiment.
In one non-limiting embodiment, the rat ES cells provided herein
have one or more of any of the following properties:
(a) have germ-line competency, meaning when the rat ES cell is
implanted into a rat host embryo, the genome of the rat ES cell
line is transmitted into an offspring;
(b) have germ-line competency following at least one targeted
genetic modification, meaning when the rat ES cell having the
targeted genetic modification is implanted into a rat host embryo,
the targeted genetic modification within the genome of the rat ES
cell line is transmitted into an offspring;
(c) have pluripotency in vitro;
(d) have totipotency in vitro;
(e) when cultured in vitro loosely adhere to a feeder cell
layer;
(f) when cultured in vitro form sphere-like colonies when plated on
a feeder cell layer in vitro;
(g) maintain pluripotency when cultured in vitro under conditions
comprising a feeder cell layer that is not genetically modified to
express leukemia inhibitory factor (LIF), wherein the culture media
comprises a sufficient concentration of LIF;
(h) maintain pluripotency when cultured in vitro under conditions
comprising a feeder cell layer, wherein the culture media comprises
mouse LIF or an active variant or fragment thereof;
(i) comprise a molecular signature that is characterized by i) the
expression of one or more of rat ES cell-specific genes comprising
Adheres Junctions Associate Protein (Ajap1), Claudin 5 (Cldn5),
Cdc42 guanine nucleotide exchange factor 9 (Arhgef9),
Calcium/calmodulin-dependent protein kinase IV (Camk4), ephrin-A1
(Efna1), EPH receptor A4 (Epha4), gap junction protein beta 5
(Gjb5), Insulin-like growth factor binding protein-like 1
(Igfbpl1), Interleukin 36 beta(Il1f8), Interleukin 28 receptor,
alpha (Il28ra), left-right determination factor 1 (Lefty1),
Leukemia inhibitory factor receptor alpha (Lifr), Lysophosphatidic
acid receptor 2 (Lpar2), Neuronal pentraxin receptor (Ntm), Protein
tyrosine phosphatase non-receptor type 18 (Ptpn18), Caudal type
homeobox 2 (Cdx2), Fibronectin type III and ankyrin repeat domains
1 (Fank1), Forkhead box E1 (thyroid transcription factor 2)
(Foxe1), Hairy/enhancer-of-split related with YRPW motif 2 (Hey2),
Forkhead box E1 (thyroid transcription factor 2) (Foxe1),
Hairy/enhancer-of-split related with YRPW motif 2 (Hey2), Lymphoid
enhancer-binding factor 1 (Lef1), Sal-like 3 (Drosophila) (Sall3),
SATB homeobox 1 (Satb1), miR-632, or a combination thereof; ii) the
expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more of the rat ES
cell-specific genes comprising Adheres Junctions Associate Protein
(Ajap1), Claudin 5 (Cldn5), Cdc42 guanine nucleotide exchange
factor 9 (Arhgef9), Calcium/calmodulin-dependent protein kinase IV
(Camk4), ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junction
protein beta 5 (Gjb5), Insulin-like growth factor binding
protein-like 1 (Igfbpl1), Interleukin 36 beta(Il1f8), Interleukin
28 receptor, alpha (Il28ra), left-right determination factor 1
(Lefty1), Leukemia inhibitory factor receptor alpha (Lifr),
Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin
receptor (Min), Protein tyrosine phosphatase non-receptor type 18
(Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III and
ankyrin repeat domains 1 (Fank1), Forkhead box E1 (thyroid
transcription factor 2) (Foxe1), Hairy/enhancer-of-split related
with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid transcription
factor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW motif
2 (Hey2), Lymphoid enhancer-binding factor 1 (Lef1), Sal-like 3
(Drosophila) (Sall3), SATB homeobox 1 (Satb1), miR-632, or a
combination thereof; iii) at least a 20-fold increase in the
expression of one or more of the rat ES cell-specific genes as set
forth in Table 9 when compared to a F1H4 mouse ES cell; iv) at
least a 20-fold increase in the expression of at least 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25 or more of the rat ES cell-specific genes as set forth in
Table 9 when compared to a F1H4 mouse ES cell; v) the expression of
one or more of rat ES cell-specific genes as set forth in Table 10;
vi) the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,
50 or more of the rat ES cell-specific genes as set forth in Table
10; vii) at least a 20-fold increase in the expression of one or
more of the rat ES cell-specific genes as set forth in Table 10
when compared to a F1H4 mouse ES cell; viii) at least a 20-fold
increase in the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,
40, 45, 50 or more of the rat ES cell-specific genes as set forth
in Table 10 when compared to a F1H4 mouse ES cell; ix) at least a
20-fold decrease in the expression of one or more of the rat ES
cell-specific genes as set forth in Table 8 when compared to a F1H4
mouse ES cell; x) at least a 20-fold decrease in the expression of
at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the
rat ES cell-specific genes as set forth in Table 8 when compared to
a F1H4 mouse ES cell; xi) any combination of expression of the rat
ES cell-specific genes of parts (i)-(x); xii) a relative expression
level of pluripotency markers as shown in Table 11 for at least 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 of the
listed pluripotency markers. See, pluripotency ranking column of
Table 11 for relative expression levels; xiii) a relative
expression level of the mesodermal markers as shown in Table 11 for
at least 2, 3, or 4 of the listed mesodermal markers. See,
mesodermal ranking column in Table 11 for relative expression
levels; xiv) a relative expression level of endodermal markers as
shown in Table 11 for at least 2, 3, 4, 5, or 6 of the listed
endodermal markers. See, endodermal ranking column in Table 11 for
relative expression levels; xv) a relative expression level of
neural markers as shown in Table 11 for at least 2 and 3 of the
listed neural markers. See, neural ranking column in Table 11 for
relative expression levels; xvi) a relative expression level of
trophectoderm markers as shown in Table 11 for the listed
trophectoderm markers. See, trophectoderm ranking column in Table
11 for relative expression levels; xvii) any relative expression
level of one or more (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30)
of the pluripotency markers, mesodermal markers, endodermal
markers, neural markers and/or trophectoderm markers set forth in
Table 11; xviii) the relative expression level of each of the
markers set forth in Table 11; xix) any combination of the
signatures set forth in xii-xiix; and/or xx) any combination of the
signature set forth in i-xiix;
(j) have the ability to produce a F0 rat;
(k) are capable of being subcultured and maintaining the
undifferentiated state;
(l) have the same number of chromosomes as a normal rat cell;
(m) maintain pluripotency in vitro without requiring paracrine LIF
signaling;
(n) have self renewal, meaning they divide indefinitely while
maintaining pluripotency;
(o) the rat ES cells express at least one pluripotency marker
comprising Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1,
LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, and/or a
combination thereof;
(p) the rat ES cells do not express one or more differentiation
markers comprising c-Myc, Ecat1, and/or Rexo1;
(q) the rat ES cells do not express one or more mesodermal markers
comprising Brachyury, Bmpr2, and/or a combination thereof;
(r) the rat ES cells do not express one or more endodermal markers
comprising Gata6, Sox17, Sox7, and/or combination thereof;
and/or
(s) the rat ES cells do not express one or more neural markers
comprising Nestin, Pax6, and/or combination thereof.
One or more of the characteristics outlined in (a)-(s) can be
present in a rat ES cell, a rat ES cell population or a rat ES cell
line employed in the methods and compositions provided herein,
wherein the rat ES cells have not undergone a targeted genetic
modification. Moreover, following the one or more genetic
modification to the rat target locus as described in detail above,
the one or more of the characteristics outlined in (a)-(s) can be
retained in the rat ES cell following the genetic modification of
the target locus.
In one embodiment, the rat ES cell exhibits a homologous
recombination efficiency of at least 2%, at least 3%, at least 4%,
at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at
least 10%, at least 11%, at least 12%, at least 13%, at least 14%,
at least 15%, at least 20%, at least 25%, at least 30%, at least
35%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, or at least
80%.
In one embodiment, the homologous recombination efficiency
employing the rat ES cell is greater than 4%.
In one embodiment, the rat ES cell has a doubling time ranging from
24 hours to 36 hours. In one embodiment, the rat ES cell has a
doubling time of 25 hours.
In one embodiment, the rat ES cell can be passaged up to at least
15 times in 2i medium (Millipore Cat. SF016-200). In one
embodiment, the rat ES cell can be passaged at least 14 times in 2i
medium (Millipore Cat. No. SF016-200). In one embodiment, the rat
ES cell can be passaged at least 13, 12, 11, 10, 9, 8, 7, 6, or 5
times in 2i medium.
In one embodiment, when transplanted into a pre-morula stage rat
embryo, the rat ES cell can contribute to at least 90% of the cells
in an F0 generation. In one embodiment, when transplanted into a
pre-morula stage rat embryo, the rat ES cell can contribute to at
least 95%, 96%, 97%, 98%, or 99% of the cells in an F0
generation.
In specific embodiments, the various rat ES cells and cell lines
employed in the various methods and compositions provided herein
are used to generate a targeted modification at a target locus. The
rat ES cell having these targeted genetic modifications can be
germ-line competent, meaning when the rat ES cell having the
targeted genetic modification is implanted into a rat host embryo,
the targeted genetic modification of the rat ES cell is transmitted
to the offspring (i.e., the F1 population). Thus, in various
aspects, the rat ES cells in the various methods and compositions
are employed to obtain a high frequency, or high efficiency, of
germline transmission of a rat cell genome from rat ES cells that
have undergone a targeted genetic modification. In various
embodiments, the frequency of germline transmission is greater than
1:600, greater than 1:500, greater than 1:400, greater than 1:300,
greater than 1:200, and greater than 1:100. In various embodiments,
the frequency of germline transmission is greater than 1%, greater
than 2%, greater than 3%, greater than 4%, greater than 5%, greater
than 6%, greater than 7%, greater than 8%, greater than 9%, greater
than 10%, up to about 16%, greater than 25%, greater than 50%,
greater than 60%, greater than 65%, greater than 70%, greater than
75% or greater. In various embodiments, the frequency of germline
transmission ranges from 9% to 16%. In various aspects, percent of
donor rESC-derived progeny in the F1 generation is 1% or more, 2%
or more, 3% or more, 10% or more, 20% or more, 30% or more, 40% or
more, 50% or more, 60% or more, from 3% to about 10% or more; from
3% or more to about 63%, from about 10% to about 30%, from about
10% to about 50%, from about 30% to about 70%, from about 30% to
about 60%, from about 20% to about 40%, from about 20% to 65%, or
from about 40% to 70%. Thus, a rat ES cell that has a targeted
genetic modification have the ability to transmit their genome into
the F1 population.
A rat ES cell that has a targeted genetic modification can be
pluripotent and/or totipotent. Various methods can be used to
determine if a rat ES cell is pluripotent. For example, the ES cell
can be assayed for the expression of various pluripotent markers
including, but not limited to, Oct-4, Sox2, alkaline phosphatase,
or a combination thereof. See, for example, Okamoto, K. et al.,
Cell, 60: 461-472 (1990), Scholer, H. R. et al., EMBO J. 9:
2185-2195 (1990)) and Nanog (Mitsui, K. et al., Cell, 113: 631-642
(2003), Chambers, I. et al., Cell, 113: 643-655 (2003) for various
methods of assaying for the presence or the level of such markers.
See, also FIGS. 2 and 3 provided herein. Other pluripotency markers
include, for example, the presence of at least 1, 2, 3, 4, or 5
pluripotency marker comprising Nanog, Klf4, Dppa2, Fgf4, Rex1,
Eras, Err-beta and/or Sall3. Other pluripotency markers include,
for example, the absence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or
10 pluripotency marker comprising T/Brachyury, Flk1, Noda1, Bmp4,
Bmp2, Gata6, Sox17, Hhex1, Sox7, and/or Pax6.
In specific embodiments, the expression and/or the level of
expression of these markers can be determined using RT-PCR. Various
kits are available to determine the level and/or presence of
alkaline phosphatase, including, for example, an ALP tissue
staining kit (Sigma) and Vector Red Alkaline Phosphatase Substrate
Kit I (Funakoshi) and the like. Additional assays include in situ
hybridization, immunohistochemistry, immunofluorescence. In
specific embodiments, the rat ES cell is characterized by
expression of at least one pluripotency marker, including for
example expression of Oct-4, Sox2, alkaline phosphatase, or a
combination thereof, and preferably all three of these markers.
The various rat ES cells employed in the method and compositions
provided herein are capable of maintaining pluripotency and/or
totipotency while being maintained in in vitro culturing
conditions. Thus, the various rat ES cells provide herein can, in
some embodiments, be subcultured while still maintaining the
undifferentiated state. Various methods of culturing the rat ES
cells are discussed in further detail elsewhere herein and in U.S.
patent application Ser. No. 14/185,103, filed on Feb. 20, 2014,
herein incorporated by reference in its entirety.
In some embodiments, the rat embryonic stem cells employed herein
have been isolated from the rat embryo employing various isolation,
purification, and culture expansion techniques which are discussed
in detail in U.S. Application in U.S. patent application Ser. No.
14/185,103, filed on Feb. 20, 2014, herein incorporated by
reference in its entirety.
An "isolated" rat ES cell or rat embryo has been removed from its
natural environment. The term "isolated" can mean free from 70%,
80%, 90%, 95%, 96%, 97%, 98% or 99% of the constituents with which
a component is found in its natural state. As used herein, a rat ES
"cell line" comprises a population of isolated rat cells that were
developed from a single rat ES cell and therefore the population of
cells within a given cell line have a uniform genetic makeup other
than for mutations or karyotypic changes occurring during
propagation or during targeted genetic modifications. For example,
rat ES cells can be characterized by a high level of euploidy.
Nevertheless, in some cell lines the level of euploidy is less than
100% due to karyotypic changes in propagation of the line from a
single cell. Moreover, a given population of rat ES cells can
comprise at least 1.times.10.sup.3, 1.times.10.sup.4,
1.times.10.sup.5, 1.times.10.sup.6, 1.times.10.sup.7,
1.times.10.sup.8, 1.times.10.sup.9, or 1.times.10.sup.10 cells or
greater. Some cell populations have sufficient cells to permit
selection of a desired modified cell but not an excessively greater
number so as to reduce the possibility of mutations or karyotypic
changes developing in the cell line. For example, some cell
populations have 1.times.10.sup.3 to 1.times.10.sup.6 cells.
As discussed elsewhere herein, various methods are provided for the
targeted genetic modification of a rat ES cell line. When such
methods are carried out, at least one cell within a rat ES cell
line contains the targeted genetic modification. Through various
culturing and/or selection techniques rat ES cell lines having one
or more desired targeted genetic modifications are produced.
In specific embodiments, a rat ES cell, a population of rat ES cell
or a rat ES cell line (that have not undergone a targeted genetic
modification and/or have a targeted genetic modification) are
euploid, and thus have a chromosome number that is an exact
multiple of the haploid number. In further embodiment, a rat ES
cell, a population of rat ES cells or a rat ES cell line (that have
not undergone a targeted genetic modification and/or have a
targeted genetic modification) are diploid, and thus have two
haploid sets of homologous chromosomes. When referring to a rat ES
cell population or a population of cells from a given population of
rat ES cells or a rat ES cell line (that have not undergone a
targeted genetic modification and/or have a targeted genetic
modification), at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or 100% of the cells with the given population are euploid
and/or diploid. In other instances, when referring to a rat ES cell
population or a population of cells from a given rat ES cell line
(that have not undergone a targeted genetic modification and/or
have a targeted genetic modification), at least about 50% to 95%,
about 60% to 90%, about 60% to 95%, about 60% to 85%, about 60% to
80%, about 70% to 80%, about 70% to 85%, about 70% to about 90%,
about 70% to about 95%, about 70% to about 100%, about 80% to about
100%, about 80% to about 95%, about 80% to about 90%, about 90% to
about 100%, about 90% to about 99%, about 90% to about 98%, about
90% to about 97%, about 90% to about 95% of the cells within the
given population are euploid and/or diploid.
In still further embodiments, a rat ES cell, a population of rat ES
cells or a rat ES cell line (that have not undergone a targeted
genetic modification and/or have a targeted genetic modification)
have 42 chromosomes. When referring to a rat ES cell population or
a population of cells from a given rat ES cell line (that have not
undergone a targeted genetic modification and/or have a targeted
genetic modification) at least about 50%, 60%, 65%, 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or 100% of the cells with the given population have 42
chromosomes. In other instances, when referring to a rat ES cell
population or a population of cells from a given rat ES cell line
(that have not undergone a targeted genetic modification and/or
have a targeted genetic modification) at least about 50% to 95%,
about 60% to 90%, about 60% to 95%, about 60% to 85%, about 60% to
80%, about 70% to 80%, about 70% to 85%, about 70% to about 90%,
about 70% to about 95%, about 70% to about 100%, about 80% to about
100%, about 80% to about 95%, about 80% to about 90%, about 90% to
about 100%, about 90% to about 99%, about 90% to about 98%, about
90% to about 97%, about 90% to about 95% of the cells within the
given population have 42 chromosomes.
In further embodiments, a rat ES cell, a population of rat ES cells
or a rat ES cell line (that have not undergone a targeted genetic
modification and/or have a targeted genetic modification) provided
herein form sphere-like colonies when plated on a feeder cell layer
in vitro. The "sphere-like" morphology refers to the shape of rat
ES cell colonies in culture, rather than the shape of individual ES
cells. The rat ES cell colonies are spherical-like. Colonies, which
are loosely attached to the feeder cells appear circular (have a
circular-like morphology). Free-floating colonies are
spherical-like. The rat ES cell colonies are spherical-like and
very compact, meaning: the boundaries between cells are very hard
to see. The edge of the colony appears bright and sharp. Individual
nuclei are difficult to distinguish because the cells are very
small (so that the nucleus takes up most of the volume of the
cell). Mouse ES Cells form elongated colonies and attach strongly
to feeder cells. mESC morphology can vary with strain; e.g. B6
colonies are rounder and more domed than F1H4 colonies but are
still more elongated than rESC. Human ES cell colonies are flatter
and more spread out than mESC colonies. The instant rat ES colonies
are not flat and do not resemble human ES cell colonies.
In still further embodiments, a rat ES cell, a population of rat ES
cells or a rat ES cell line (that have not undergone a targeted
genetic modification and/or have a targeted genetic modification)
have a circular morphology. A morphology scale for a circle is
provided below, where a score of a 10 represents a perfect circle
and a score of a 1 represents an ellipse.
Morphology scale of a circle:
10=A circle with a structure having a longitudinal axis and a
vertical axis that run through the center of the structure and are
of equal length.
9=A structure having a longitudinal axis and vertical axis that run
through the center of the structure, wherein one of the axis is
between 0.9999 to 0.9357 the length of the other axis.
8=A structure having a longitudinal axis and vertical axis that run
through the center of the structure, wherein one of the axis is
between 0.9357 to 0.875 the length of the other axis.
7=A structure having a longitudinal axis and vertical axis that run
through the center of the structure, wherein one of the axis is
between 0.875 to about 0.8125 the length of the other axis.
6=A structure having a longitudinal axis and vertical axis that run
through the center of the structure, wherein one of the axis is
between 0.8125 to 0.750 the length of the other axis.
5=A structure having a longitudinal axis and vertical axis that run
through the center of the structure, wherein one of the axis is
between 0.750 to 0.6875 the length of the other axis.
4=A structure having a longitudinal axis and vertical axis that run
through the center of the structure, wherein one of the axis is
between 0.6875 to 0.625 the length of the other axis.
3=A structure having a longitudinal axis and vertical axis that run
through the center of the structure, wherein one of the axis is
between 0.625 to 0.5625 the length of the other axis.
2=A structure having a longitudinal axis and vertical axis that run
through the center of the circle, wherein one of the axis is
between 0.5625 to 0.523 the length of the other axis.
1=An ellipse is defined as having a longitudinal axis and vertical
axis that run through the center of the structure, wherein one of
the axis is between 0.523 to 0.500 the length of the other
axis.
In one non-limiting embodiment, the rat ES cell population or a
population of cells from a given rat ES cell line (that have not
undergone a targeted genetic modification and/or have a targeted
genetic modification) have at least about 50%, 60%, 65%, 70%, 75%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% of the cells with the given population have a
circular morphology score of a 10, 9 or 8. In other embodiments,
the rat ES cell population or a population of cells from a given
rat ES cell line (that have not undergone a targeted genetic
modification and/or have a targeted genetic modification) have at
least about 50% to 95%, about 60% to 90%, about 60% to 95%, about
60% to 85%, about 60% to 80%, about 70% to 80%, about 70% to 85%,
about 70% to about 90%, about 70% to about 95%, about 70% to about
100%, about 80% to about 100%, about 80% to about 95%, about 80% to
about 90%, about 90% to about 100%, about 90% to about 99%, about
90% to about 98%, about 90% to about 97%, about 90% to about 95% of
the cells within the given population have a circular morphology
score of a 10, 9, or 8.
In another non-limiting embodiment, the rat ES cell population or a
population of cells from a given rat ES cell line (that have not
undergone a targeted genetic modification and/or have a targeted
genetic modification) have at least about 50%, 60%, 65%, 70%, 75%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% of the cells with the given population have a
circular morphology score of a 7, 6, 5, 4 or 3. In other
non-limiting embodiments, the rat ES cell population or a
population of cells from a given rat ES cell line (that have not
undergone a targeted genetic modification and/or have a targeted
genetic modification) have at least about 50% to 95%, about 60% to
90%, about 60% to 95%, about 60% to 85%, about 60% to 80%, about
70% to 80%, about 70% to 85%, about 70% to about 90%, about 70% to
about 95%, about 70% to about 100%, about 80% to about 100%, about
80% to about 95%, about 80% to about 90%, about 90% to about 100%,
about 90% to about 99%, about 90% to about 98%, about 90% to about
97%, about 90% to about 95% of the cells within the given
population have a circular morphology score of a 7, 6, 5, 4, or
3.
In still further embodiments, sphere-like colonies form when the
rat ES cells (that have not undergone a targeted genetic
modification and/or have a targeted genetic modification) are
plated on a feeder cell layer in vitro. A morphology scale for a
sphere is provided below, where a score of a 10 represents a
perfect sphere and a score of a 1 represents a three dimensional
elliptical structure.
Morphology Scale of a Sphere-Like Structure:
10=A sphere is a structure having an X-axis and a Y-axis and a
Z-axis each of which runs through the center of the structure and
are of equal length.
9=A structure having an X axis and a Y-axis and a Z-axis that run
through the center of the structure, wherein one of the axis is
between 0.9999 to 0.9357 the length of at least one of the other
axes.
8=A structure having an X axis and a Y-axis and a Z-axis that run
through the center of the structure, wherein one of the axis is
between 0.9357 to 0.875 the length of at least one or both of the
other axes.
7=A structure having an X axis and a Y-axis and a Z-axis that run
through the center of the structure, wherein one of the axis is
between 0.875 to 0.8125 the length of at least one or both of the
other axes.
6=A structure having an X axis and a Y-axis and a Z-axis that run
through the center of the structure, wherein one of the axis is
between 0.8125 to 0.750 the length of at least one or both of the
other axes.
5=A structure having an X axis and a Y-axis and a Z-axis that run
through the center of the structure, wherein one of the axis is
0.750 to 0.6875 the length of at least one or both of the other
axes.
4=A structure having an X axis and a Y-axis and a Z-axis that run
through the center of the structure, wherein one of the axis is
0.6875 to 0.625 the length of at least one or both of the other
axes.
3=A structure having an X axis and a Y-axis and a Z-axis that run
through the center of the structure, wherein one of the axis is
between 0.625 to 0.5625 the length of at least one or both of the
other axes.
2=A structure having an X axis and a Y-axis and a Z-axis that run
through the center of the structure, wherein one of the axis is
between 0.5625 to 0.523 the length of at least one or both of the
other axes.
1=A structure having an X axis and a Y-axis and a Z-axis that run
through the center of the structure, wherein one of the axis is
between 0.523 to 0.500 the length of at least one or both of the
other axes.
In one non-limiting embodiment, the rat ES cell population or a
population of cells from a given rat ES cell line (that have not
undergone a targeted genetic modification and/or have a targeted
genetic modification) have at least about 50%, 60%, 65%, 70%, 75%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% of the colonies that form when the cells are
plated on a feeder cell layer in vitro have a sphere-like
morphology of a 10, 9 or 8. In other embodiments, the rat ES cell
population or a population of cells from a given rat ES cell line
(that have not undergone a targeted genetic modification and/or
have a targeted genetic modification) have at least about 50% to
95%, about 60% to 90%, about 60% to 95%, about 60% to 85%, about
60% to 80%, about 70% to 80%, about 70% to 85%, about 70% to about
90%, about 70% to about 95%, about 70% to about 100%, about 80% to
about 100%, about 80% to about 95%, about 80% to about 90%, about
90% to about 100%, about 90% to about 99%, about 90% to about 98%,
about 90% to about 97%, about 90% to about 95% of the colonies that
form when the cells are plated on a feeder cell layer in vitro have
a sphere-like morphology of a 10, 9 or 8.
In another non-limiting embodiment, the rat ES cell population or a
population of cells from a given rat ES cell line (that have not
undergone a targeted genetic modification and/or have a targeted
genetic modification) have at least about 50%, 60%, 65%, 70%, 75%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% of the colonies that form when the cells are
plated on a feeder cell layer in vitro have a sphere-like
morphology of a 7, 6, 5, 4, or 3. In other embodiments, the rat ES
cell population or a population of cells from a given rat ES cell
line (that have not undergone a targeted genetic modification
and/or have a targeted genetic modification) have at least about
50% to 95%, about 60% to 90%, about 60% to 95%, about 60% to 85%,
about 60% to 80%, about 70% to 80%, about 70% to 85%, about 70% to
about 90%, about 70% to about 95%, about 70% to about 100%, about
80% to about 100%, about 80% to about 95%, about 80% to about 90%,
about 90% to about 100%, about 90% to about 99%, about 90% to about
98%, about 90% to about 97%, about 90% to about 95% of the colonies
that form when the cells are plated on a feeder cell layer in vitro
have a sphere-like morphology of a 7, 6, 5, 4, or 3.
A given rat ES cell, employed in the various methods and
compositions provided herein can be a male (XY) rat ES cell, a male
(XY) population of rat ES cells, or a male (XY) rat ES cell line.
In other embodiments, a population of rat ES cells or a rat ES cell
line employed herein can be a female (XX) rat ES cell, a female
(XX) population of rat ES cells, or a female (XX) rat ES cell line.
Any such rat ES cell, population of rat ES cells or rat ES cell
line can comprise the euploidy and/or diploidy as described
above.
The various rat ES cells employed in the methods and compositions
can be from any rat strain, including but not limited to, an ACI
rat strain, a Dark Agouti (DA) rat strain, a Wistar rat strain, a
LEA rat strain, a Sprague Dawley (SD) rat strain, or a Fischer rat
strain such as Fisher F344 or Fisher F6. The various rat ES cells
can also be obtained from a strain derived from a mix of two or
more strains recited above. In one embodiment, the rat ES cell is
derived from a strain selected from a DA strain and an ACI strain.
In a specific embodiment, the rat ES cell is derived from an ACI
strain. The ACI rat strain is are characterized as having black
agouti, with white belly and feet and an RT1av1 haplotype. Such
strains are available from a variety of sources including Harlan
Laboratories. In other embodiments, the various rat ES cells are
from a Dark Agouti (DA) rat strain, which is characterized as
having an agouti coat and an RT1av1 haplotype. Such rats are
available from a variety of source including Charles River and
Harlan Laboratories. In a further embodiment, the various rat ES
cells employed herein are from an inbred rat strain.
In specific embodiments the rat ES cell line is from an ACI rat and
comprises the ACI.G1 rat ES cell as described in detail in U.S.
patent application Ser. No. 14/185,103, filed on Feb. 20, 2014,
herein incorporated by reference in its entirety. In another
embodiment, the rat ES cell line is from a DA rat and comprises the
DA.2B rat ES cell line or the DA.2C rat ES cell line as described
in detail in U.S. patent application Ser. No. 14/185,103, filed on
Feb. 20, 2014, herein incorporated by reference in its
entirety.
A given rat ES cell provided herein can be obtained from a rat
embryo at various stages of rat embryo development. The rat embryos
employed to derive the rat ES cells can be a morula-stage embryo, a
blastocyst-stage embryo, or a rat embryo at a developmental stage
between a morula-stage embryo and a blastocyst-stage embryo. Thus,
in specific embodiments, the rat embryo employed is at or between
the Witschi stages of 5 and 7. In other embodiments, the rat embryo
employed is at the Witschi stage 5, 6, or 7.
In one embodiment, the rat ES cell is obtained from a rat
blastocyst. In other embodiments, the rat ES cell is obtained from
a blastocyst from a superovulated rat. In other embodiments, the
rat ES cells are obtained from an 8-cell stage embryo, which is
then cultured in vitro until it develops into a morula-stage,
blastocyst stage, an embryo between the Witschi stages 5 and 7, or
into an embryo at the Witschi stage 5, 6, or 7. At which time the
embryos are then plated. Morula-stage embryos comprise a compact
ball of cells with no internal cavity. Blastocyst-stage embryos
have a visible internal cavity (the blastocoel) and contain an
inner cell mass (ICM). The ICM cells form ES cells.
B. Derivation and Propagation of Rat Embryonic Stem (ES) Cells
Methods of derivation and propagation of rat embryonic stem cells
are known in the art and are disclosed, for example, in U.S. patent
application Ser. No. 14/185,103, filed on Feb. 20, 2014, herein
incorporated by reference in its entirety. In specific embodiments,
such methods comprise (a) providing an in vitro culture comprising
a feeder cell layer and a population of isolated rat embryonic stem
(ES) cells; (b) culturing in vitro under conditions which are
sufficient to maintain pluripotency and/or totipotency of the
isolated rat ES cell. Such methods thereby allow for the
propagation of a rat ES cell population and/or a rat ES cell
line.
Methods for culturing a rat embryonic stem cell line is provided.
Such methods comprise culturing in vitro a feeder cell layer and a
rat ES cell line, wherein the culture conditions maintain
pluripotency of the rat ES cells and comprise a media having mouse
leukemia inhibitory factor (LIF) or an active variant or fragment
thereof. The methods can further comprise passaging and culturing
in vitro the cells of the rat ES cell line, wherein each subsequent
in vitro culturing comprises culturing the rat ES cells on the
feeder cell layer under conditions that maintain pluripotency of
the rat ES cells and comprises a media having mouse LIF or an
active variant or fragment thereof.
The culture media employed in the various methods and compositions
can maintain the rat ES cells. The terms "maintaining" and
"maintenance" refer to the stable preservation of at least one or
more of the characteristics or phenotypes of the rat ES cells
outline herein. Such phenotypes can include maintaining
pluripotency and/or totipotency, cell morphology, gene expression
profiles and the other functional characteristics of the rat stem
cells described herein. The term "maintain" can also encompass the
propagation of stem cells, or an increase in the number of stem
cells being cultured. The term further contemplates culture
conditions that permit the stem cells to remain pluripotent, while
the stem cells may or may not continue to divide and increase in
number.
The term "feeder cell" or "feeder cell layer" comprises a culture
of cells that grow in vitro and secrete at least one factor into
the culture medium that is used to support the growth of another
cell of interest in the culture. The feeder cells employed herein
aid in maintaining the pluripotency of the rat ES cells, and in
specific embodiments, one or more of the other characteristics or
phenotypes described herein. Various feeder cells can be used
including, for example, mouse embryonic fibroblasts, including
mouse embryonic fibroblasts obtained between the 12.sup.th and
16.sup.th day of pregnancy. In specific embodiments, feeder cell
layer comprises a monolayer of mitotically inactivated mouse
embryonic fibroblasts (MEFs).
The in vitro cultures of the rat ES cells further comprise an
effective amount of Leukemia Inhibitory Factor (LIF) or an active
variant or fragment thereof. Leukemia inhibitory factor (LIF)
belongs to the IL-6 receptor family. LIF binds to a heterodimeric
membrane receptor made up of a LIF-specific subunit, gp190 or LIFR,
and the subunit gp130, which is shared with the other members of
the IL-6 family. LIF inhibits the differentiation of embryonic stem
cells in mice and contribute to stem cell self-renewal. Human and
mouse LIF share 79% sequence homology and exhibit cross-species
activity. Rat LIF (rtLIF) is a 22.1 kDa protein containing 202
amino acid residues that exhibits 91% amino acid sequence identity
with murine LIF (Takahama et al. 1998). There are six possible
asparagine-linked glycosylation (N-glycosylation) sites which are
conserved among the LIF polypeptide from the various species and an
additional site of Asn150 which is specific for rat LIF. The
tertiary structure of the mouse LIF and its function is described
in further detail in Aikawa et al. (1998) Biosci. Biotechnol.
Biochem. 62 1318-1325 and Senturk et al. (2005) Immunology of
Pregnancy, editor Gil Mor., U.S. Pat. No. 5,750,654 and D P Gearing
(1987) EMBO Journal 1987-12-20, each of which is herein
incorporated by reference in their entirety. A partial mouse LIF
sequence is reported on the SwissProt website under the accession
number P09056.
Mouse LIF activity is assessed by its ability to induce
differentiation of M1 myeloid leukemia cells. The specific activity
is 1.times.10.sup.6 units/ml (Cat. No. 03-0011 from Stemgent) and
1.times.10.sup.7 units/ml (Cat. No. 03-0011-100 from Stemgent),
where 50 units is defined as the amount of mouse LIF required to
induce differentiation in 50% of the M1 colonies in 1 ml of medium.
See, also, Williams, R. L. et al. (1988) Nature 336: 684-687.;
Metcalf, D. et al. (1988) Leukemia 2: 216-221; Niwa, H. et al.
(2009) Nature 460: 118-122; Xu, J. et al. (2010) Cell Biol Int. 34:
791-797; Fukunaga, N. et al. (2010) Cell Reprogram. 12: 369-376;
and, Metcalf D. (2003) Stem Cells 21: 5-14, each of which is herein
incorporated by reference in their entirety. An "effective amount
of LIF" comprises a concentration of LIF that allows the rat ES
cells of an in vitro culture to remain in an undifferentiated
pluripotent state. Various markers that can be used to assay for
the cells remaining in a pluripotent state are discussed elsewhere
herein.
The LIF polypeptide employed in the various methods and
compositions provided herein can be from any organism, including
from a mammal, a rodent, a human, a rat or a mouse. In one
embodiment, the LIF polypeptide is from a mouse. In still further
embodiments, the mouse LIF polypeptide comprises the amino acid
sequence set forth in SwissProt Accession number: P09056, which is
herein incorporated by reference in its entirety and is also set
forth in SEQ ID NO: 9.
In other embodiments, an active variant or fragment of the mouse
LIF polypeptide as set forth in SEQ ID NO: 9 or in SwissProt
Accession number: P09056 can be used. Such active variants and
fragments (including active variants having at least 75%, 80%, 85%
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to SEQ ID NO: 9 are discussed in further detail elsewhere
herein.
LIF polypeptide or the active variant or fragment thereof can be
provided to the in vitro culture in a variety of ways. In one
embodiment, the effective amount of the LIF polypeptide or the
active variant or fragment thereof is added to the culture media.
In other embodiments, the feeder cells have been genetically
modified to overexpress the LIF polypeptide or the active variant
or fragment thereof. Such feeder cells include feeder cells
prepared from gamma-irradiated or mitomycin-C treated DIA-M mouse
fibroblasts that express matrix-associated LIF. Method of
generating and using such genetically modified feeder cells can be
found, for example, in See, Buehr et al. (2003) Biol Reprod
68:222-229, Rathjen et al. (1990) Cell 62 1105-4115, and Buehr et
al. (2008) Cell 135:1287-1298, each of which is herein incorporated
by reference. The heterologous LIF expressed in the feeder cells
can be from the same organism as the feeder cells or from an
organism that is different from that of the feeder cell. In
addition, the heterologous LIF expressed in the feeder cells can be
from the same or from a different organism than the ES cells the
feeder layer is supporting.
In still other embodiments, the feeder cells employed in the
various methods disclosed herein are not genetically modified to
express a heterologous LIF polypeptide or an active variant or
fragment thereof. Thus, in particular embodiments, the monolayer of
mitotically inactivated mouse embryonic fibroblast employed in the
methods has not been genetically modified to express a heterologous
LIF polypeptide.
In other embodiments, the LIF polypeptide or the active variant or
fragment thereof is added to the culture media. When LIF is added
to the culture media, the LIF can be from any organism, including
from a mammal, a rodent, a human, a rat or a mouse. In one
embodiment, the LIF present in the culture media is from a mouse.
In still further embodiments, the mouse LIF polypeptide comprises
the amino acid sequence set forth in SEQ ID NO:9. In other
embodiments, an active variant or fragment of the mouse LIF
polypeptide as set forth in SEQ ID NO:9 can be used. Such active
variants and fragments (including active variants having at least
75%, 80%, 85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to SEQ ID NO: 9) are discussed in further detail
elsewhere herein.
In specific embodiments, the rat ES cells and rat ES cell lines
provided herein maintain pluripotency in vitro without requiring
paracrine LIF signaling.
In specific embodiments, LIF or an active variant or fragment
thereof is present in the culture media at any concentration that
maintains the rat ES cells. LIF polypeptide or active variant or
fragment thereof is present in the culture media at about 25 U/ml
to about 50 U/ml, at about 50 U/ml to about 100 U/ml, at about 100
U/ml to about 125 U/ml, at about 125 U/ml to about 150 U/ml, at
about 150 U/ml to about 175 U/ml, at about 175 U/ml to about 200
U/ml, at about 200 U/ml to about 225 U/ml, at about 225 U/ml to
about 250 U/ml, at about 250 U/ml to about 300 U/ml, to about 300
U/ml to about 325 U/ml, at about 325 U/ml to about 350 U/ml, at
about 350 U/ml to about 400 U/ml, at about 400 U/ml to about 425
U/ml, at about 425 U/ml to about 450 U/ml, at about 450 U/ml to
about 475 U/ml, at about 475 U/ml to about 500 U/ml, at about 75
U/ml to about 500 U/ml or greater. In other embodiments, LIF
polypeptide or active variant or fragment thereof is present in the
culture media at about 25 U/ml to about 50 U/ml, at about 25 U/ml
to about 100 U/ml, at about 75 U/ml to about 125 U/ml, at about 50
U/ml to about 150 U/ml, at about 90 U/ml to about 125 U/ml, at
about 90 U/ml to about 110 U/ml, at about 80 U/ml to about 150
U/ml, or at about 80 U/ml to about 125 U/ml. In a specific
embodiment, LIF polypeptide or active variant or fragment thereof
is present in the culture media at about 100 U/ml.
When mouse LIF is employed, the mouse LIF polypeptide or active
variant or fragment thereof is present in the culture media at any
concentration that maintains the rat ES cells. Mouse LIF
polypeptide or active variant or fragment thereof is present at
about 25 U/ml to about 50 U/ml, at about 50 U/ml to about 100 U/ml,
at about 100 U/ml to about 125 U/ml, at about 125 U/ml to about 150
U/ml, at about 150 U/ml to about 175 U/ml, at about 175 U/ml to
about 200 U/ml, at about 200 U/ml to about 225 U/ml, at about 225
U/ml to about 250 U/ml, at about 250 U/ml to about 300 U/ml, to
about 300 U/ml to about 325 U/ml, at about 325 U/ml to about 350
U/ml, at about 350 U/ml to about 400 U/ml, at about 400 U/ml to
about 425 U/ml, at about 425 U/ml to about 450 U/ml, at about 450
U/ml to about 475 U/ml, at about 475 U/ml to about 500 U/ml, at
about 75 U/ml to about 500 U/ml or greater. In other embodiments,
mouse LIF polypeptide or active variant or fragment thereof is
present at about 25 U/ml to about 50 U/ml, at about 25 U/ml to
about 100 U/ml, at about 75 U/ml to about 125 U/ml, at about 50
U/ml to about 150 U/ml, at about 90 U/ml to about 125 U/ml, at
about 90 U/ml to about 110 U/ml, at about 80 U/ml to about 150
U/ml, or at about 80 U/ml to about 125 U/ml. In a specific
embodiment, mouse LIF polypeptide or active variant or fragment
thereof is present in the culture media at about 100 U/ml.
The culture media employed maintains rat ES cells. As such, in
specific embodiments, the culture media employed in the various
method and compositions will maintain the pluripotency of all or
most of (i.e., over 50%) of the rat ES cells in a cell line for a
period of a at least 5, 10 or 15 passages. In one embodiment, the
culture media comprises one or more compounds that assist in
maintaining pluripotency. In one embodiment, the culture media
comprises a MEK pathway inhibitor and a glycogen synthase kinase-3
(GSK-3) inhibitor. The media can further comprise additional
components that aid in maintaining the ES cells, including for
example, FGF receptor inhibitors, ROCK inhibitors, and/or ALK (TGFb
receptor) inhibitors. A non-limiting example of an FGF receptor
inhibitors includes PD184352. A non-limiting example of a ROCK
inhibitor includes Y-27632, and non-limiting example of an ALK
(TGFb receptor) inhibitor includes A-83-01. In specific
embodiments, 2i media is used with 10 uM ROCKi when thawing
cryopreserved rESC or when re-plating rESC after dissociation with
trypsin.
In other embodiments, the media comprises a combination of
inhibitors consisting of a MEK pathway inhibitor and a glycogen
synthase kinase-3 (GSK-3) inhibitor.
In one non-limiting embodiment, the culture media comprises a GSK-3
inhibitor comprising CHIR99021 and/or comprises a MEK inhibitor
comprising PD0325901. In other embodiments, the media comprises a
combination of inhibitors consisting of CHIR99021 and PD0325901.
Either of these compounds can be obtained, for example, from
Stemgent. In specific embodiments, CHIR99021 is present in the
culture media at a concentration of about 0.5 .mu. to about 3
.mu.M, about 0.5 .mu. to about 3.5 .mu.M, about 0.5 .mu.M to about
4 .mu.M, about 0.5 .mu.M to about 1 .mu.M, about 1 .mu.M to about
1.5 .mu.M, about 1.5 .mu.M to about 2 .mu.M, about 2 .mu.M to about
2.5 .mu.M, about 2.5 to about 3 .mu.M, 3 .mu.M to about 3.5 .mu.M.
In further embodiments, CHIR99021 is present in the culture media
at a concentration of about 3 .mu.M. In other embodiments,
PD0325901 is present in the culture media at a concentration of
about 0.4 .mu.M to about 1 uM, about 0.4 .mu.M to about 1.5 uM,
about 0.4 .mu.M to about 2 .mu.M, about 0.4 .mu.M to about 0.8
.mu.M, 0.8 .mu.M to about 1.2 .mu.M, about 1.2 to about 1.5 .mu.M.
In further embodiments, PD0325901 is present in the culture media
at a concentration of about 1 .mu.M. In specific embodiments,
CHIR99021 is present in the culture media at a concentration of
about 3 .mu.M and PD0325901 is present at a concentration of about
1 .mu.M.
In one non-limiting embodiment, the culture media employed in the
various methods and compositions disclosed herein is a 2i media
which comprises: DMEM/F12 basal media (at a concentration of
1.times. (50%)); Neurobasal media (at a concentration of 1.times.
(50%)); Penicillin/streptomycin (at a concentration of 1%);
L-Glutamine (at a concentration of 4 mM); 2-Mercaptoethanol (at a
concentration of 0.1 mM); N2 supplement (at a concentration of
1.times.); B27 supplement (at a concentration 1.times.); LIF (at a
concentration of 100 U/ml); PD0325901 (MEK inhibitor) (at a
concentration of 1 .mu.M) and CHIR99021 (GSK inhibitor) (at a
concentration of 3 .mu.M).
Additional media that can be employed include those disclosed in Li
et al. (2008) Cell 135:1299-1310, Yamamoto et al. (2012) Transgenic
Rats 21:743-755, Ueda et al. (2008) PLoS ONE 3(6):e2800, Meek et
al. (2010) PLoS ONE 4 (12): e14225; Tong et al. (2010) Nature
467:211-213; US Patent Publication 2012/0142092, Buehr et al.
(2008) Cell 135:1287-1298, Li et al. (135) Cell 1299-1310, each of
which is herein incorporated by reference in their entirety. When
employing such media, the concentration and the source of LIF can
be modified as outlined herein. In specific embodiments, the
various culture medias are used in combination with mouse LIF or an
active variant or fragment thereof, and in even further
embodiments, the various culture medias comprise a mouse LIF or an
active variant or fragment thereof at a concentration of about 50
U/ml to about 100 U/ml, about 50 U/ml to about 150 U/ml, or about
100 U/ml.
The temperature of the cultures of rat ES cells, both for the
production of the ES cell line and for the culturing and
maintaining of the ES line it typically carried out at about
35.degree. C. to about 37.5.degree. C. In specific embodiment, the
temperature is 37.0.degree. C. The culture is typically carried out
at 7.5% CO.sub.2.
7. Sequence Identity
The methods and compositions provided herein employ a variety of
different components of the targeted genomic integration system
(i.e. nuclease agents, recognition sites, insert nucleic acids,
polynucleotides of interest, targeting vectors, selection markers
and other components). It is recognized throughout the description
that some components of the targeted genomic integration system can
have active variants and fragments. Such components include, for
example, nuclease agents (i.e. engineered nuclease agents),
nuclease agent recognition sites, polynucleotides of interest,
target sites and corresponding homology arms of the targeting
vector. Biological activity for each of these components is
described elsewhere herein.
As used herein, "sequence identity" or "identity" in the context of
two polynucleotides or polypeptide sequences makes reference to the
residues in the two sequences that are the same when aligned for
maximum correspondence over a specified comparison window. When
percentage of sequence identity is used in reference to proteins it
is recognized that residue positions which are not identical often
differ by conservative amino acid substitutions, where amino acid
residues are substituted for other amino acid residues with similar
chemical properties (e.g., charge or hydrophobicity) and therefore
do not change the functional properties of the molecule. When
sequences differ in conservative substitutions, the percent
sequence identity may be adjusted upwards to correct for the
conservative nature of the substitution. Sequences that differ by
such conservative substitutions are said to have "sequence
similarity" or "similarity". Means for making this adjustment are
well known to those of skill in the art. Typically this involves
scoring a conservative substitution as a partial rather than a full
mismatch, thereby increasing the percentage sequence identity.
Thus, for example, where an identical amino acid is given a score
of 1 and a non-conservative substitution is given a score of zero,
a conservative substitution is given a score between zero and 1.
The scoring of conservative substitutions is calculated, e.g., as
implemented in the program PC/GENE (Intelligenetics, Mountain View,
Calif.).
As used herein, "percentage of sequence identity" means the value
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using GAP Version 10
using the following parameters: % identity and % similarity for a
nucleotide sequence using GAP Weight of 50 and Length Weight of 3,
and the nwsgapdna.cmp scoring matrix; % identity and % similarity
for an amino acid sequence using GAP Weight of 8 and Length Weight
of 2, and the BLOSUM62 scoring matrix; or any equivalent program
thereof. "Equivalent program" means any sequence comparison program
that, for any two sequences in question, generates an alignment
having identical nucleotide or amino acid residue matches and an
identical percent sequence identity when compared to the
corresponding alignment generated by GAP Version 10.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein also can be used in the practice or testing of the described
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
It must be noted that as used herein and in the appended claims,
the singular forms "a", "and", and "the" include plural references
unless the context clearly dictates otherwise. All technical and
scientific terms used herein have the same meaning.
The publications discussed herein are provided solely for their
disclosure prior to the filing date of the present application.
Nothing herein is to be construed as an admission that the
described invention is not entitled to antedate such publication by
virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates, which
may need to be independently confirmed.
The described invention may be embodied in other specific forms
without departing from the spirit or essential attributes thereof
and, accordingly, reference should be made to the appended claims,
rather than to the foregoing specification, as indicating the scope
of the invention.
Non-limiting Embodiments Include:
1. A method for targeted modification of a genomic locus of
interest in a pluripotent rat cell, comprising (a) introducing into
the pluripotent rat cell a large targeting vector (LTVEC)
comprising an insert nucleic acid flanked with a 5' rat homology
arm and a 3' rat homology arm, wherein the sum total of the 5' and
the 3' homology arms is at least 10 kb but less than 150 kb; and
(b) identifying a genetically modified pluripotent rat cell
comprising the targeted genetic modification at the genomic locus
of interest, wherein the targeted genetic modification is capable
of being transmitted through the germline.
2. The method of embodiment 1, wherein the targeted genetic
modification is biallelic.
3. The method of embodiment 1 or 2, wherein the pluripotent rat
cell is a rat embryonic stem (ES) cell.
4. The method of embodiment 1, 2 or 3, wherein the pluripotent rat
cell is derived from a DA strain or an ACI strain.
5. The method of any one of embodiments 1-4, wherein the
pluripotent rat cell is characterized by expression of at least one
pluripotency marker comprising Dnmt3L, Eras, Err-beta, Fbxo15,
Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15,
Sox2, Utf1, or a combination thereof.
6. The method of any one of embodiments 1-4 wherein the pluripotent
rat cell is characterized by one of more of the following
characteristics:
(a) lack of expression of one or more pluripotency markers
comprising c-Myc, Ecat1, and/or Rexo1; (b) lack of expression of
mesodermal markers comprising Brachyury and/or Bmpr2; (c) lack of
expression of one or more endodermal markers comprising Gata6,
Sox17 and/or Sox7; or (d) lack of expression of one or more neural
markers comprising Nestin and/or Pax6.
7. The method of any one of embodiments 1-6, wherein the sum total
of the 5' and the 3' homology arms of the LTVEC is from about 10 kb
to about 30 kb, from about 20 kb to about 40 kb, from about 40 kb
to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb
to about 100 kb, from about 100 kb to about 120 kb, or from about
120 kb to 150 kb.
8. The method of any one of embodiments 1-6, wherein the sum total
of the 5' and the 3' homology arms of the LTVEC is from about 16 Kb
to about 150 Kb.
9. The method of any one of embodiments 1-8, wherein the targeted
genetic modification comprises: (a) a replacement of an endogenous
rat nucleic acid sequence with a homologous or an orthologous
nucleic acid sequence; (b) a deletion of an endogenous rat nucleic
acid sequence; (c) a deletion of an endogenous rat nucleic acid
sequence, wherein the deletion ranges from about 5 kb to about 10
kb, from about 10 kb to about 20 kb, from about 20 kb to about 40
kb, from about 40 kb to about 60 kb, from about 60 kb to about 80
kb, from about 80 kb to about 100 kb, from about 100 kb to about
150 kb, or from about 150 kb to about 200 kb, from about 200 kb to
about 300 kb, from about 300 kb to about 400 kb, from about 400 kb
to about 500 kb, from about 500 kb to about 1 Mb, from about 1 Mb
to about 1.5 Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb
to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb; (d) an
exogenous nucleic acid sequence ranging from about 5 kb to about 10
kb, from about 10 kb to about 20 kb, from about 20 kb to about 40
kb, from about 40 kb to about 60 kb, from about 60 kb to about 80
kb, from about 80 kb to about 100 kb, from about 100 kb to about
150 kb, from about 150 kb to about 200 kb, from about 200 kb to
about 250 kb, from about 250 kb to about 300 kb, from about 300 kb
to about 350 kb, or from about 350 kb to about 400 kb; (e) an
exogenous nucleic acid sequence comprising a homologous or an
orthologous nucleic acid sequence; (f) a chimeric nucleic acid
sequence comprising a human and a rat nucleic acid sequence; (g) a
conditional allele flanked with site-specific recombinase target
sequences; or, (h) a reporter gene operably linked to a promoter
active in a rat cell.
10. The method of any one of embodiments 1-9, wherein the genomic
locus of interest comprises (i) a first nucleic acid sequence that
is complementary to the 5' rat homology arm; and (ii) a second
nucleic acid sequence that is complementary to the 3' rat homology
arm.
11. The method of embodiment 10, wherein the first and the second
nucleic acid sequence is separated by at least 5 kb but less than 3
Mb.
12. The method of embodiment 10, wherein the first and the second
nucleic acid sequence is separated by at least 5 kb but less than
10 kb, at least 10 kb but less than 20 kb, at least 20 kb but less
than 40 kb, at least 40 kb but less than 60 kb, at least 60 kb but
less than 80 kb, at least about 80 kb but less than 100 kb, at
least 100 kb but less than 150 kb, or at least 150 kb but less than
200 kb, at least about 200 kb but less than about 300 kb, at least
about 300 kb but less than about 400 kb, at least about 400 kb but
less than about 500 kb, at least about 500 kb but less than about 1
Mb, at least about 1 Mb but less than about 1.5 Mb, at least about
1.5 Mb but less than about 2 Mb, at least about 2 Mb but less than
about 2.5 Mb, or at least about 2.5 Mb but less than about 3
Mb.
13. The method of any one of embodiment 1-12, wherein introducing
step (a) further comprises introducing a second nucleic acid
encoding a nuclease agent that promotes a homologous recombination
between the targeting construct and the genomic locus of interest
in the pluripotent rat cell.
14. The method of embodiment 13, wherein the nuclease agent
comprises (a) a chimeric protein comprising a zinc finger-based DNA
binding domain fused to a FokI endonuclease; or, (b) a chimeric
protein comprising a Transcription Activator-Like Effector Nuclease
(TALEN) fused to a FokI endonuclease.
15. The method of any one of embodiments 1-12, wherein introducing
step (a) further comprises introducing into the pluripotent rat
cell: (i) a first expression construct comprising a first promoter
operably linked to a first nucleic acid sequence encoding a
Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR)-associated (Cas) protein, (ii) a second expression
construct comprising a second promoter operably linked to a genomic
target sequence linked to a guide RNA (gRNA), wherein the genomic
target sequence is immediately flanked on the 3' end by a
Protospacer Adjacent Motif (PAM) sequence.
16. The method of embodiment 15, wherein the genomic locus of
interest comprises the nucleotide sequence of SEQ ID NO: 1.
17. The method of embodiment 15 or 16, wherein the gRNA comprises a
third nucleic acid sequence encoding a Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR) RNA (crRNA) and a
trans-activating CRISPR RNA (tracrRNA).
18. The method of embodiment 15, 16 or 17, wherein the Cas protein
is Cas9.
19. The method of embodiment 15, 16, 17, or 18, wherein the gRNA
comprises: (a) the chimeric RNA of the nucleic acid sequence of SEQ
ID NO: 2; or, (b) the chimeric RNA of the nucleic acid sequence of
SEQ ID NO: 3.
20. The method of embodiment 17, wherein the crRNA comprises SEQ ID
NO: 4; SEQ ID NO: 5; or SEQ ID NO: 6.
21. The method of embodiment 17, wherein the tracrRNA comprises SEQ
ID NO: 7 or SEQ ID NO: 8.
22. A modified rat genomic locus comprising: (i) an insertion of a
homologous or orthologous human nucleic acid sequence; (ii) a
replacement of an endogenous rat nucleic acid sequence with the
homologous or orthologous human nucleic acid sequence; or (iii) a
combination thereof, wherein the modified rat genomic locus is
capable of being transmitted through the germline.
23. The modified rat genomic locus of embodiment 22, wherein the
size of the insertion or replacement is from about 5 kb to about
400 kb.
24. The rat genomic locus of embodiment 22, wherein the size of the
insertion or replacement is from about 5 kb to about 10 kb, from
about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from
about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from
about 80 kb to about 100 kb, from about 100 kb to about 150 kb,
from about 150 kb to about 200 kb, from about 200 kb to about 250
kb, from about 250 kb to about 300 kb, from about 300 kb to about
350 kb, or from about 350 kb to about 400 kb.
25. A method for making a humanized rat, comprising: (a) targeting
a genomic locus of interest in a pluripotent rat cell with a
targeting construct comprising a human nucleic acid to form a
genetically modified pluripotent rat cell; (b) introducing the
genetically modified pluripotent rat cell into a host rat embryo;
and (c) gestating the host rat embryo in a surrogate mother;
wherein the surrogate mother produces rat progeny comprising a
modified genomic locus that comprises: (i) an insertion of a human
nucleic acid sequence; (ii) a replacement of the rat nucleic acid
sequence at the genomic locus of interest with a homologous or
orthologous human nucleic acid sequence; (iii) a chimeric nucleic
acid sequence comprising a human and a rat nucleic acid sequence;
or (iv) a combination thereof, wherein the modified genomic locus
is capable of being transmitted through the germline.
26. The method of embodiment 25, wherein the targeting construct is
a large targeting vector (LTVEC), and the sum total of the 5' and
the 3' homology arms of the LTVEC is at least 10 kb but less than
150 kb.
27. The method of embodiment 26, wherein the sum total of the 5'
and the 3' homology arms of the targeting construct is from about
10 kb to about 30 kb, from about 20 kb to 40 kb, from about 40 kb
to about 60 kb, from about 60 kb to about 80 kb, or from about 80
kb to about 100 kb, from about 100 kb to about 120 kb, or from
about 120 kb to 150 kb.
28. The method of embodiment 25, 26 or 27, wherein the human
nucleic acid sequence is at least 5 kb but less than 400 kb.
29. The method of embodiment 25, 26, or 27, wherein the human
nucleic acid sequence is at least 5 kb but less than 10 kb, at
least 10 kb but less than 20 kb, at least 20 kb but less than 40
kb, at least 40 kb but less than 60 kb, at least 60 kb but less
than 80 kb, at least about 80 kb but less than 100 kb, at least 100
kb but less than 150 kb, at least 150 kb but less than 200 kb, at
least 200 kb but less than 250 kb, at least 250 kb but less than
300 kb, at least 300 kb but less than 350 kb, or at least 350 kb
but less than 400 kb.
30. The method of any one of embodiments 25-29, wherein the
pluripotent rat cell is a rat embryonic stem (ES) cell.
31. The method of any one of embodiments 25-30, wherein the
pluripotent rat cell is derived from a DA strain or an ACI
strain.
32. The method of any one of embodiments 25-31, wherein the
pluripotent rat cell is characterized by expression of at least one
pluripotency marker comprising Dnmt3L, Eras, Err-beta, Fbxo15,
Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15,
Sox2, Utf1, or a combination thereof.
33. The method of any one of embodiment 25-31, wherein the
pluripotent rat cell is characterized by one or more of the
following features: (a) lack of expression of one or more
pluripotency markers comprising c-Myc, Ecat1, and/or Rexo1; (b)
lack of expression of one or more mesodermal markers comprising
Brachyury and/or Bmpr2; (c) lack of expression of one or more
endodermal markers comprising Gata6, Sox17, and/or Sox7; or (d)
lack of expression of one or more neural markers comprising Nestin
and/or Pax6.
34. A modified rat comprising a humanized genomic locus, wherein
the humanized genomic locus comprises: (i) an insertion of a
homologous or orthologous human nucleic acid sequence; (ii) a
replacement of a rat nucleic acid sequence at an endogenous genomic
locus with a homologous or orthologous human nucleic acid sequence;
(iii) a chimeric nucleic acid sequence comprising a human and a rat
nucleic acid sequence or, (iv) a combination thereof, wherein the
humanized genomic locus is capable of being transmitted through the
germline.
35. A rat or rat cell comprising a targeted genetic modification in
its genomic locus, wherein the genomic locus is an Interleukin-2
receptor gamma locus, an ApoE locus, a Rag1 locus, a Rag2 locus, or
a Rag2/Rag1 locus, wherein the targeted genetic modification
comprises: (a) a deletion of an endogenous rat nucleic acid
sequence at the genomic locus; (b) an insertion of a homologous
nucleic acid, an orthologous nucleic acid, or a chimeric nucleic
acid comprising a human and a rat nucleic acid sequence, or (c) a
combination thereof, wherein the targeted genetic modification is
transmissible through the germline of the rat or a rat propagated
from the rat cell.
36. The rat or rat cell of embodiment 35, wherein (a) the deletion
of the endogenous rat nucleic acid at the genomic locus is at least
about 10 kb; or, (b) the deletion of the endogenous rat nucleic
acid at the genomic locus is from about 5 kb to about 10 kb, from
about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from
about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from
about 80 kb to about 100 kb, from about 100 kb to about 150 kb, or
from about 150 kb to about 200 kb, from about 200 kb to about 300
kb, from about 300 kb to about 400 kb, from about 400 kb to about
500 kb, from about 500 kb to about 1 Mb, from about 1 Mb to about
1.5 Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb to about
2.5 Mb, or from about 2.5 Mb to about 3 Mb; (c) the insertion of
the exogenous nucleic acid sequence at the genomic locus is at
least about 5 kb; or, (d) the insertion of the exogenous nucleic
acid sequence at the genomic locus is from about 5 kb to about 10
kb, from about 10 kb to about 20 kb, from about 20 kb to about 40
kb, from about 40 kb to about 60 kb, from about 60 kb to about 80
kb, from about 80 kb to about 100 kb, from about 100 kb to about
150 kb, from about 150 kb to about 200 kb, from about 200 kb to
about 250 kb, from about 250 kb to about 300 kb, from about 300 kb
to about 350 kb, or from about 350 kb to about 400 kb.
37. The rat or rat cell of embodiment 35 or 36, wherein (a) the
targeted genetic modification at the Interleukin-2 receptor gamma
locus results in a decrease in or absence of Interleukin-2 receptor
gamma protein activity; (b) the targeted genetic modification at
the ApoE locus results in a decrease in or absence of ApoE protein
activity; (c) the targeted genetic modification at the Rag1 locus
results in a decrease in or absence of Rag1 protein activity; (d)
the targeted genetic modification at the Rag2 locus results in a
decrease in or absence of Rag2 protein activity; or, (e) the
targeted genetic modification at the Rag2/Rag1 locus results in a
decrease in or absence of Rag2 protein activity and Rag1
activity.
38. The rat or rat cell of embodiment 35, 36, or 37, wherein the
targeted genetic modification of the Interleukin-2 receptor gamma
locus comprises: (a) a deletion of the entire rat Interleukin-2
receptor gamma coding region or a portion thereof; (b) a
replacement of the entire rat Interleukin-2 receptor gamma coding
region or a portion thereof with a human Interleukin-2 receptor
gamma coding region or a portion thereof; (c) a replacement of an
ecto-domain of the rat Interleukin-2 receptor gamma coding region
with the ecto-domain of a human Interleukin-2 receptor gamma; or,
(d) at least a 3 kb deletion of the Interleukin-2 receptor gamma
locus.
39. The rat or rat cell of any one of embodiments 35-37, wherein
the targeted genetic modification of the ApoE locus comprises: (a)
a deletion of the entire ApoE coding region or a portion thereof;
or, (b) at least a 1.8 kb deletion of the ApoE locus comprising the
ApoE coding region.
40. The rat or rat cell of any one of embodiments 35-37, wherein
the targeted genetic modification of the Rag2 locus comprises: (a)
a deletion of the entire Rag2 coding region or a portion thereof;
(b) at least a 5.7 kb deletion of the Rag2 locus comprising the
Rag2 coding region.
41. The rat or rat cell of any one of embodiments 35-37, wherein
the targeted genetic modification of the Rag2/Rag1 locus comprises:
(a) a deletion of the entire Rag2 coding region or a portion
thereof and a deletion of the entire Rag1 coding region or portion
thereof; or, (b) a deletion of at least 16 kb of the Rag2/Rag1
locus comprising the Rag2 coding region.
42. The rat or rat cell of any one of embodiment 35-41, wherein the
targeted genetic modification comprises an insertion of an
expression cassette comprising a selective marker at the
Interleukin-2 receptor gamma locus, the ApoE locus, the Rag1 locus,
the Rag2 locus, or the Rag2/Rag1 locus.
43. The rat or rat cell of any one of embodiments 42, wherein the
expression cassette comprises a lacZ gene operably linked to the
endogenous promoter at the genomic locus and a human ubiquitin
promoter operably linked to a selective marker.
44. The rat or rat cell of any one of embodiments 35-43, wherein
the targeted genetic modification in the Interleukin-2 receptor
gamma locus, the ApoE locus, the Rag1 locus, the Rag2 locus or the
Rag2/Rag1 locus comprises the insertion of a self-deleting
selection cassette.
45. The rat or rat cell of embodiment 44, wherein the self-deleting
selection cassette comprises a selective marker gene operably
linked to a promoter active in the rat cell and a recombinase gene
operably linked to a male germ cell-specific promoter, wherein the
self-deleting cassette is flanked by recombination recognition
sites recognized by the recombinase.
46. The rat or rat cell of embodiment 45, wherein (a) the male germ
cell-specific promoter is a Protamine-1 promoter; or, (b) the
recombinase gene encodes Cre, and the recombination recognition
sites are loxP sites.
47. The rat or rat cell of any one of embodiments 35-46, wherein
the insertion of the exogenous nucleic acid sequence at the genomic
locus comprises a reporter nucleic acid operably linked to an
endogenous Interleukin-2 receptor gamma promoter, an endogenous
ApoE promoter, an endogenous Rag1 promoter, or an endogenous Rag2
promoter.
48. The rat or rat cell of embodiment 47, wherein the reporter
nucleic acid encodes a reporter comprising .beta.-galactosidase,
mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO,
mCitrine, Venus, YPet, enhanced yellow fluorescent protein (EYFP),
Emerald, enhanced green fluorescent protein (EGFP), CyPet, cyan
fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase,
alkaline phosphatase, or a combination thereof.
49. The rat cell of any one of embodiments 35-48, wherein the rat
cell is a pluripotent rat cell or a rat embryonic stem (ES)
cell.
50. The rat cell of embodiment 49, wherein the pluripotent rat cell
or the rat embryonic stem (ES) cell (a) is derived from a DA strain
or an ACI strain; (b) is characterized by expression of at least
one pluripotency marker comprising Dnmt3L, Eras, Err-beta, Fbxo15,
Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15,
Sox2, Utf1, or a combination thereof; or (c) is characterized by
one or more of the following characteristics: (i) lack of
expression of one or more pluripotency markers comprising c-Myc,
Ecat1, and/or Rexo1; (ii) lack of expression of mesodermal markers
comprising Brachyury and/or Bmpr2; (iii) lack of expression of one
or more endodermal markers comprising Gata6, Sox17 and/or Sox7; or
(iv) lack of expression of one or more neural markers comprising
Nestin and/or Pax6.
51. A method for modifying a target genomic locus in an
Interleukin-2 receptor gamma locus, an ApoE locus, a Rag1 locus, a
Rag2 locus or a Rag2/Rag1 locus in a pluripotent rat cell, the
method comprising: (a) introducing into the pluripotent rat cell a
targeting vector comprising an insert nucleic acid flanked with 5'
and 3' rat homology arms homologous to the target genomic locus,
(b) identifying a genetically modified pluripotent rat cell
comprising a targeted genetic modification at the target genomic
locus, wherein the targeted genetic modification is capable of
being transmitted through the germline of a rat propagated from the
pluripotent rat cell.
52. The method of embodiment 51, wherein the targeting vector is a
large targeting vector (LTVEC) wherein the sum total of the 5' and
the 3' rat homology arms is at least about 10 kb but less than
about 150 kb.
53. The method of embodiment 51 or 52, wherein introducing the
targeting vector into the pluripotent rat cell leads to: (i) a
deletion of an endogenous rat nucleic acid sequence at the target
genomic locus; (ii) an insertion of an exogenous nucleic acid
sequence at the target genomic locus; or (iii) a combination
thereof.
54. The method of embodiment 53, wherein (a) the deletion of the
endogenous rat nucleic acid at the genomic locus is at least about
10 kb; or, (b) the deletion of the endogenous rat nucleic acid at
the genomic locus is from about 5 kb to about 10 kb, from about 10
kb to about 20 kb, from about 20 kb to about 40 kb, from about 40
kb to about 60 kb, from about 60 kb to about 80 kb, from about 80
kb to about 100 kb, from about 100 kb to about 150 kb, or from
about 150 kb to about 200 kb, from about 200 kb to about 300 kb,
from about 300 kb to about 400 kb, from about 400 kb to about 500
kb, from about 500 kb to about 1 Mb, from about 1 Mb to about 1.5
Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb to about 2.5
Mb, or from about 2.5 Mb to about 3 Mb; (c) the insertion of the
exogenous nucleic acid sequence at the genomic locus is at least
about 5 kb; or. (d) the insertion of the exogenous nucleic acid
sequence at the genomic locus is from about 5 kb to about 10 kb,
from about 10 kb to about 20 kb, from about 20 kb to about 40 kb,
from about 40 kb to about 60 kb, from about 60 kb to about 80 kb,
from about 80 kb to about 100 kb, from about 100 kb to about 150
kb, from about 150 kb to about 200 kb, from about 200 kb to about
250 kb, from about 250 kb to about 300 kb, from about 300 kb to
about 350 kb, or from about 350 kb to about 400 kb.
55. The method of any one of embodiment 51-54, wherein (a) the
targeted genetic modification at the Interleukin-2 receptor gamma
locus results in a decrease in or absence of Interleukin-2 receptor
gamma protein activity; (b) the targeted genetic modification at
the ApoE locus results in a decrease in or absence of ApoE protein
activity; (c) the targeted genetic modification at the Rag1 locus
results in a decrease in or absence of Rag1 protein activity; (d)
the targeted genetic modification at the Rag2 locus results in a
decrease in or absence of Rag2 protein activity; or, (e) the
targeted genetic modification at the Rag2/Rag1 locus results in a
decrease in or absence of Rag2 protein activity and i Rag1 protein
activity.
56. The method of any one of embodiment 51-54, wherein the targeted
genetic modification of the Interleukin-2 receptor gamma locus
comprises (a) a deletion of the entire rat Interleukin-2 receptor
gamma coding region or a portion thereof; (b) a replacement of the
entire rat Interleukin-2 receptor gamma coding region or a portion
thereof with a human Interleukin-2 receptor gamma coding region or
a portion thereof; (c) a replacement of an ecto-domain of the rat
Interleukin-2 receptor gamma coding region with the ecto-domain of
a human Interleukin-2 receptor gamma; or, (d) at least a 3 kb
deletion of the Interleukin-2 receptor gamma locus comprising the
Interleukin-2 receptor gamma coding region.
57. The method of any one of embodiment 51-55, wherein the targeted
genetic modification of the ApoE locus comprises: (a) a deletion of
the entire ApoE coding region or a portion thereof; or, (b) at
least a 1.8 kb deletion of the ApoE locus comprising the ApoE
coding region.
58. The method of any one of embodiment 51-55, wherein the targeted
genetic modification of the Rag2 locus comprises: (a) a deletion of
the entire Rag2 coding region or a portion thereof; or, (b) at
least a 5.7 kb deletion of the Rag2 locus comprising the Rag2
coding region.
59. The method of any one of embodiment 51-55, wherein the targeted
genetic modification of the Rag1/Rag2 locus comprises: (a) a
deletion of the entire Rag2 coding region or a portion thereof and
a deletion of the entire Rag1 coding region or portion thereof; or,
(b) a deletion of at least 16 kb of the Rag2/Rag1 locus comprising
the Rag2 and Rag1 coding regions.
60. The method of any one of embodiment 51-59, wherein the insert
nucleic acid comprises an expression cassette comprising a
polynucleotide encoding a selective marker.
61. The method embodiment 60, wherein the expression cassette
comprises a lacZ gene operably linked to an endogenous promoter at
the genomic locus and a human ubiquitin promoter operably linked to
a selective marker gene.
62. The method of any one of embodiments 51-60, wherein the insert
nucleic acid comprises a self-deleting selection cassette.
63. The method of embodiment 62, wherein the self-deleting
selection cassette comprises a selective marker operably linked to
a promoter active in the rat pluripotent cell and a polynucleotide
encoding a recombinase operably linked to a male germ cell-specific
promoter, wherein the self-deleting cassette is flanked by
recombination recognition sites recognized by the recombinase.
64. The method of embodiment 63, wherein (a) the male germ
cell-specific promoter is a Protamine-1 promoter; or, (b) the
recombinase gene encodes Cre and the recombination recognition
sites are loxP sites.
65. The method of embodiment 53, wherein the insertion of the
exogenous nucleic acid sequence at the genomic locus comprises a
reporter nucleic acid sequence operably linked to an endogenous
Interleukin-2 receptor gamma promoter, an endogenous ApoE promoter,
an endogenous Rag1 promoter, or an endogenous Rag2 promoter.
66. The method of embodiment 65, wherein the reporter nucleic acid
sequence encodes a reporter comprising .beta.-galactosidase, mPlum,
mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO,
mCitrine, Venus, YPet, enhanced yellow fluorescent protein (EYFP),
Emerald, enhanced green fluorescent protein (EGFP), CyPet, cyan
fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase,
alkaline phosphatase, or a combination thereof.
67. The method of any one of embodiment 51-66, wherein the
pluripotent rat cell is a rat embryonic stem (ES) cell.
68. The method of any one of embodiment 51-67, wherein the
pluripotent rat cell (a) is derived from a DA strain or an ACI
strain; or, (b) is characterized by expression of a pluripotency
marker comprising Oct-4, Sox-2, alkaline phosphatase, or a
combination thereof; or, (c) is characterized by one or more of the
following characteristics: (i) lack of expression of one or more
pluripotency markers comprising c-Myc, Ecat1, and/or Rexo1; (ii)
lack of expression of mesodermal markers comprising Brachyury
and/or Bmpr2; (iii) lack of expression of one or more endodermal
markers comprising Gata6, Sox17 and/or Sox7; or (iv) lack of
expression of one or more neural markers comprising Nestin and/or
Pax6.
69. The method of any one of embodiment 51-68, further comprising
identifying the targeted genetic modification at the target genomic
locus, wherein the identification step employs a quantitative assay
for assessing a modification of allele (MOA) at the target genomic
locus.
70. The method of any one of embodiment 51-69, wherein introducing
step (a) further comprises introducing a second nucleic acid
encoding a nuclease agent that promotes a homologous recombination
between the targeting vector and the target genomic locus in the
pluripotent rat cell.
71. The method of embodiment 70, wherein the nuclease agent
comprises a chimeric protein comprising a zinc finger-based DNA
binding domain fused to a FokI endonuclease.
72. The method of embodiment 71, wherein the method results in
bi-allelic modification of the target genomic locus.
73. The method of any one of embodiment 51-70, wherein introducing
step (a) further comprises introducing into the pluripotent rat
cell: (i) a first expression construct comprising a first promoter
operably linked to a first nucleic acid sequence encoding a
Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR)-associated (Cas) protein, (ii) a second expression
construct comprising a second promoter operably linked to a genomic
target sequence linked to a guide RNA (gRNA), wherein the genomic
target sequence is immediately flanked on the 3' end by a
Protospacer Adjacent Motif (PAM) sequence.
74. The method of embodiment 73, wherein the genomic locus of
interest comprises the nucleotide sequence of SEQ ID NO: 1.
75. The method of embodiment 73 or 74, wherein the gRNA comprises a
third nucleic acid sequence encoding a Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR) RNA (crRNA) and a
trans-activating CRISPR RNA (tracrRNA).
76. The method of embodiment 73, wherein the Cas protein is
Cas9.
77. The method of embodiment 73, 74, or 75, wherein the gRNA
comprises: (a) the chimeric RNA of the nucleic acid sequence of SEQ
ID NO: 2; or, (b) the chimeric RNA of the nucleic acid sequence of
SEQ ID NO: 3.
78. The method of embodiment 75, wherein the crRNA comprises SEQ ID
NO: 4; SEQ ID NO: 5; or SEQ ID NO: 6.
79. The method of embodiment 75, wherein the tracrRNA comprises SEQ
ID NO: 7 or SEQ ID NO: 8.
EXAMPLES
The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Example 1
Rat ES Cell Derivation and Characterization
1.1. Rat ES Cell Characterization
As shown in FIG. 1, rat ESCs grow as compact spherical colonies,
which routinely detach and float in the dish (close-up, FIG. 7).
Rat ESCs express pluripotency markers including Oct-4 (FIG. 2A) and
Sox2 (FIG. 2B), and express high levels of alkaline phosphatase
(FIG. 3, left panel). Karyotype for line DA.2B is 42X,Y (FIG. 3,
right panel). Rat ESCs often become tetraploid; thus, lines were
pre-screened by counting metaphase chromosome spreads; lines with
mostly normal counts were then formally karyotyped.
ACI blastocysts were collected from super-ovulated females obtained
commercially. DA blastocysts were cultured from frozen 8-cell
embryos obtained commercially. Zona pellucidae were removed with
Acid Tyrodes; and blastocysts were plated onto mitotically
inactivated MEFs. Outgrowths were picked and expanded using
standard methods. All blastocysts were plated, cultured and
expanded using 2i media (Li et al. (2008) Germline competent
embryonic stem cells derived from rat blastocysts, Cell
135:1299-1310; incorporated herein by reference in its
entirety).
TABLE-US-00001 TABLE 1 Rat ES Cell Derivation ACI DA Embryo source
Blastocysts Frozen 8-cell embryos (Superovulation) cultured to
blastocyst Blastocysts plated: 107 22 Outgrowths: 32 (30% of
blasts) 10 (45% of blasts) Lines: 16 (50% of outgrowths) 9 (90% of
outgrowths) Karyotyped: 3; all 42X, Y 6: 3 42X, X 3 42X, Y GLT
validated: 1 (ACI.G1) 1 42X, X (DA.2C) 1 42X, Y (DA.2B)
1.2.: Rat Production
Chimeric rats were produced by blastocyst injection and
transmission of the rat ESC genome. Chimeras produced by blastocyst
microinjection using parental ACI.G1 rat ESCs are shown in FIG. 8.
F1 agouti pups with albino littermates, sired by the ACI/SD chimera
labeled with an asterisk (*) in FIG. 8 are shown in FIG. 9.
Germline Transmission of Parental Rat ESC.
Three euploid rat ESC lines were evaluated for pluripotency by
microinjection into albino SD blastocysts. Chimeras were identified
by agouti coat color, which indicates rat ESC contribution. For
each line, a majority of chimeras transmitted the rESC genome to F1
offspring (Table 2).
TABLE-US-00002 TABLE 2 Germline Transmission of Parental rESC
Germline Total pups rESC- GLT Chimeras trans- from GLT derived
efficiency Line bred mitters chimeras pups (%) ACI.G1 5 3 (60%) 103
11 11 DA.2B 5 4 (80%) 129 11 9 DA.2C (XX) 3 2 (66%) 45 7 16
1.3.: Derivation of Rat Embryonic Stem Cells. Superovulation
Protocol, Rats Day 0: injected with pregnant mare serum: IP, 20 U
(0.4 ml). Day 1: no action Day 2: (46 hr. later): injected with
hCG, IP, 50 U (1 ml). set up single female matings. Day 3: checked
plugs. Females were plugged. This is day 0.5. Day 6 (e3.5):
Euthanized females and flushed embryos. ES Cell Derivation Protocol
(Superovulation) Day 0: 1) Euthanized female rat with CO.sub.2. 2)
Swabbed ventral abdomen with 70% ethanol; using scissors, opened
the ventral body wall to expose the viscera. 3) Dissected out the
oviducts and uterine horns and placed them into a tissue culture
dish containing warm N2B27 media. Washed out as much blood as
possible and transferred to a new dish with N2B27. 4) Using a 1 ml
syringe and a blunt 27 g needle, flushed media through the uterine
horns and oviducts to eject blastocysts into the media. 5)
Collected the blastocysts with a mouth pipet and transfer to embryo
culture dish containing KSOM+2i (1.mu.MPD0325901, 3 .mu.M
CHIR99021). KSOM is a culture medium produced by Millipore. Catalog
number is MR-106-D. 6) Cultured overnight at 37.degree.; 7.5%
CO.sub.2. ES Cell Derivation Protocol (Frozen Embryos) Day 0: 1)
Thawed frozen 8-cell embryos (commercially obtained) into M2
medium. Cultured 10 minutes at room temperature. 2) Transferred to
KSOM+2i and culture overnight. ES Cell Derivation Protocol (Same
for Both) Day 1: 1) Transferred cavitated embryos to 2i medium
& culture overnight. 2) Continued culturing un-cavitated
embryos in KSOM+2i Day 2: 1) Transferred all remaining embryos to
2i medium (whether or not they've cavitated). 2) Cultured
overnight; continued culturing earlier embryos in 2i medium. Day 3:
1) Transferred embryos for 30-60 seconds with Acid Tyrodes to
remove the zona pellucida. 2) Washed embryos 3.times. in 2i medium
to remove Acid Tyrodes. 3) Deposited each embryo into a separate
well of a 96-well feeder plate (the well contains a monolayer of
mitotically inactivated mouse embryonic fibroblasts (MEFs). 4)
Cultured overnight in 2i medium. Day 4-5: 1) Monitored plated
embryos for the presence of an outgrowth (an amorphous
undifferentiated mass of cells). Outgrowths are ready for transfer
when they are approximately twice the size of the plated embryo. 2)
Each day: remove spent media with a mircropipet and replace with
fresh 2i media. 3) Transferred outgrowths to new feeder wells: a.
Removed spent media and gently wash well with PBS. b. Removed PBS
and add 30 .mu.l 0.05% trypsin; incubate for 10 minutes. c. Stopped
trypsin reaction by adding 30 .mu.l 2i+10% FBS. d. Gently
dissociated the cells with a micropipettor and transferred entire
contents of the well to a new well in a 24-well feeder plate. This
was Passage 1 (P1). e. Cultured overnight in 2i medium. Day 5-8:
(timing depends on how fast each line expands) 1) Changed media
each day (2i media) and monitored for the presence of colonies with
an ESC morphology. 2) When colonies appear, continued culturing
until colonies expand to .about.50% confluency. 3) Trypsinized and
passaged colonies as before; plated on feeders, 1 well per line, in
a 6-well dish. This was Passage 2 (P2). Ongoing: 1) Continued
feeding and monitoring each line until approximately 50% confluent.
2) Trypsinized cells as usual. 3) stopped trypsin with 2i+10% FBS;
pelleted the cells by centrifugation (5', 1200 rpm in
Beckman-Coulter tabletop centrifuge). 4) Aspirated the supernatant
and gently resuspend the cells in 400 .mu.l Freezing Medium (70%
2i, 20% FBS, 10% DMSO). 5) Distributed the cells into 2 vials and
freeze at -80.degree.. This was Passage 3 (P3). 6) For long-term
storage, transferred the vials to liquid N2 storage. The 2i media
was prepared as follows in Table 3.
TABLE-US-00003 Reagent Vendor Concentration DMEM/F12 basal media
Invitrogen/Life 1x Technologies Neurobasal media Invitrogen/Life 1x
Technologies Penicillin/streptomycin Invitrogen/Life 1%
Technologies L-Glutamine Invitrogen/Life 4 mM Technologies
2-Mercaptoethanol Invitrogen/Life 0.1 mM Technologies N2 supplement
Invitrogen/Life 1x Technologies B27 supplement Invitrogen/Life 1x
Technologies LIF Millipore 100 U/ml PD0325901 (MEK Stemgent 1 uM
inhibitor). CHIR99021 (GSK Stemgent 3 uM inhibitor).
Materials: Pregnant Mare's Serum Gonadotropin (PMSG) Human
Pregnancy Urine Chorionic Gonadotropin (HCG) Female Rats (5-12
weeks old) Male rats (12 wks. to 8 mos. old), one per cage
Syringes/needles Animal room with lights on 6:00-18:00 Procedure:
Day 1: 8:00-10:00 AM Inject females with 20 IU PMSG (0.4 ml), IP
Discard unused PMSG. Day 3: 8:00-10:00 AM (48 hours after PMSG
injection) Inject females with 50 IU HCG (1 ml), IP Place one
female per male in mating cage. Discard unused HCG. Day 4:
8:00-10:00 AM (24 hrs. after HCG injection) Check females for
plugs. Hormone Suppliers
PMSG: Sigma #G-4877 (1000 IU). Resuspend in PBS to a final [ ] of
50 IU/ml. Store at -20.degree. in 1 ml aliquots.
HCG: Sigma #CG-5 (5000 IU). Resuspend in PBS to a final [ ] of 50
IU/ml. Store at -20.degree. in 1 ml aliquots.
1.4.: Karyotyping of Rat Embryonic Stem Cell Lines
The rat ES cell lines generated herein were karyotyped, and the
results are summarized in Tables 4-7.
TABLE-US-00004 TABLE 4 ACI.G1 Karyotyping Results Number of cells
Number of cells karyotyped 7 Number of cells analyzed 20 Number of
42, XY cells 18 Number of abnormal cells 2 40, XY, -5, -9 1 41, XY,
-14 1 42, XY 18 Other notes: Two analyzed cells were missing
different autosomes, which may be a sporadic occurrence due to
technical artifact. 90% of analyzed cells had a normal male 42, XY
karyotype. FIG. 4 provides a photograph showing the analysis of the
chromosome number of the ACI.G1 rat ES cell line.
TABLE-US-00005 TABLE 5 DA.2B Karyotyping Results Number of cells
Number of cells karyotyped 6 Number of cells analyzed 20 Number of
42, XY cells 20 Number of abnormal cells 0 42, XY 20 Other notes:
All analyzed cells had a normal diploid 42, XY karyotype. FIG. 5
provides a photograph showing the analysis of the chromosome number
of the DA.2B rat ES cell line.
TABLE-US-00006 TABLE 6 DA.C2 Karyotyping Results Number of cells
Number of cells karyotyped 5 Number of cells analyzed 20 Number of
42, XY cells 20 Number of abnormal cells 0 42, XX Other notes: 100%
of analyzed cells had normal female XX rat karyotype. FIG. 6
provides a photograph showing the analysis of the chromosome number
of the DA.C2 rat ES cell line.
TABLE-US-00007 TABLE 7 Blasto- Lines cysts Lines Karyo- strain
plated established typed Karyotypes BN x SD 41 8 (20%) 5 all lines
were high % F1 complex polyploid ACI 27 16 (60%) 3 G1: 90% 42 XY;
others were 70-85% euploid DA 20 9 (45%) 6 2B: 100% 42 XY; 2C: 100%
42 XX; others were 95-100% euploid F344 4 1 (25%) 0 Totals 92 34
(37%)
1.5.: Electroporation of Vector into Rat Embryonic Stem Cell
1. Passaged rat ES cells 24-48 hrs prior to electroporation.
2. Changed media to RVG2i+ROCKi (10 .mu.M Y-27632) 24 hr. prior to
electroporation
3. Changed media 30' prior to trypsinization.
4. Aliquoted DNA to be electroporated.
5. Allowed DNA to warm at RT for >10 min.
6. Heated DNA for 5' @ 62.degree. C. Place DNA on ice.
7. Trypsinized cells: a. Collected floating colonies. Washed plate
to collect as many floaters as possible. b. Pelleted colonies: 3' @
750 rpm. c. Washed pellet 1.times. with 5-10 ml PBS and
re-spin/pellet d. Aspirated supernatant; add 500.lamda. trypsin,
0.05%+1% chicken serum. i. Did not pool more than 1 10 cm plate of
colonies per tube. If there are too many colonies packed into the
bottom of the tube during trypsinization they will clump and most
of the cells will be lost. e. 4' @ 37.degree.. Pipeted colonies
several times to minimize clumping. f. Repeated steps 1-2 X: 4' @
37.degree.. g. Stopped trypsin with 500.lamda. RVG2i+10% FBS.
8. Pelleted cells: 5' @ 1200 rpm.
9. Resuspend cells in 10 ml PBS. Count two 20.lamda. aliquots to
determine total cell number.
10. Pelleted cells (5'/1200 rpm); calculate total cell number and
total resuspension volume to achieve correct cell concentration
(target #/75 .mu.l EP buffer).
11. Resuspend in a minimal volume of EP buffer; measure total
volume and adjust to target volume with EP buffer. Electroporation
buffer is sold by Millipore. The catalog # is ES-003-D. See,
Valenzuela et al. (2003) Nature Biotechnology 21:652-659, which is
herein incorporated by reference.
12. Add 75& cells to 50.lamda. DNA; transfer the 125.lamda.
cells/DNA solution to one well of a BTX 48-well cuvette. a. Filled
the empty wells in the same column with 125.lamda. EP buffer.
13. Pulsed the cuvette once in the BTX electroporator: a. Settings:
400V; .OMEGA.; 100 .mu.F (settings may vary)
14. Placed cuvette on ice for 15' to recover.
15. Removed cells into 5 ml RVG2i+10 .mu.M ROCKi.
16. Added to 15 cm plate with 20 ml RVG2i+10 .mu.M ROCKi. Plate has
2.times. neoR MEFs (or other MEFs depending on project). The neoR
selectable marker is the neomycin phosphotransferase (neo) gene of
Beck et al. (1982) Gene, 19:327-36 or in U.S. Pat. No. 7,205,148 or
6,596,541, each of which are herein incorporated by reference.
17. Incubated @ 37.degree.. Begin selection 48 hrs later.
ROCK inhibitor used was Y-27632.
1.6: Selecting a Targeted Genetic Modification in a Rat Embryonic
Stem Cell.
1. Passaged cells for 24-48 hrs prior to electroporation.
2. Changed media to RVG2i+ROCKi (10 .mu.M Y-27632) 24 hr. prior to
electroporation
3. Changed media 30' prior to trypsinization.
4. Aliquoted DNA to be electroporated.
5. Allowed DNA warm at RT for >10 min.
6. Heated DNA for 5' @ 62.degree. C. Place DNA on ice.
7. Trypsinized cells: a. Collected floating colonies. Washed plate
to collect as many floaters as possible. b. Pelleted colonies: 3' @
750 rpm. c. Washed pellet 1.times. with 5-10 ml PBS and
re-spin/pellet d. Aspirated supernatant; add 500.lamda. trypsin,
0.05%+1% chicken serum. i. Did not pool more than 1 10 cm plate of
colonies per tube. If there are too many colonies packed into the
bottom of the tube during trypsinization they will clump and most
of the cells will be lost. e. 4' @ 37.degree.. Pipeted colonies
several times to minimize clumping f. Repeated 1-2 X: 4' @
37.degree.. g. Stopped trypsin with 500.lamda. RVG2i+10% FBS.
8. Pelleted cells: 5' @ 1200 rpm.
9. Resuspended cells in 10 ml PBS. Count two 20.lamda. aliquots to
determine total cell number.
10. Pelleted cells (5'/1200 rpm); calculate total cell number and
total resuspension volume to achieve correct cell concentration
(target #/75 .mu.l EP buffer).
11. Resuspend in a minimal volume of EP buffer; measured total
volume and adjusted to target volume with EP buffer.
12. Added 75& cells to 50.lamda. DNA; transfer the 125.lamda.
cells/DNA solution to one well of a BTX 48-well cuvette. a. Filled
the empty wells in the same column with 125.lamda. EP buffer.
13. Pulsed the cuvette once in the BTX electroporator: a. Settings:
400V; 100 .mu.F (settings may vary)
14. Placed cuvette on ice for 15' to recover.
15. Removed cells into 5 ml RVG2i+10 .mu.M ROCKi.
16. Added to 15 cm plate with 20 ml RVG2i+10 .mu.M ROCKi. Plate had
2.times. neoR MEFs (or other MEFs depending on project).
17. Incubated @ 37.degree.. Began selection 48 hrs later.
18. G418 selection protocol was as follows: a. Day 2 (2.sup.nd day
after EP): incubated cells in 2i media+G418, 75 .mu.g/ml. b. Day 3:
incubated cells in 2i media without G418 c. Day 4: incubated cells
in 2i media+G418, 75 .mu.g/ml. d. Day 5: incubated cells in 2i
media without G418 e. Day 6: incubated cells in 2i media+G418, 75
.mu.g/ml. f. Day 7: incubated cells in 2i media without G418 g. Day
8: incubated cells in 2i media+G418, 75 .mu.g/ml. h. Day 9:
incubated cells in 2i media without G418 i. Day 10: incubated cells
in 2i media+G418, 75 .mu.g/ml. j. Day 11: incubated cells in 2i
media without G418 k. Day 12: picked colonies to expand for
screening. Each colony was dissociated in 0.05% trypsin+1% chicken
serum for 10 minutes and then plated into 1 well of a 96-well
feeder plate.
19. Expanded colonies for 3 days in 2i media.
20. Passaged clones 1:1 to new 96-well feeder plates.
21. Expanded clones for 3 days in 2i media.
22. For each clone, dissociated colonies in trypsin. Froze 2/3 of
each clone and store at -80.degree.; plated the remaining 1/3 onto
laminin plates (96-well plates coated with 10 .mu.g/ml
laminin).
23. When the laminin plates were confluent, passed off to the
screening lab for genotyping of the clones.
1.7. Molecular Signature of the Rat Embryonic Stem Cells
The genes listed in Table 8 were expressed at 20-fold lower in rat
ES cells than the corresponding genes in mouse ES cells. The genes
listed in Table 9 were expressed at levels 20-fold higher in rat ES
cells than the corresponding genes in mouse ES cells.
The microarray data in Tables 8 and 9 were generated as follows.
Rat ES cells (ACI.G2 and DA.2B) and mouse ES cells (F1H4) were
cultured in 2i media for 3 passages until confluent. F1H4 cells
were cultured on gelatin-coated plates in the absence of feeders.
F1H4 mouse ES cells were derived from 12956/SvEvTac and C57BL/6NTac
heterozygous embryos (see, e.g., U.S. Pat. No. 7,294,754 and
Poueymirou, W. T., Auerbach, W., Frendewey, D., Hickey, J. F.,
Escaravage, J. M., Esau, L., Dore, A. T., Stevens, S., Adams, N.
C., Dominguez, M. G., Gale, N. W., Yancopoulos, G. D., DeChiara, T.
M., Valenzuela, D. M. (2007), incorporated by reference herein in
its entirety).
The following protocol was used for sample prep: The 1.5 mL
Eppendorf tubes were labeled with the Sample ID. Cells grown on a
plate were rinsed in 37.degree. C. Phosphate-Buffered Saline (PBS).
PBS was removed and 300 ul of Trizol.RTM. was added. A scraper was
used to break the cells in Trizol.RTM. (Life Technology). The lysed
cells were collected in Trizol.RTM. in a 1.5 mL Epperdorf tube. For
cells grown on suspension, the cells were rinsed in 37.degree. C.
PBS and collected in a 1.5 mL tube. The cells were spun down; PBS
was removed; and 300 ul of Trizol.RTM. was added to the cells. The
cell membranes were broken by pipetting. Samples were sorted for
FACS with 10 to 10.sup.5 cells, the volume was concentrated to less
than 100 uL. 4 volumes of RNA Lysis buffer were added and mixed by
pipetting. For sample, 320 uL RNA Lysis buffer was added to 80 uL
sample. Samples were stored at -20.degree. C.
RNA-Seq was used to measure the expression level of mouse and rat
genes. Sequencing reads were mapped to mouse and rat reference
genome by Tophat, and RPKM (fragments per kilobase of exon per
million fragments mapped) were calculated for mouse and rat genes.
Homology genes based on gene symbol were selected, and then used
t-test to compare the expression level of each gene between mouse
and rat. miR-632 was in the top 10 highest expressed in rat ESCs
but was not expressed in mouse ES cells. Although no comparative
data exist from miR-632, based on the level of its expression
compared to other genes expressed in rat ESCs and their known
function in embryonic development, miR-632 was selected as a marker
for rat ES cells.
TABLE-US-00008 TABLE 8 The genes listed were expressed at levels
20-fold lower in rat ES cells than the corresponding genes in mouse
ES cells. ID Symbol Entrez Gene Name Location Type(s) Abcb1b Abcb1b
ATP-binding Plasma transporter cassette, sub- Membrane family B
(MDR/TAP), member 1B Acta2 ACTA2 actin, alpha 2, Cytoplasm other
smooth muscle, aorta Actg2 ACTG2 actin, gamma 2, Cytoplasm other
smooth muscle, enteric Aebp1 AEBP1 AE binding protein 1 Nucleus
peptidase Angptl2 ANGPTL2 angiopoietin-like 2 Extracellular other
Space Ankrd1 ANKRD1 ankyrin repeat Cytoplasm transcription domain 1
(cardiac regulator muscle) Anxa1 ANXA1 annexin A1 Plasma other
Membrane Anxa6 ANXA6 annexin A6 Plasma other Membrane Anxa8 ANXA8L2
annexin A8-like 2 Plasma other Membrane Arhgef25 ARHGEF25 Rho
guanine Cytoplasm other nucleotide exchange factor (GEF) 25 Axl AXL
AXL receptor Plasma kinase tyrosine kinase Membrane Basp1 BASP1
brain abundant, Nucleus transcription membrane attached regulator
signal protein 1 Bgn BGN biglycan Extracellular other Space Bst2
BST2 bone marrow Plasma other stromal cell antigen 2 Membrane Btf3
BTF3 basic transcription Nucleus transcription factor 3 regulator
Btg2 BTG2 BTG family, Nucleus transcription member 2 regulator
Capsl CAPSL calcyphosine-like Other other Cav1 CAV1 caveolin 1,
Plasma transmembrane caveolae protein, Membrane receptor 22 kDa
Ccdc80 CCDC80 coiled-coil domain Nucleus other containing 80 Ccnd2
CCND2 cyclin D2 Nucleus other Cd248 CD248 CD248 molecule, Plasma
other endosialin Membrane Cd44 CD44 CD44 molecule Plasma enzyme
(Indian blood Membrane group) Cd97 CD97 CD97 molecule Plasma
G-protein Membrane coupled receptor Cdc42ep5 CDC42EP5 CDC42
effector Cytoplasm other protein (Rho GTPase binding) 5 Cdh11 CDH11
cadherin 11, type 2, Plasma other OB-cadherin Membrane (osteoblast)
Cdkn2a CDKN2A cyclin-dependent Nucleus transcription kinase
inhibitor 2A regulator Cdo1 CDO1 cysteine Cytoplasm enzyme
dioxygenase type 1 Clip3 CLIP3 CAP-GLY domain Cytoplasm other
containing linker protein 3 Cln5 CLN5 ceroid- Cytoplasm other
lipofuscinosis, neuronal 5 Cnn1 CNN1 calponin 1, basic, Cytoplasm
other smooth muscle Col1a1 COL1A1 collagen, type I, Extracellular
other alpha 1 Space Col1a2 COL1A2 collagen, type I, Extracellular
other alpha 2 Space Col3a1 COL3A1 collagen, type III, Extracellular
other alpha 1 Space Col5a2 COL5A2 collagen, type V, Extracellular
other alpha 2 Space Col6a2 COL6A2 collagen, type VI, Extracellular
other alpha 2 Space Cryab CRYAB crystallin, alpha B Nucleus other
Csf1 CSF1 colony stimulating Extracellular cytokine factor 1 Space
(macrophage) Cth CTH cystathionase Cytoplasm enzyme (cystathionine
gamma-lyase) Cthrc1 CTHRC1 collagen triple Extracellular other
helix repeat Space containing 1 Ctsc CTSC cathepsin C Cytoplasm
peptidase Cyr61 CYR61 cysteine-rich, Extracellular other angiogenic
inducer, Space 61 Ddx58 DDX58 DEAD (Asp-Glu- Cytoplasm enzyme
Ala-Asp) box polypeptide 58 Dkk3 DKK3 dickkopf WNT Extracellular
cytokine signaling pathway Space inhibitor 3 Dmc1 DMC1 DNA meiotic
Nucleus enzyme recombinase 1 Dpysl3 DPYSL3 dihydropyrimidinase-
Cytoplasm enzyme like 3 Dse DSE dermatan sulfate Cytoplasm enzyme
epimerase Dusp1 DUSP1 dual specificity Nucleus phosphatase
phosphatase 1 Dusp27 DUSP27 dual specificity Other phosphatase
phosphatase 27 (putative) Dusp9 DUSP9 dual specificity Nucleus
phosphatase phosphatase 9 Ece2 ECE2 endothelin Plasma peptidase
converting enzyme 2 Membrane Ecm1 ECM1 extracellular matrix
Extracellular transporter protein 1 Space Egr1 EGR1 early growth
Nucleus transcription response 1 regulator Emp1 EMP1 epithelial
Plasma other membrane protein 1 Membrane Emp3 EMP3 epithelial
Plasma other membrane protein 3 Membrane Ephx2 EPHX2 epoxide
hydrolase Cytoplasm enzyme 2, cytoplasmic F3 F3 coagulation factor
Plasma transmembrane III (thromboplastin, Membrane receptor tissue
factor) Fau FAU Finkel-Biskis- Cytoplasm other Reilly murine
sarcoma virus (FBR-MuSV) ubiquitously expressed Fbn1 FBN1 fibrillin
1 Extracellular other Space Fbxo15 FBXO15 F-box protein 15 Other
transcription regulator Fhl2 FHL2 four and a half Nucleus
transcription LIM domains 2 regulator Flnc FLNC filamin C, gamma
Cytoplasm other Fos FOS FBJ murine Nucleus transcription
osteosarcoma viral regulator oncogene homolog Fundc2 FUNDC2 FUN14
domain Cytoplasm other containing 2 Gjb3 GJB3 gap junction Plasma
transporter protein, beta 3, Membrane 31 kDa Gpa33 GPA33
glycoprotein A33 Plasma other (transmembrane) Membrane Gpbp1l1
GPBP1L1 GC-rich promoter Other other binding protein 1- like 1 Gpc3
GPC3 glypican 3 Plasma other Membrane Grb10 GRB10 growth factor
Cytoplasm other receptor-bound protein 10 Gstm1 GSTM5 glutathione
S- Cytoplasm enzyme transferase mu 5 Hap1 HAP1 huntingtin-
Cytoplasm other associated protein 1 Hist1h2bc HIST2H2BE histone
cluster 2, Nucleus other (includes H2be others) Hmga2 HMGA2 high
mobility Nucleus enzyme group AT-hook 2 Hmgn3 Hmgn3 high mobility
Nucleus other group nucleosomal binding domain 3 Hormad1 HORMAD1
HORMA domain Nucleus other containing 1 Hsd17b14 HSD17B14
hydroxysteroid Cytoplasm enzyme (17-beta) dehydrogenase 14 Hspb1
HSPB1 heat shock 27 kDa Cytoplasm other protein 1 Hspb8 HSPB8 heat
shock 22 kDa Cytoplasm kinase protein 8 Htra1 HTRA1 HtrA serine
Extracellular peptidase peptidase 1 Space Ifi204 Ifi204 interferon
activated Nucleus transcription (includes gene 204 regulator
others) Ifi44 IFI44 interferon-induced Cytoplasm other protein 44
Ifit1 IFIT1B interferon-induced Cytoplasm other protein with
tetratricopeptide repeats 1B Ifitm3 IFITM2 interferon induced
Cytoplasm other transmembrane protein 2 Igf2 IGF2 insulin-like
growth Extracellular growth factor 2 Space factor (somatomedin A)
Igfbp7 IGFBP7 insulin-like growth Extracellular transporter factor
binding Space protein 7 Il1rl1 IL1RL1 interleukin 1 Plasma
transmembrane receptor-like 1 Membrane receptor Inhba INHBA
inhibin, beta A Extracellular growth Space factor Inhbb INHBB
inhibin, beta B Extracellular growth Space factor Irf7 IRF7
interferon Nucleus transcription regulatory factor 7 regulator
Isg15 ISG15 ISG15 ubiquitin- Extracellular other like modifier
Space Itga5 ITGA5 integrin, alpha 5 Plasma transmembrane
(fibronectin Membrane receptor receptor, alpha polypeptide) Jun JUN
jun proto-oncogene Nucleus transcription regulator Junb JUNB jun B
proto- Nucleus transcription oncogene regulator Lgals3bp LGALS3BP
lectin, galactoside- Plasma transmembrane binding, soluble, 3
Membrane receptor binding protein Lgals9 LGALS9 lectin,
galactoside- Extracellular other binding, soluble, 9 Space Lmna
LMNA lamin A/C Nucleus other Lox LOX lysyl oxidase Extracellular
enzyme Space Loxl2 LOXL2 lysyl oxidase-like 2 Extracellular enzyme
Space Loxl3 LOXL3 lysyl oxidase-like 3 Extracellular enzyme Space
Lrp1 LRP1 low density Plasma transmembrane lipoprotein Membrane
receptor receptor-related protein 1 Mageb16 MAGEB16 melanoma
antigen Other other family B, 16 Mcam MCAM melanoma cell Plasma
other adhesion molecule Membrane Mgp MGP matrix Gla protein
Extracellular other Space Mmp2 MMP2 matrix Extracellular
peptidase
metallopeptidase 2 Space (gelatinase A, 72 kDa gelatinase, 72 kDa
type IV collagenase) Mxra8 MXRA8 matrix-remodelling Other other
associated 8 Myl9 MYL9 myosin, light chain Cytoplasm other 9,
regulatory Mylpf MYLPF myosin light chain, Cytoplasm other
phosphorylatable, fast skeletal muscle Nab2 NAB2 NGFI-A binding
Nucleus transcription protein 2 (EGR1 regulator binding protein 2)
Ndufb4 NDUFB4 NADH Cytoplasm transporter dehydrogenase (ubiquinone)
1 beta subcomplex, 4, 15 kDa Npm1 NPM1 nucleophosmin Nucleus
transcription (nucleolar regulator phosphoprotein B23, numatrin)
Nr0b1 NR0B1 nuclear receptor Nucleus ligand- subfamily 0, group
dependent B, member 1 nuclear receptor Nr4a1 NR4A1 nuclear receptor
Nucleus ligand- subfamily 4, group dependent A, member 1 nuclear
receptor Nrp2 NRP2 neuropilin 2 Plasma kinase Membrane Oas1a OAS1
2'-5'-oligoadenylate Cytoplasm enzyme synthetase 1, 40/46 kDa Oasl2
Oasl2 2'-5' oligoadenylate Other enzyme synthetase-like 2 P4ha2
P4HA2 prolyl 4- Cytoplasm enzyme hydroxylase, alpha polypeptide II
Parp3 PARP3 poly (ADP-ribose) Nucleus enzyme polymerase family,
member 3 Pcolce PCOLCE procollagen C- Extracellular other
endopeptidase Space enhancer Pcyt1b PCYT1B phosphate Cytoplasm
enzyme cytidylyltransferase 1, choline, beta Pdgfc PDGFC platelet
derived Extracellular growth growth factor C Space factor Phlda1
PHLDA1 pleckstrin Cytoplasm other homology-like domain, family A,
member 1 Phlda2 PHLDA2 pleckstrin Cytoplasm other homology-like
domain, family A, member 2 Pla2g1b PLA2G1B phospholipase A2,
Extracellular enzyme group IB Space (pancreas) Pla2g4a PLA2G4A
phospholipase A2, Cytoplasm enzyme group IVA (cytosolic, calcium-
dependent) Porcn PORCN porcupine homolog Cytoplasm other
(Drosophila) Postn POSTN periostin, Extracellular other osteoblast
specific Space factor Prrx1 PRRX1 paired related Nucleus
transcription homeobox 1 regulator Prss23 PRSS23 protease, serine,
23 Extracellular peptidase Space Psmb8 PSMB8 proteasome Cytoplasm
peptidase (prosome, macropain) subunit, beta type, 8 Ptgs2 PTGS2
prostaglandin- Cytoplasm enzyme endoperoxide synthase 2
(prostaglandin G/H synthase and cyclooxygenase) Ptn PTN
pleiotrophin Extracellular growth Space factor Ptrf PTRF polymerase
I and Nucleus transcription transcript release regulator factor
Rarg RARG retinoic acid Nucleus ligand- receptor, gamma dependent
nuclear receptor Rgs16 RGS16 regulator of G- Cytoplasm other
protein signaling 16 Rn45s Rn45s 45S pre-ribosomal Other other RNA
Rpl10a RPL10A ribosomal protein Other other L10a Rpl31 RPL31
ribosomal protein Other other L31 Rpl37a RPL37A ribosomal protein
Cytoplasm other L37a Rps10 RPS10- RPS10-NUDT3 Cytoplasm other NUDT3
readthrough Rps14 RPS14 ribosomal protein Cytoplasm translation S14
regulator Rps20 Rps20 ribosomal protein Cytoplasm other S20 Rps26
RPS26 ribosomal protein Cytoplasm other S26 Rps9 RPS9 ribosomal
protein Cytoplasm translation S9 regulator S100a4 S100A4 S100
calcium Cytoplasm other binding protein A4 S100a6 S100A6 S100
calcium Cytoplasm transporter binding protein A6 Schip1 SCHIP1
schwannomin Cytoplasm other interacting protein 1 Sdc2 SDC2
syndecan 2 Plasma other Membrane Serpine1 SERPINE1 serpin peptidase
Extracellular other inhibitor, clade E Space (nexin, plasminogen
activator inhibitor type 1), member 1 Serpine2 SERPINE2 serpin
peptidase Extracellular other inhibitor, clade E Space (nexin,
plasminogen activator inhibitor type 1), member 2 Serpinf1 SERPINF1
serpin peptidase Extracellular other inhibitor, clade F Space
(alpha-2 antiplasmin, pigment epithelium derived factor), member 1
Sh3gl2 SH3GL2 SH3-domain Plasma enzyme GRB2-like 2 Membrane Slc19a2
SLC19A2 solute carrier Plasma transporter family 19 Membrane
(thiamine transporter), member 2 Slc25a5 SLC25A5 solute carrier
Cytoplasm transporter family 25 (mitochondrial carrier; adenine
nucleotide translocator), member 5 Slc29a1 SLC29A1 solute carrier
Plasma transporter family 29 Membrane (equilibrative nucleoside
transporter), member 1 Slc35f2 SLC35F2 solute carrier Other other
family 35, member F2 Snrpn SNRPN small nuclear Nucleus other
ribonucleoprotein polypeptide N Snx22 SNX22 sorting nexin 22 Other
transporter Sparc SPARC secreted protein, Extracellular other
acidic, cysteine- Space rich (osteonectin) Spp1 SPP1 secreted
Extracellular cytokine phosphoprotein 1 Space Sult4a1 SULT4A1
sulfotransferase Cytoplasm enzyme family 4A, member 1 Tagln TAGLN
transgelin Cytoplasm other Tcea3 TCEA3 transcription Nucleus
transcription elongation factor A regulator (SII), 3 Tgfb3 TGFB3
transforming Extracellular growth growth factor, beta 3 Space
factor Thbs1 THBS1 thrombospondin 1 Extracellular other Space Thbs2
THBS2 thrombospondin 2 Extracellular other Space Tm4sf1 TM4SF1
transmembrane 4 L Plasma other six family member 1 Membrane Tmbim1
TMBIM1 transmembrane Cytoplasm other BAX inhibitor motif containing
1 Tmem176b TMEM176B transmembrane Other other protein 176B Tnc TNC
tenascin C Extracellular other Space Tpd52l1 TPD52L1 tumor protein
D52- Cytoplasm other like 1 Tpm2 TPM2 tropomyosin 2 Cytoplasm other
(beta) Usp18 USP18 ubiquitin specific Cytoplasm peptidase peptidase
18 Vim VIM vimentin Cytoplasm other Wfdc2 WFDC2 WAP four-
Extracellular other disulfide core Space domain 2 Wisp2 WISP2 WNT1
inducible Extracellular growth signaling pathway Space factor
protein 2 Ybx1 YBX1 Y box binding Nucleus transcription protein 1
regulator
TABLE-US-00009 TABLE 9 The genes listed were expressed at levels
20-fold higher in rat ES cells than the corresponding genes in
mouse ES cells. ID Symbol Entrez Gene Name Location Type(s) Ajap1
Ajap1 adherens junction Other other associated protein 1 Amd1 AMD1
adenosylmethionine Cytoplasm enzyme decarboxylase 1 Ankrd2 ANKRD2
ankyrin repeat Nucleus transcription domain 2 (stretch regulator
responsive muscle) Arhgef9 ARHGEF9 Cdc42 guanine Cytoplasm other
nucleotide exchange factor (GEF) 9 Atp5h Atp5h ATP synthase, H+
Cytoplasm enzyme transporting, mitochondrial F0 complex, subunit d
Btg3 BTG3 BTG family, Nucleus other member 3 Car6 CA6 carbonic
anhydrase Extracellular enzyme VI Space Camk4 CAMK4
calcium/calmodulin- Nucleus kinase dependent protein kinase IV
Capn12 CAPN12 calpain 12 Other peptidase Cct6b CCT6B chaperonin
Cytoplasm transporter containing TCP1, subunit 6B (zeta 2) Cdx2
CDX2 caudal type Nucleus transcription homeobox 2 regulator Cldn5
CLDN5 claudin 5 Plasma other Membrane Clec3a CLEC3A C-type lectin
Other other domain family 3, member A Clic6 CLIC6 chloride
intracellular Plasma ion channel channel 6 Membrane Dhrsx DHRSX
dehydrogenase/reductase Other enzyme (SDR family) X-linked Dpysl2
DPYSL2 dihydropyrimidinase- Cytoplasm enzyme like 2 Dusp26 DUSP26
dual specificity Cytoplasm enzyme phosphatase 26 (putative) Eci3
Eci3 enoyl-Coenzyme A Other enzyme delta isomerase 3 Eef2k EEF2K
eukaryotic Cytoplasm kinase elongation factor-2 kinase Efna1 EFNA1
ephrin-A1 Plasma other Membrane Epha4 EPHA4 EPH receptor A4 Plasma
kinase Membrane Fank1 FANK1 fibronectin type III Nucleus
transcription and ankyrin repeat regulator domains 1 Fhit FHIT
fragile histidine Cytoplasm enzyme triad Filip1 FILIP1 filamin A
Cytoplasm other interacting protein 1 Fmod FMOD fibromodulin
Extracellular other Space Foxe1 FOXE1 forkhead box E1 Nucleus
transcription (thyroid regulator transcription factor 2) Fry FRY
furry homolog Extracellular other (Drosophila) Space Gjb5 GJB5 gap
junction protein, Plasma transporter beta 5, 31.1 kDa Membrane Gpx2
GPX2 glutathione Cytoplasm enzyme peroxidase 2 (gastrointestinal)
Grxcr2 GRXCR2 glutaredoxin, Other other cysteine rich 2 Hecw2 HECW2
HECT, C2 and WW Extracellular enzyme domain containing Space E3
ubiquitin protein ligase 2 Hey2 HEY2 hairy/enhancer-of- Nucleus
transcription split related with regulator YRPW motif 2 Icos Icos
inducible T-cell co- Plasma other stimulator Membrane Ifitm1 IFITM1
interferon induced Plasma transmembrane transmembrane Membrane
receptor protein 1 Il1f8 IL1F8 Interleukin-1 Extracellular cytokine
(IL36B) family member space (Interleukin 36 beta) Il28ra IL-28RA
Interleukin 28 receptor, Plasma Cytokine alpha membrane receptor
Igfbpl1 IGFBPL1 insulin-like growth Other other factor binding
protein-like 1 Ipcef1 IPCEF1 interaction protein Cytoplasm enzyme
for cytohesin exchange factors 1 Lctl Lctl lactase-like Cytoplasm
other Ldhd LDHD lactate Cytoplasm enzyme dehydrogenase D Lef1 LEF1
lymphoid enhancer- Nucleus transcription binding factor 1 regulator
Lefty1 LEFTY1 left-right Extracellular growth factor determination
factor 1 Space Lifr LIFR leukemia inhibitory Plasma transmembrane
factor receptor alpha Membrane receptor Lpar2 LPAR2
lysophosphatidic Plasma G-protein acid receptor 2 Membrane coupled
receptor Mog MOG myelin Extracellular other oligodendrocyte Space
glycoprotein Morn5 MORN5 MORN repeat Other other containing 5 Pigz
NCBP2 nuclear cap binding Nucleus other protein subunit 2, 20 kDa
Nptxr NPTXR neuronal pentraxin Plasma transmembrane receptor
Membrane receptor Ntm NTM neurotrimin Plasma other Membrane Nutf2
NUTF2 nuclear transport Nucleus transporter factor 2 Ocln OCLN
occludin Plasma enzyme Membrane Olr1 OLR1 oxidized low density
Plasma transmembrane lipoprotein (lectin- Membrane receptor like)
receptor 1 Pabpc4 PABPC4 poly(A) binding Cytoplasm translation
protein, cytoplasmic regulator 4 (inducible form) Pde11a PDE11A
phosphodiesterase Cytoplasm enzyme 11A Pdyn PDYN prodynorphin
Extracellular transporter Space Per3 PER3 period circadian Nucleus
other clock 3 Pllp PLLP plasmolipin Plasma transporter Membrane
Ppp1r14c PPP1R14C protein phosphatase Cytoplasm other 1, regulatory
(inhibitor) subunit 14C Pramel6 Pramel6 preferentially Other other
expressed antigen in melanoma like 6 Ptpn18 PTPN18 protein tyrosine
Nucleus phosphatase phosphatase, non- receptor type 18
(brain-derived) Pycr1 PYCR1 pyrroline-5- Cytoplasm enzyme
carboxylate reductase 1 Rab26 RAB26 RAB26, member Plasma enzyme RAS
oncogene Membrane family Ramp2 RAMP2 receptor (G protein- Plasma
transporter coupled) activity Membrane modifying protein 2 Rbm24
RBM24 RNA binding motif Other other protein 24 Rhag RHAG
Rh-associated Plasma peptidase glycoprotein Membrane Rpl3 RPL3
ribosomal protein Cytoplasm other L3 Sall3 SALL3 sal-like 3 Nucleus
other (Drosophila) Satb1 SATB1 SATB homeobox 1 Nucleus
transcription regulator Scg2 SCG2 secretogranin II Extracellular
cytokine Space Slc15a1 SLC15A1 solute carrier family Plasma
transporter 15 (oligopeptide Membrane transporter), member 1 Slc1a1
SLC1A1 solute carrier family 1 Plasma transporter
(neuronal/epithelial Membrane high affinity glutamate transporter,
system Xag), member 1 Slc24a5 Slc24a5 solute carrier family Other
other 24 (sodium/potassium/calcium exchanger), member 5 Slc37a2
SLC37A2 solute carrier family Other transporter 37 (glucose-6-
phosphate transporter), member 2 40424 SNTB1 syntrophin, beta 1
Plasma other (dystrophin- Membrane associated protein A1, 59 kDa,
basic component 1) St6galnac3 ST6GALNAC3 ST6 (alpha-N- Cytoplasm
enzyme acetyl-neuraminyl- 2,3-beta-galactosyl- 1,3)-N-
acetylgalactosaminide alpha-2,6- sialyltransferase 3 Tex12 TEX12
testis expressed 12 Nucleus other Tex15 TEX15 testis expressed 15
Extracellular other Space Tfap2a TFAP2A transcription factor
Nucleus transcription AP-2 alpha regulator (activating enhancer
binding protein 2 alpha) Tmc1 TMC1 transmembrane Plasma other
channel-like 1 Membrane Tmem130 TMEM130 transmembrane Other other
protein 130 Tmem30b TMEM30B transmembrane Other other protein 30B
Tomm20 TOMM20 translocase of outer Cytoplasm transporter
mitochondrial membrane 20 homolog (yeast) Tox3 TOX3 TOX high
mobility Other other group box family member 3 Ttc25 TTC25
tetratricopeptide Cytoplasm other repeat domain 25 Tymp TYMP
thymidine Extracellular growth factor phosphorylase Space Ubb Ubb
ubiquitin B Cytoplasm other Vamp7 VAMP7 vesicle-associated
Cytoplasm transporter membrane protein 7 Wfdc12 Wfdc12 WAP
four-disulfide Extracellular other core domain 12 Space Wfdc15a
Wfdc15a WAP four-disulfide Other other core domain 15A Wfdc6a
Wfdc6a WAP four-disulfide Other other core domain 6A
TABLE-US-00010 TABLE 10 A subset of genes from Table 9, which are
expressed at levels 20-fold higher in rat ES cells than the
corresponding genes in mouse ES cells. ID Entrez Gene Name Ajap1
Adheres Junctions Associate Protein Cldn5 Claudin 5 Arhgef9 Cdc42
guanine nucleotide exchange facter 9 Camk4
Calcium/calmodulin-dependent protein kinase IV Efna1 ephrin-A1
Epha4 EPH receptor A4 Gjb5 gap junction protein beta 5 Igfbpl1
Insulin-like growth factor binding protein-like 1 Il1f8 Interleukin
36 beta Il28ra Interleukin 28 receptor, alpha Lefty1 left-right
determination factor 1 Lifr Leukemia inhibitory factor receptor
alpha Lpar2 Lysophosphatidic acid receptor 2 Ntm Neuronal pentraxin
receptor Ptpn18 Protein tyrosine phosphatase non-receptor type 18
Cdx2 Caudal type homeobox 2 Fank1 Fibronectin type III and ankyrin
repeat domains 1 Foxe1 Forkhead box E1 (thyroid transcription
factor 2) Hey2 Hairy/enhancer-of-split related with YRPW motif 2
Lef1 Lymphoid enhancer-binding factor 1 Sall3 Sal-like 3
(Drosophila) Satb1 SATB homeobox 1
An additional molecular signature employing the pluripotency
markers/genes for the rat ES cells has also been developed. Table
11 provides a gene list and their expression ranks from the RNA
profiling data. mRNA was isolated from rat ES cells and the
expression level of various markers were compared relative to each
other. The term "rank" means the comparative expression levels of
individual genes: the higher the rank (1 is highest), the higher
the expression. For example, Oct4's rank of 13 means that, of all
the genes assayed, it was expressed higher than all but 12 genes.
Background in this experiment was any expression value below 30;
6107 genes had expression values of 30 or higher.
TABLE-US-00011 TABLE 11 Rat ES cell molecular signature employing
various pluripotency, mesodermal, endodermal, neural and
trophectoderm markers/genes. Pluripotency Mesodermal Endodermal
Neural Trophectoderm Pluripotency Rank Mesodermal Rank Endodermal
Rank Neural Rank Trophectoder- m Rank c-Myc 8248 Brachyury 7542
Gata6 11195 Nestin 7761 Cdx2 739 Dnmt3L 127 Flk1 Not tested Sox17
11418 Pax6 13570 Dppa2 Not tested Nodal 3050 Hhex1 4571 Sox2 681
Dppa5 Not tested Bmp4 3072 Nodal 3050 Ecat1 9714 Bmpr2 6382 Ext1
6091 Eras 2541 Sox7 10284 Err-beta 1368 Fbxo15 1369 Fgf4 3440
Fthl17 Not tested Gdf3 2771 Rank > 6107 = bkg expression Klf4
836 Lef1 1313 LIF receptor 724 Lin28 828 Nanog 774 Oct4 13 Rexo1
6119 Sox15 4524 Sox2 681 SSEA1 Not tested SSEA4 Not tested Stella
Not tested Tcl1 Not tested Utf1 1501
Example 2
Inactivation of Genomic Loci in Rats
2.1: Inactivation of Endogenous Genomic Loci Using an Endonuclease
Agent
In order to introduce a mutant allele at an endogenous rat genomic
locus, the rat ES cells described herein are electroporated with
expression vectors (or mRNA) that express ZFNs 1 and 2 (or TALENs 1
and 2). These proteins bind their target sequences on opposite
strands, separated by about 6 bp to about 40 bp. A double-stranded
break is formed within the target locus, which the cell attempts to
repair by Non-Homologous End-Joining (NHEJ). In many cases, NHEJ
results in creation of a deletion, which often disrupts the
function of the gene (most often by producing a frameshift
mutation). In order to identify a positive clone comprising a
mutant allele, the electroporated cells are plated at low density,
because no drug selection is done. Colonies are picked and assayed
at the target site to see if a mutation was produced (e.g., using a
modification of allele (MOA) assay described above). The selected
ES cells comprising the mutant allele are then introduced into a
host rat embryo, for example, a pre-morula stage or blastocyst
stage rat embryo, and implanted in the uterus of a surrogate mother
to generate a founder rat (F0 rat). Subsequently, the founder rat
is bred to a wild-type rat to create F1 progeny heterozygous for
the mutant allele. Mating of the heterozygous F1 rat can produce
progeny homozygous for the mutant allele.
2.2.: Rat ESC Targeting for the Inactivation of the Rat
Apolipoprotein E (ApoE) Gene Using Zinc Finger Nucleases
Zinc finger nucleases use sequence specific modular DNA binding
domains to direct endonuclease activity to unique target sequence
in the genome. ZFNs are engineered as a pair of monomers. Each
monomer contains nonspecific cleavage domain from FokI endonuclease
fused to 3 or more zinc finger DNA-binding domains. Each zinc
finger binds a 3 bp subsite and specificity is achieved by the
combined target sites of both monomers. ZFNs produce
double-stranded breaks (DSB'S) in DNA, and mutations (insertions or
deletions) frequently occur during non-homologous end joining
(NHEJ). DSBs also stimulate homology-directed repair (HDR) by
homologous recombination if a donor sequence is provided with
ZFN.
Such ZFNs were employed in combination with the various methods and
compositions described herein to improve targeting efficiency. The
rat Apolipoprotein E (ApoE) locus was targeted as described in
Example 3.2(a)(i), except expression vectors that express ZFNs 1
and 2 were also introduced into the rat ES cells. See FIG. 10 which
provides a schematic of the ApoE targeting event in combination
with rTZFN1P and rTZFN2P. The targeting efficiency was determined
as discussed below in Example 6 and results are shown in FIG. 11.
Surprisingly, the targeting efficiency went up 8-10 fold.
A plasmid targeting vector was built with a self-deleting drug
selection cassette and a lacZ gene as a reporter gene. Good
targeting efficiency was achieved and a high % chimeras were
produced. Zinc finger nucleases (ZFNs) were also tested in
combination with targeting vectors to examine its effect on
improving targeting efficiency. The targeting vector was
co-expressed with the expression vectors for 2 ZFN pairs that cut
the ApoE locus. The rat ESC clones electroporated with both the
targeting vector and a set of the ZFNs showed a targeting
efficiency of 8-10 fold higher than that of rat ESC clones
electroporated with a targeting vector alone. Moreover, bi-allelic
homozygous targeting in about 2% of our clones was detected. High %
chimeras from two of these targeted clones were obtained.
The ApoE-targeted (with ZFN assistance) rat ESC clones were
microinjected into SD blastocysts, which were then transferred to
pseudopregnant SD recipient females, using standard techniques.
Chimeras were identified by coat color; male F0 chimeras were bred
to SD females. Germline F1 pups were genotyped for the presence of
the targeted ApoE allele (FIG. 17). There was a high % chimeras
from two of these targeted clones.
An ApoE knockout rat provides a means to study various types of
disorders and diseases. In humans, Apolipoprotein is found in
chylomicron, HDL, LDL and VLDL. ApoE is essential for the normal
catabolism of triglyceride-rich lipoprotein constituents. Defects
in APOE result in numerous disease states including, for example,
familial hypercholesterolemia, hyperlipemia, betalipoproteinemia,
familial dysbetalipoproteinemia, type III hyperlipoproteinemia (HLP
III), risk of coronary artery disease. One isoform (ApoE4) is
associated with late-onset familial and sporadic Alzheimer's
disease, possibly with MS as well.
In mice, ApoE is primarily found in HDL; transports cholesterol, as
in humans. ApoE-deficient mice (2 independent KOs) have 5 times
normal plasma cholesterol; developed foam cell-rich depositions in
their proximal aortas by age 3 months (comparable to human
syndrome).
ApoE knockouts in rats offer an animal model to study endothelial
function, including, but not limited to, plaque formation,
transcriptional changes (RNA-Seq), ex vivo function. Moreover,
larger size of rats would facilitate all these assays and
potentially improve the quality of the RNA-Seq data.
2.3. Inactivation of the Rat Interleukin-2 Receptor Gamma
(IL2r-.gamma.) Locus Using Zinc Finger Nucleases
The rat Interleukin-2 receptor gamma (IL2r-.gamma.) locus was
targeted as described in Example 3.3(a), except that expression
vectors that express ZFN U (ZFN upstream) and ZFN D (ZFN
downstream) were also introduced into the rat ES cells. FIG. 18
provides a schematic of the IL2r-.gamma. targeting event in
combination with ZFN U and ZFN D. The sequence of the IL2r-.gamma.
locus which these zinc fingers bind is denoted in FIG. 18. The
targeting efficiency was determined as discussed below in Example
3.3(a) and the results are shown in FIG. 18. Briefly, homozygously
targeted clones were confirmed by PCR. For the ZFN1 pair: 173
mutant clones out of 192 screened (90%) and for the ZFN2 pair: 162
clones out of 192 (84%) screened.
The IL2r-.gamma.-targeted (with ZFN assistance) rat ESC clones were
microinjected into SD blastocysts, which were then transferred to
pseudopregnant SD recipient females, using standard techniques.
Chimeras were identified by coat color; male F0 chimeras were bred
to SD females. Germline F1 pups were genotyped for the presence of
the targeted IL2r-.gamma. allele.
2.4.: Inactivation of the Rat Interleukin-2 Receptor Gamma
(IL2r-.gamma.) Using CRISPR/Cas9
The rat IL2r-.gamma. locus was targeted as described in Example
3.3(a), except that the CRISPR/Cas9 system was also introduced into
the rat ES cells to aid in targeting efficiency. SBI: System
Biosciences Cas9 "SmartNuclease" all-in-one vectors were employed
and Cas9 expression was driven by CAG, EF1a, PGK, or CMV promoter.
Custom gRNA was ligated into a vector and expressed by H1 promoter.
4 gRNAs against Il2rg were designed. The targeting efficiency when
employing the various guide RNAs is shown in FIG. 19.
Example 3
Targeted Modification of Rat Genomic Loci
3.1: Rat ESC Targeting: The Rat Rosa 26 Locus.
The rat Rosa26 locus lies between the Setd5 and Thumpd3 genes as in
mouse, with the same spacing. The rat Rosa 26 locus (FIG. 12, Panel
B) differs from the mouse Rosa 26 locus (FIG. 12, Panel A). The
mouse Rosa26 transcripts consist of 2 or 3 exons. The rat locus
contains a 2nd exon 1 (Ex1b) in addition to the homologous exon to
mouse exon1 (Ex1a). No 3rd exon has been identified in rat.
Targeting of a rat Rosa26 allele is depicted in FIG. 12 (bottom),
where homology arms of 5 kb each were cloned by PCR using genomic
DNA from DA rat ESC. The targeted allele contains a SA (splicing
acceptor)-lacZ-hUb-neo cassette replacing a 117 bp deletion in the
rat Rosa26 intron.
Targeting efficiency at the rat Rosa 26 locus was determined (Table
12). Linearized vector was electroporated into DA or ACI rat ESCs,
and transfected colonies were cultured in 2i media+G418, using
standard techniques. Individual colonies were picked and screened
using a Loss of Allele (LOA) assay (Valenzuela, D. et al. (2003)
High-throughput engineering of the mouse genome coupled with
high-resolution expression analysis, Nature Biotech. 21:652-660,
incorporated herein by reference).
TABLE-US-00012 TABLE 12 rat Rosa26 Targeting Efficiency Colonies
Reconfirmed Targeting Cell line picked positives efficiency (%)
DA.2B 192 4 2.1 ACI.G1 96 4 4.2
Chimera production and germline transmission using Rosa26-targeted
Rat ESC Clones. Reconfirmed Rosa26-targeted rat ESC clones were
microinjected into SD blastocysts, which were then transferred to
pseudopregnant SD recipient females, using standard techniques.
Chimeras were identified by coat color; male F0 chimeras were bred
to SD females. Germline (agouti) F1 pups were genotyped for the
presence of the targeted Rosa26 allele; nine of 22 agouti pups
genotyped as heterozygous at the Rosa26 locus (Table 13).
TABLE-US-00013 TABLE 13 Germline Transmission Using Targeted Rosa26
rESC Clones Germline rESC- ESC- R26 clones producing Transmitting
Total derived derived Cell line injected Chimeras Clones Pups Pups
pups (%) DA.2B 4 3 2 AH7: 64 AH7: 41 AH7: 63 AE3: 112 AE3: 6 AE3: 3
ACI.G1 4 4 1 DE9: 39 DE9: 4 10
3.2.(a)(i): Targeting of the Rat Apolipoprotein E (ApoE) Locus.
The rat Apolipoprotein E (ApoE) locus was targeted to disrupt ApoE
function. Targeting of the ApoE locus was done using a targeting
vector comprising a lacZ-hUb-neo cassette flanked with a 5' and 3'
homology arms homologous to the ApoE locus. FIG. 20 depicts a
genetically modified rat ApoE locus that has been disrupted by a
1.8 kb deletion and the insertion of a lacZ-hUb-neo cassette, which
further includes a self-deleting Cre cassette comprising a Crei
gene driven by a protamine promoter. The electroporation conditions
were as follows: 6 ug DNA; 2.05.times.10.sup.6 cells; 400V; 200 uF:
342 V, 593 usec; plate on 15 cm 2.times. dense neoR MEFs in 2i+10
uM ROCKi.
Targeting efficiency at the ApoE locus was determined and is shown
in Table 14. Linearized vector was electroporated into DA.2B rat
ESCs derived from the DA strain, and transfected colonies were
cultured using standard techniques. Individual colonies were picked
and screened using a Loss of Allele (LOA) assay.
TABLE-US-00014 TABLE 14 rat ApoE Targeting Efficiency Colonies
Targeting Cell line Vector picked Targeted efficiency (%) DA.2B
ApoE-mSDC 192 7 3.7 DA.2B ApoE-mSDC 192 15 7.8
Chimera production and germline transmission using ApoE-targeted
rat ESC clones was performed. ApoE-targeted rat ESC clones were
microinjected into SD blastocysts, which were then transferred to
pseudopregnant SD recipient females, using standard techniques.
Chimeras were identified by coat color; male F0 chimeras were bred
to SD females. F1 pups were genotyped for the presence of the
targeted ApoE allele (Table 15).
TABLE-US-00015 TABLE 15 Microinjection Results Exp Clone pups
Chimeras 1 ApoE-AF5 4 3 (90, 90, 90) 2 ApoE-BC4 5 0
Additional targeting data for ApoE is also provided in FIG. 21.
3.2.(a)(ii). Targeting ApoE in Rats with a Targeting Vector
FIG. 20 provides a schematic of the rat ApoE locus and a targeting
plasmid. The upper schematic of FIG. 20 shows the genomic structure
of the rat ApoE locus and the genomic regions corresponding to 5'
and 3' homology arms (5 kb and 5.4 kb, respectively; dark grey
boxes). Exon 1 of ApoE is non-coding and is shown as an open box
closest to the 5' homology arm. The 3 introns of ApoE are denoted
as lines and exons 2 and 3 comprise coding regions and are shown as
stippled grey boxes. Exon 4 contains both coding and non-coding
sequences as denoted by the stippled grey shading and the open
box.
The lower schematic in FIG. 20 is the targeting vector. The 5' and
3' homology arms (5 kb and 5.4 kb respectively) are denoted by the
dark grey boxes. The targeting vector comprises a reporter gene
(lacZ) and a self-deleting cassette flanked by loxP sites (open
arrows). The self-deleting cassette comprises the Crei gene
operably linked to a mouse Prm1 promoter and a selection cassette
comprising a neomycin resistance gene operably linked to a human
ubiquitin promoter.
The Crei gene comprises two exons encoding a Cre recombinase, which
are separated by an intron (Crei) to prevent its expression in a
prokaryotic cell. See, See, for example, U.S. Pat. No. 8,697,851
and U.S. Application Publication 2013-0312129, which describe the
self-deleting cassette in detail and are hereby incorporated by
reference in their entirety. By employing the Prm1 promoter, the
self-deleting cassette can be deleted specifically in male germ
cells of F0 rats. The targeting vector was electroporated into the
rat ES cells obtained in Example 1 and the cells were plated on 15
cm 2.times. dense neomycin-resistant MEFs in 2i+10 uM ROCKi. The
transformed rat ES cells were cultured, selected, and maintained as
described in Example 1.
As shown in Table 23, 384 colonies were screened and 23 targeted
clones were obtained. The targeting efficiency was 5.99%. 3 clones
were injected into blastocysts as described herein in Example 1. 3
clones producing chimeras were obtained and 2 of the clones
transmitted the targeted modification through the germline.
3.2.(a)(iii). Targeting ApoE in Rats with a Targeting Vector in
Combination with Zinc Finger Nucleases
The targeting vector employed in Example 3.2(a)(ii) was used in
combination with zinc finger nucleases to target the rat ApoE
locus. Table 16 provides a summary of the genomic organization of
the rat ApoE locus. The positions shown in the Table 16 were taken
from build 5.0 of the Reference Sequence of the rat genome
(ENSMBL). ApoE is on chromosome 1 on the (-) strand.
TABLE-US-00016 TABLE 16 Summary of the rat ApoE locus and the
positions of the zinc finger nuclease binding sites and cutting
sites. Feature Start End length Notes Exon 1 81881110 81881182 73
5' non-coding Exon2 81880269 81880332 64 contains ATG ATG 81880309
81880311 3 start codon Exon3 81879607 81879775 169 ZFN1a binding
site 81879707 81879693 15 CAGGCCCTGAACCGC (SEQ ID NO: 10) ZFN1
cutting site 81879692 81879687 6 TTCTGG (SEQ ID NO: 11) ZFN1b
binding site 81879686 81879671 16 GATTACCTGCGCTGGG (SEQ ID NO: 12)
Intron 3-4 81879776 81879207 400 ZF21a binding site 81879591
81879577 15 TTCACCCTCCGCACC (SEQ ID NO: 13) ZFN2 cutting site
81879576 81879570 7 TGCTGAG (SEQ ID NO: 14) ZF21b binding site
81879569 81879552 18 TATCCAGATCCAGGGGTT (SEQ ID NO: 15) Exon 4
81878371 81879208 838 contains TGA TGA 81878482 81878484 3 ApoE
deletion 81878482 81880311 1830
FIG. 10 provides a schematic of the rat ApoE locus and denotes with
grey bars the cutting site for ZFN1 and ZFN2. The cutting site for
ZFN1 is in exon 3 and the cutting site for ZNF2 is in intron 3. The
exact position of the both ZFN sites is set forth in Table 16. The
genomic regions corresponding to the 5' and 3' homology arms (5 kb
and 5.4 kb, respectively) are denoted by the dark grey boxes. Exon
1 of ApoE is non-coding and is shown as an open box closest to the
5' homology arm. The three introns of the ApoE gene are denoted as
lines and exons 2 and 3 comprise coding regions and are shown as
stippled grey boxes. Exon 4 contains both coding and non-coding
sequences as denoted by the stippled grey shading and the open
box.
The employed targeting vector was the same as that in Example
3.2(a)(ii) and shown in FIG. 20. The ZFNs were introduced as two
expression plasmids, one for each half of the ZFN pair. 20 ug of
the plasmid for ZFN1 and 20 ug of the plasmid for ZFN2 was used.
ZFNs were purchased from Sigma. The expression of each ZFN was
driven by the CMV promoter.
The targeting vector were electroporated into the rat ES cells
obtained in Example 1 and the cells were plated on 15 cm 2.times.
dense neoR MEFs in 2i+10 uM ROCKi. The transformed rat ES cells
were cultured, selected and maintained as described in Example
1.
As shown in Table 23, 384 colonies were screened and 290 targeted
clones were obtained. The targeting efficiency was 75.52%. 2 clones
were injected into blastocysts as described herein in Example 1.
Two clones producing chimeras were obtained and one of the clones
transmitted the targeted modification through the germline.
Moreover, employing ZFN1 and ZFN2 produced 8 biallelic targeted
clones with an efficiency of 2.08%.
3.2.(b)(i): Targeted Modification of the Rat Apolipoprotein E
(ApoE) Locus Using a Large Targeting Vector (LTC).
Targeting of the ApoE locus is done using a large targeting vector
(LTVEC) comprising a lacZ-mouse Prm1-Crei cassette flanked with a
5' homology arm to the ApoE locus of about 45 kb and a 3' homology
arm to the ApoE locus of about 23 Kb. FIG. 22 depicts the rat ApoE
locus in which the ApoE locus has been disrupted by a 1.83 kb
deletion and the insertion of the lacZ gene and a self-deleting
cassette comprising mPrm1-Crei cassette and a hUb-neo selection
cassette. Methods employed in example 3.2(a)(i) can be used to
introduce this vector into rat ES cells.
Example 3.2.(b)(ii)
Targeting of the Rat ApoE Locus with a Large Targeting Vector
(LTVEC)
FIG. 22 provides a schematic of the rat ApoE locus and a large
targeting vector (LTVEC). The upper schematic of FIG. 22 shows the
genomic organization of the rat ApoE locus and the genomic regions
corresponding to the 5' and 3' homology arms (45 kb and 23 kb,
respectively; dark grey boxes). Exon 1 of ApoE is non-coding and is
shown as an open box closest to the 5' homology arm. The 3 introns
of ApoE are denoted as lines and exons 2 and 3 comprise coding
regions and are shown as stippled grey boxes. Exon 4 contains both
coding and non-coding sequences as denoted by the stippled grey
shading and the open box.
The lower schematic in FIG. 22 is the LTVEC. The 5' and 3' homology
arms (45 kb and 23 kb, respectively) are denoted by the dark grey
boxes. The targeting vector comprises a reporter gene (lacZ) and a
self-deleting cassette flanked by loxP sites (open arrows), which
comprises the Crei gene operably linked to a mouse Prm1 promoter
and a drug selection cassette comprising a neomycin resistance gene
operably linked to a human ubiquitin promoter. The Crei comprises
two exons encoding the Cre recombinase which are separated by an
intron (Crei) to prevent its expression in a prokaryotic cell. See,
for example, U.S. Pat. No. 8,697,851 and U.S. Application
Publication 2013-0312129, which describes the self-deleting
cassette in detail and is hereby incorporated by reference in their
entirety. By employing a mouse Prm1 promoter, the self-deleting
cassette can be deleted specifically in male germ cells of F0
rat.
The LTVEC was electroporated into the rat ES cells obtained in
Example 1 and the cells were plated on 15 cm 2.times. dense neoR
MEFs in 2i+10 uM ROCKi. The transformed rat ES cells were cultured,
selected, and maintained as described in Example 1.
As shown in Table 23, 288 colonies were screened and 8 targeted
clones were obtained. The targeting efficiency was 2.78%. 3 clones
were injected into a host embryo at a blastocyst stage as described
herein in Example 2 to produce chimeric rats (F0). Moreover, one
biallelic targeted clone was produced providing a biallelic
efficiency of 0.35%.
3.2.(b)(iii). Targeting ApoE in Rats with a Large Targeting Vector
(LTVEC) in Combination with Zinc Finger Nucleases
The LTVEC employed in Example 3.2.(b)(ii) was used in combination
with zinc finger nucleases to target the rat ApoE locus. Table 16
provides a summary of the genomic organization of the rat ApoE
locus and the positions shown were taken from build 5.0 of the
Reference Sequence of the rat genome (ENSMBL).
FIG. 23 provides a schematic of the rat ApoE locus and denotes with
grey bars the cutting site for ZFN1 and ZFN2. The cutting site for
ZFN1 is in t exon 3 and the cutting site for ZNF2 is in intron 3.
The exact position of the both ZFN sites is set forth in Table 16.
The 5' and 3' homology arms (45 kb and 23 kb, respectively) are
denoted by the dark grey boxes. Exon 1 of the ApoE gene is
non-coding and is shown as an open box closest to the 5' homology
arm. The three introns of the ApoE gene are denoted as lines. Exons
2 and 3 comprise coding regions and are shown as stippled grey
boxes. Exon 4 contains both coding and non-coding sequences as
denoted by the stippled grey shading and the open box.
The LTVEC employed was the same as that in Example 3.2(b)(ii) and
shown in FIG. 22. The ZFNs were introduced as two expression
plasmids, one for each half of the ZFN pair. 20 ug of the plasmid
for ZFN 1 and 20 ug of the plasmid for ZFN2 was used. ZFNs were
purchased from Sigma. The expression of each ZFN was driven by the
CMV promoter.
The targeting vector was electroporated into the rat ES cells
obtained in Example 1 and the cells were plated on 15 cm 2.times.
dense neoR MEFs in 2i+10 uM ROCKi. The transformed rat ES cells
were cultured, selected, and maintained as described in Example
1.
As shown in Table 23, 288 colonies were screened and 16 targeted
clones were obtained. The targeting efficiency was 5.56%. One clone
was injected into blastocysts as described herein in Example 2.
Moreover, the employment of ZFN1 and ZFN2 produced one biallelic
targeted clone, with an efficiency of 0.35%.
3.3(a): Targeting of the Rat Interleukin-2 Receptor Gamma
(IL2r-.gamma.) Locus
The rat Interleukin-2 receptor gamma (IL2r-.gamma.) locus was
targeted to disrupt IL2r-.gamma. function. IL2r-.gamma. plays an
important role for signaling by IL-2, IL-4, IL-7, IL-9, IL-15,
IL-21 and mutations in IL2r-.gamma. are associated with severe
defects in T, B and NK cell development.
Targeting of the IL2r-.gamma. locus was done using a targeting
vector comprising an eGFP-hUb-neo cassette flanked with a 5' and 3'
homology arms homologous to the IL2r-.gamma. locus. FIG. 26 depicts
the genomic structure of the rat IL2r-.gamma. locus in which the
IL2r-.gamma. locus has been disrupted by a 3.2 kb deletion. The
targeted IL2r-.gamma. locus also comprised an eGFP gene and a
self-deleting cassette containing Crei operably linked to a mouse
Protaminel promoter and a drug selection cassette comprising a hUb
promoter operably linked to a neomycin resistance gene.
Targeting efficiency at the IL2r-.gamma. locus was determined and
shown in Table 17. Linearized vector was electroporated into DA.2B
rat ESCs, and transfected colonies were cultured using standard
techniques. Individual colonies were picked and screened using a
Loss of Allele (LOA) assay.
TABLE-US-00017 TABLE 17 rat IL2r-.gamma. Targeting Efficiency
Colonies Targeting Cell line Vector picked Targeted efficiency (%)
DA.2B II2rg-floxed neo 136 1 0.7 DA.2B II2rg-mSDC 96 4 4.2
Chimera production and germline transmission using
IL2r-.gamma.-targeted rat ESC clones was performed.
IL2r-.gamma.-targeted rat ESC clones were microinjected into SD
blastocysts, which were then transferred to pseudopregnant SD
recipient females, using standard techniques. Chimeras were
identified by coat color; male F0 chimeras were bred to SD females.
Germline F1 pups were genotyped for the presence of the targeted
IL2r-.gamma. allele (Table 18).
TABLE-US-00018 TABLE 18 Microinjection Results Exp Clone pups
Chimeras 1 Il2rg-AA1 5 2 (90, 70) 2 Il2rg-AA1 10 3 (90, 90, 80)
The phenotype of Il2rg.sup.-/Y chimera #3 was further studied. The
peripheral blood mononuclear cells (PBMCs) were stained with
antibodies that recognize antigens in several lymphoid lineages.
GFP-positive PBMCs were detected from 2 of the chimeras. Moreover,
the GFP+ cells were negative for the T-cell marker CD3, and were
mostly negative for the B-cell marker B220 and the NK cell marker
CD161a. See, FIG. 30. The small double-positive populations are
consistent with the published Il2rg knockout phenotype in mice.
These data were obtained from a chimeric rat, which contains IL2
receptor gamma-positive cells, and this may complicate the analysis
of the phenotype.
3.3(b): Targeted Modification of the Rat Interleukin-2 Receptor
Gamma (IL2r-.gamma.) Locus
The rat Interleukin-2 receptor gamma (IL2r-.gamma.) locus was
targeted to disrupt the IL2r-.gamma. function in rats. FIG. 26
shows the genomic structure of the rat Il2rg locus and the
targeting vector introduced into the locus. eGFP was chosen as a
reporter so that the immunophenotype of the genetically modified
rats could be examined using FACS. The self-deleting cassette
(hUb-Neo; Prm1-Cre) was used to delete the drug section cassette
and the Cre gene specifically in male germ cells of the F0 rat.
Additionally, the targeting vector was designed to delete the
entire coding region (about 3.2 kb) of the rat Il2rg gene.
The size of the deletion in rat ESCs was confirmed by PCR using
primers specific to the rat Il2rg locus. Upon microinjection of the
targeted clones into host embryos at a blastocyst stage, high
percentage of chimeras were obtained. Those chimeras have been set
up for breeding. To determine if the targeting worked as expected,
the peripheral blood from the chimeras were collected prior to
breeding, and the phenotype of the immune cells in the peripheral
blood was analyzed via FACS. As shown in FIG. 30, GFP-positive
cells were detected in the peripheral blood in 2 of the 3 chimeras
examined (upper right panel), and the chimeric rats contained less
than 1% of T cells, less than 1% of B cells, and less than 1% of
NK-cells, which are positive for GFP (i.e., Il2rg KO cells).
3.4(a). Targeting the Rag2 Locus in Rats with a Large Targeting
Vector (LTVEC)
Table 19 provides a summary of the genomic organization of the rat
Rag2 locus and the positions shown were taken from build 5.0 of the
Reference Sequence of the rat genome (ENSMBL). Rag2 is on
chromosome 3 on the (+) strand.
TABLE-US-00019 TABLE 19 Genomic organization summary of the rat
Rag2 locus. Feature Start End length Notes Exon 1 97,851,317
97,851,448 132 Exon 2 97,854,635 97,854,693 59 Exon 3 97,858,260
97,859,615 1,356 contains entire coding sequence ATG 97,856,286
97,856,288 3 start codon TGA 97,857,867 97,857,869 3 stop codon
Rag2 97,856,289 97,859,784 3,496 deletion
FIG. 27 provides a schematic of the rat Rag2 locus and a large
targeting vector (LTVEC). The upper schematic of FIG. 27 shows the
genomic organization of the rat ApoE locus and the genomic regions
corresponding to the 5' and 3' homology arms (48 Kb and 15 Kb,
respectively; dark grey boxes). Rag2 comprises a single exon
denoted by the stippled grey shading.
The lower schematic in FIG. 27 is the LTVEC. The 5' and 3' homology
arms (48 kb and 15 kb, respectively) are denoted by the dark grey
boxes. The LTVEC comprises a reporter gene (lacZ) and a
self-deleting cassette flanked by loxP sites (open arrows). The
self-deleting cassette comprises a mouse Prm1 promoter operably
linked to the Crei gene and a drug selection cassette comprising a
human ubiquitin promoter operably linked to a neomycin resistance
gene. The Crei comprises two exons encoding the Cre recombinase are
separated by an intron (Crei) to prevent its expression in a
prokaryotic cell. See, for example, U.S. Pat. No. 8,697,851 and
U.S. Application Publication 2013-0312129, which describe the
self-deleting cassette in detail and are hereby incorporated by
reference in their entirety. By employing a mouse Prm1 promoter,
the self-deleting cassette can be deleted specifically in male germ
cells of F0 rats.
The LTVEC was electroporated into the rat ES cells obtained in
Example 1 and the cells were plated on 15 cm 2.times. dense neoR
MEFs in 2i+10 uM ROCKi. The transformed rat ES cells were cultured
and maintained as described in Example 1. Colonies are screened as
described elsewhere herein and targeted clones are obtained. The
targeted clones are then injected into a host embryo as described
elsewhere herein to produce an F0 rat.
3.4.(b). Targeting the Rag1 and the Rag 2 Locus in Rats
FIG. 28 provides the genomic structure of the rat Rag1/Rag2 locus.
CDS denotes the coding sequence and grey boxes represent exons.
Rag2 is on the "plus" strand with transcription to the right. Rag1
is on the "minus" strand with transcription to the left.
Mbp=million base pairs.
Table 20 provides a summary of the genomic organization of the rat
Rag2 and Rag1 locus and the positions shown were taken from build
5.0 of the Reference Sequence of the rat genome (ENSMBL). Rag1 is
on chromosome 3 on the (-) strand.
TABLE-US-00020 TABLE 20 Genomic organization summary of the rat
Rag1 locus. Feature Start End length Notes Exon 1 97,877,145
97,877,066 80 Exon 2 97,872,503 97,866,047 6,457 contains entire
coding sequence ATG 97,872,489 97,872,487 3 start codon TAA
97,869,369 97,869,367 3 stop codon Rag1-2 97,856,289 97,872,486
16,198 deletion
FIG. 29 provides a schematic of the rat Rag2 and Rag1 locus and a
large targeting vector (LTVEC). The upper schematic of FIG. 29
shows the genomic organization of the Rag1 and Rag2 loci and the
genomic regions corresponding to the 5' and 3' homology arms (48 kb
and 84 kb, respectively; dark grey boxes). Rag2 and Rag 1 each
comprises a single exon denoted by the stippled grey shading. The
lower schematic in FIG. 29 is the LTVEC. The 5' and 3' homology
arms (48 kb and 84 kb, respectively) are denoted by the dark grey
boxes. The LTVEC comprises a reporter gene (lacZ) and a
self-deleting cassette flanked by loxP sites (open arrows). The
self-deleting cassette comprises a rat Prm1 promoter operably
linked to the Crei gene and a drug selection cassette comprising a
human ubiquitin promoter operably linked to a neomycin resistance
gene. The Crei comprises two exons encoding the Cre recombinase are
separated by an intron (Crei) to prevent its expression in a
prokaryotic cell. See, for example, U.S. Pat. No. 8,697,851 and
U.S. Application Publication 2013-0312129, which describes the
self-deleting cassette in detail and is hereby incorporated by
reference in their entirety. By employing a rat Prm1 promoter that
drives expression of Crei specifically in male germ cells, the
self-deleting cassette can be deleted from the male germ cells of
F0 rats.
The LTVEC was electroporated into the rat ES cells obtained in
Example 1 and the cells were plated on 15 cm 2.times. dense neoR
MEFs in 2i+10 uM ROCKi. The transformed rat ES cells were cultured
and maintained as described in Example 1.
Colonies are screened as described elsewhere herein and targeted
clones are obtained. The targeted clones are then injected into a
host embryo as described elsewhere herein to produce an F0 rat.
Example 4
Humanization
4.1. Humanization of Rat Genomic Loci
Humanization of rat genomic loci is carried out employing the rat
ES cells described herein, which are capable of sustaining their
pluripotency following one or more electroporations in vitro, and
are capable of transmitting the targeted genetic modifications to
subsequent generations. In addition, in order to circumvent the
limitations of plasmids in accommodating a large genomic DNA
fragment, and to overcome the low efficiency of introducing a
targeted genetic modification into an endogenous locus in rat ES
cells, one or more targeted genetic modifications are carried out
in bacteria, e.g., E. coli, by utilizing bacterial homologous
recombination (BHR) and employing a large targeting vector (LTVEC).
The LTVEC described herein, for example, includes a large fragment
of an endogenous rat genomic sequence with one or more
modifications or comprises an exogenous nucleic acid (e.g., a
homologous or orthologous human nucleic acid) flanked with rat
homology arms complementary to specific genomic regions.
4.2. Humanization of Rat Immunoglobulin Loci
Humanization of an endogenous rat immunoglobulin heavy chain locus
is carried out by removing one or more endogenous rat
immunoglobulin heavy chain nucleic acid sequences (e.g., one or
more endogenous V.sub.H gene segments, one or more human D gene
segments, and one or more human J.sub.H gene segments); and
introducing into the modified immunoglobulin locus a targeting
vector, e.g., a large targeting vector (LTVEC) comprising: (i) one
or more unrearranged human variable region nucleic acid sequences
(e.g., one or more human V.sub.H gene segments, one or more human D
gene segments, and one or more human J.sub.H gene segments), or one
or more rearranged human variable region nucleic acid sequences
(e.g., one or more human rearranged V-D-J gene segments); (ii) a
selection cassette (e.g., neomycin resistance gene flanked with
loxP sites); and (iii) 5' and 3' rat homology arms.
Briefly, one or more endogenous rat immunoglobulin heavy chain
variable region gene segments (i.e., one or more V.sub.H gene
segments, one or more human D gene segments, and one or more human
J.sub.H gene segments) in a rat BAC clone are removed or
inactivated by targeting the endogenous rat immunoglobulin heavy
chain locus with a selection cassette flanked by rat homology arms.
More specifically, a targeting vector is constructed to contain a
selection cassette (e.g., a neomycin resistance gene flanked with
loxP sites) flanked with 5' and 3' rat homology arms that are
complementary to target rat genomic sequences (e.g., upstream and
downstream rat genomic DNA sequences encompassing one or more rat
V.sub.H gene segments, one or more human D gene segments, and one
or more human J.sub.H gene segments).
Next, bacterial cells containing a large rat genomic DNA fragment
encompassing a rat immunoglobulin heavy chain locus are selected
and introduced with a plasmid (e.g., pABG) encoding a recombinase
operably linked to a transiently inducible promoter. The targeting
vector constructed above is then introduced into the
recombination-competent bacterial cells. Following electroporation,
the bacterial cells are treated with an inducer (e.g., arabinoside)
to initiate homologous recombination between the targeting vector
and the target rat genomic sequence in the BAC clone. Transformed
cells are plated at a high density and subjected to drug selection
to find colonies that are drug-resistant. Drug-resistant colonies
are picked and screened for the targeted modification.
In order to facilitate identification of the targeted genetic
modification, a high-throughput quantitative assay, namely,
modification of allele (MOA) assay, is employed, which allows a
large-scale screening of a modified allele(s) in a parental
chromosome following a genetic modification. The MOA assay can be
carried out via various analytical techniques, including, but not
limited to, a quantitative PCR, e.g., a real-time PCR (qPCR). For
example, the real-time PCR comprises a first primer set that
recognizes the target locus and a second primer set that recognizes
a non-targeted reference locus. In addition, the primer set can
comprise a fluorescent probe that recognizes the amplified
sequence. Alternatively, the quantitative assay can be carried out
via a variety of analytical techniques, including, but not limited
to, fluorescence-mediated in situ hybridization (FISH), comparative
genomic hybridization, isothermic DNA amplification, quantitative
hybridization to an immobilized probe(s), Invader Probes.RTM., MMP
Assays.RTM., TaqMan.RTM. Molecular Beacon, and Eclipse.TM. probe
technology. (See, for example, US2005/0144655, incorporated by
reference herein in its entirety).
The bacterial cells comprising the modified rat BAC clone, i.e., a
BAC clone containing a rat genomic DNA sequence wherein one or more
endogenous heavy chain variable region gene segments (V.sub.H, D,
and/or J.sub.H gene segments) have been deleted or inactivated, are
then electroporated with a large targeting vector (LTVEC)
comprising: (i) one or more unrearranged human variable region
nucleic acid sequences (e.g., one or more unrearranged human
V.sub.H gene segments, one or more human D gene segments, and one
or more human J.sub.H gene segments), or one or more rearranged
human variable region nucleic acid sequences (e.g., one or more
rearranged human V-D-J gene segments).
Initiation of homologous recombination in the bacterial cells and
the selection of positive clones are performed as described above.
The unrearranged or rearranged human immunoglobulin heavy chain
variable region nucleic acid sequences, when targeted into the
endogenous immunoglobulin heavy chain locus, become operably linked
to an endogenous rat immunoglobulin heavy chain constant region
nucleic acid sequence. Alternatively, endogenous rat heavy chain
constant region locus can be inactivated, for example, by deleting
one or more rat heavy chain constant region gene segments (CH) from
the endogenous heavy chain constant region locus, and can be
replaced with a human heavy chain constant region nucleic acid
sequence.
Likewise, humanization of an endogenous rat immunoglobulin .kappa.
or .lamda. light chain locus is carried out by removing one or more
endogenous rat immunoglobulin .kappa. and/or .lamda. light chain
variable region nucleic acid sequences (e.g., one or more
endogenous rat V.sub..kappa. gene segments and one or more
endogenous rat J.sub..kappa. gene segments); and targeting the
modified immunoglobulin light chain locus with a targeting vector,
e.g., a large targeting vector (LTVEC), comprising: (i) one or more
unrearranged human immunoglobulin light chain variable region
nucleic acid sequences (e.g., one or more human V.sub..kappa. gene
segments and one or more human J.sub..kappa. gene segments), or one
or more rearranged human variable region nucleic acid sequences
(e.g., one or more human rearranged V.sub..kappa.-J.sub..kappa.
gene segments); (ii) a selection cassette (e.g., neomycin
resistance gene flanked with loxP sites); and (iii) 5' and 3' rat
homology arms.
The unrearranged or rearranged human immunoglobulin light chain
variable region nucleic acid sequences, when targeted into the
endogenous immunoglobulin light chain locus, become operably linked
to the endogenous rat immunoglobulin light chain constant region
nucleic acid sequence.
The LTVEC so produced in the bacterial cells comprises, for
example, an insert nucleic acid that contains a humanized rat
immunoglobulin heavy chain or light chain locus in which one or
more endogenous rat heavy or light chain variable region gene
segments have been replaced with one or more human heavy or light
chain variable region gene segments; and rat homologous arms (e.g.,
ranging from 5 kb to 150 kb) complementary to specific genomic
target sequences. The LTVEC comprising the genetic modification
described above is then linearized and electroporated into the rat
ES cells. Electroporated rat ES cells are plated at a high density
to select drug-resistant ES cells comprising the targeting vector.
The drug selection process removes the majority of the plated cells
(.about.99%), leaving behind individual colonies, each of which is
a clone derived from a single cell. Of the remaining cells, most
cells (.about.80-100%) contain the targeting vector integrated at a
random location in the genome. Therefore, the colonies are picked
and genotyped individually in order to identify rat ES cells
comprising the targeting vector at the correct genomic location
(e.g., using the modification of allele (MOA) assay described
above).
In order to increase the efficiency of the targeted genetic
modification, the rat ES cells are electroporated with expression
vectors (or mRNA) that express ZFNs 1 and 2 (or TALENs 1 and 2)
together with the LTVEC. The targeting vector's homology arms lie
outside the ZFN target site, therefore, the targeting vector is not
cleaved by the ZFNs. The double strand break produced by the ZFNs
stimulates homology-directed repair (HDR), which otherwise accounts
for a very small percentage of repairs occurred normally in
mammalian cells (compared to non-homologous end-joining; NHEJ).
Alternatively, expression vectors containing a type II
CRISPR-associated nuclease (e.g., Cas9), a guide RNA (including
CRISPR-RNA (cr-RNA) and trans-activating CRISPR RNA (tracrRNA)), as
described herein, can be introduced into the bacterial cells
together with the LTVEC to increase the efficiency of homologous
recombination at the target genomic locus. Electroporated cells are
plated at a high density and subjected to drug selection to find
colonies that are drug-resistant. Drug-resistant colonies are
picked and screened for the targeted modification using the
modification of allele (MOA) assay as described herein. Following
these procedures, improvement in the targeting efficiency can be
achieved. For example, the amount of improvement can be small
(e.g., improve from 10% to 15%) or large (e.g., improve from 10% to
80%).
The selected rat ES cells comprising the targeted genetic
modification are then introduced into a host rat embryo, for
example, a pre-morula stage or blastocyst stage rat embryo, and
implanted in the uterus of a surrogate mother to generate a founder
rat (F0 rat). Subsequently, the founder rat is bred to a wild-type
rat to create F1 progeny heterozygous for the genetic modification.
Mating of the heterozygous F1 rat can produce progeny homozygous
for the genetic modification.
4.3(a). Replacing Rat IL2rg with Human IL2 Receptor Gamma
Table 21 provides a summary of the genomic organization of the rat
Interleukin 2 receptor gamma locus and the positions shown were
taken from build 5.0 of the Reference Sequence of the rat genome
(ENSMBL). IL2rg is on chromosome X on the (-) strand.
TABLE-US-00021 TABLE 21 Summary of the genomic organization of the
rat Il2rg locus Feature Start End length Notes Exon 1 72,021,388
72,021,516 129 contains ATG ATG 72,017,500 72,017,502 3 start codon
Exon2 72,021,007 72,021,160 154 ZFN1a binding site 72,021,014
72,021,028 15 CAGGCCCTGAACCGC (SEQ ID NO: 17) ZFN1 cutting site
72,021,008 72,021,013 6 TTCTGG (SEQ ID NO: 18) ZFN1b binding site
72,020,993 72,021,007 15 GATTACCTGCGCTGGG (SEQ ID NO: 20) Exon3
72,020,606 72,020,790 185 Exon4 72,020,274 72,020,413 140 Exon5
72,019,662 72,019,824 163 Exon6 72,019,101 72,019,197 97 Exon7
72,018,844 72,018,910 67 Exon8 72,017,856 72,018,506 651 contains
TGA TGA 72,018,321 72,018,323 3 stop codon Il2rg deletion
72,018,323 72,021,502 3,180
The lower schematic in FIG. 26 is the targeting vector for the
IL2rg 3.2 kb deletion. The targeting vector comprises a reporter
gene (eGFP) operably linked to the endogenous promoter and a
self-deleting cassette flanked by loxP sites (open arrows). The
self-deleting cassette comprises the Crei gene operably linked to a
mouse Prm1 promoter and a selection cassette comprising a neomycin
resistance gene operably linked to a human ubiquitin promoter.
The Crei gene comprises two exons encoding a Cre recombinase, which
are separated by an intron (Crei) to prevent its expression in a
prokaryotic cell. See, See, for example, U.S. Pat. No. 8,697,851
and U.S. Application Publication 2013-0312129, which describe the
self-deleting cassette in detail and are hereby incorporated by
reference in their entirety. By employing the mouse Prm1 promoter
the Cre expression cassette and the drug selection cassette can be
deleted specifically in male germ cells of F0 rats. The targeting
vector was electroporated into the rat ES cells obtained in Example
1 and the cells were plated on 15 cm 2.times. dense
neomycin-resistant MEFs in 2i+10 uM ROCKi. The transformed rat ES
cells were cultured, selected, and maintained as described in
Example 1.
As shown in Table 23, 168 colonies were screened and 6 targeted
clones were obtained. The targeting efficiency was 3.57%.
Clones are injected into blastocysts as described herein in Example
1. Clones producing F0 rats are obtained and F0 rats that transmit
the targeted modification through the germline are obtained.
Example 4.3(b)
Replacing Rat IL2rg Ecto-Domain with Human IL2rg Ecto-Domain
The full-length humanization of IL 2 receptor gamma is useful
because rats having this modified locus will produce human Il2rg;
and this would allow for the detection of human Il2rg in rats with
antibodies specific to human Il2rg.
The ecto-humanization (i.e., replacing the rat ecto-domain of Il2rg
with the human ecto-domain of Il2rg) will result in an Il2rg
polypeptide that will bind the human ligands for Il2rg, but because
the cytoplasmic domain is still rat, it ecto-humanized form of
Il2rg will also interact with the rat signaling machinery. FIG. 33
provides a sequence alignment of the human IL-2rg protein (SEQ ID
NO: 20; NP_000197.1); the rat IL-2rg protein (SEQ ID NO: 21;
NP_543165.1); and the chimeric IL-2rg protein (SEQ ID NO: 22)
comprising the human ecto-domain of IL-2rg fused to the remainder
of the rat IL-2rg protein. The junction between the human and rat
IL-2rg is noted by the vertical line.
Table 22 provides a summary of the genomic organization of the rat
Interleukin 2 receptor gamma locus and the positions shown were
taken from build 5.0 of the Reference Sequence of the rat genome
(ENSMBL). IL2rg is on chromosome X on the (-) strand. Further noted
is the position of the ecto-domain of IL2rg.
TABLE-US-00022 TABLE 22 Summary of the genomic organization of the
rat Il2rg locus Feature Start End length Notes Exon 1 71,111,444
71,111,543 100 contains ATG ATG 71,111,537 71,111,539 3 start codon
Exon2 71,110,897 71,111,050 154 Exon3 71,110,504 71,110,688 185
Exon4 71,110,156 71,110,295 140 Exon5 71,109,228 71,109,390 163
Exon6 71,108,599 71,108,645 47 contains transmembrane domain Exon7
71,108,277 71,108,346 70 Exon8 71,107,404 71,107,921 518 contains
TGA TGA 71,108,736 71,108,738 3 stop codon full-length 71,107,404
71,111,539 4,136 (ATG to TGA humaniza- plus 3' poly-A) tion: ecto-
71,108,679 71,111,539 2,861 (ATG to beginning humaniza- of
transmembrane tion domain)
A plasmid targeting vectors were constructed to replace the rat
ecto-domain of the interleukin 2 receptor gamma coding region with
the human ecto domain as shown in FIG. 31. The targeting vector was
electroporated into the rat ES cells obtained in Example 1 and the
cells were plated on 15 cm 2.times. dense neomycin-resistant MEFs
in 2i+10 uM ROCKi. The transformed rat ES cells were cultured,
selected, and maintained as described in Example 1.
As shown in Table 23, 192 colonies were screened and 13 targeted
clones were obtained. The targeting efficiency was 6.77%.
Clones are injected into blastocysts as described herein in Example
1. Clones producing F0 rats are obtained and F0 rats that transmit
the targeted modification through the germline are obtained.
Example 5
Summary
Table 23. Summary of rat targeting with various vector types and
nuclease agents discussed in Examples 3 and 4.
TABLE-US-00023 TABLE 23 Targeting Summary Clones Clones
transmitting Example Colonies Targeted Targeting Biallelic
Biallelic Clones producing- through # Locus Vector screened Clones
efficiency targeted efficiency Injected chi- meras germline Notes
3.2(a)(ii) ApoE plasmid 384 23 5.99% 3 3 2 3.2(a)(iii) ApoE +
plasmid 384 290 75.52% 8 2.08% 2 2 1 These 2 clones are ZFN
biallelic targeted 3.3(a) Il2rg plasmid 232 5 2.16% 6 5 3.2(b)(ii)
ApoE LTVEC 288 8 2.78% 1 0.35% 3 1 LTVEC 3.2(b)(iii) ApoE LTVEC 288
16 5.56% 1 0.35% 1 N/A This clone is LTVEC + biallelic targeted ZFN
4.3(a) Il2rg plasmid 168 6 3.57% replaces entire rat Humaniza-
Il2rg with human tion 1 Il2rg 4.3(b) Il2rg plasmid 192 13 6.77% 2
N/A replaces rat Il2rg Humaniza- ecto-domain with tion 2 human
Il2rg ecto- domain 3.4(a) Rag2 LTVEC 270 N/A Predicted 5.7 KB
deletion 3.4(b) Rag1-2 LTVEC 256 N/A Predicted 16.2 kb deletion
All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference. Unless otherwise apparent from the context of any
embodiment, aspect, step or feature of the invention can be used in
combination with any other. Reference to a range includes any
integers within the range, any subrange within the range. Reference
to multiple ranges includes composites of such ranges.
SEQUENCE LISTINGS
1
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 23 <210>
SEQ ID NO 1 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: a genomic target sequence that is
linked to a guide RNA (gRNA) <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 <223> OTHER
INFORMATION: n = A,T,C or G <400> SEQUENCE: 1 gnnnnnnnnn
nnnnnnnnnn ngg 23 <210> SEQ ID NO 2 <211> LENGTH: 80
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: a guide RNA
(gRNA) <400> SEQUENCE: 2 guuuuagagc uagaaauagc aaguuaaaau
aaggcuaguc cguuaucaac uugaaaaagu 60 ggcaccgagu cggugcuuuu 80
<210> SEQ ID NO 3 <211> LENGTH: 42 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: a guide RNA (gRNA) <400>
SEQUENCE: 3 guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc cg 42
<210> SEQ ID NO 4 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: a crRNA <400> SEQUENCE: 4
guuuuagagc uagaaauagc aaguuaaaau 30 <210> SEQ ID NO 5
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: a crRNA <400> SEQUENCE: 5 guuuuagagc uagaaauagc
aaguuaaaau aag 33 <210> SEQ ID NO 6 <211> LENGTH: 26
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: a crRNA
<400> SEQUENCE: 6 gaguccgagc agaagaagaa guuuua 26 <210>
SEQ ID NO 7 <211> LENGTH: 12 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: a tracrRNA <400> SEQUENCE: 7
aaggcuaguc cg 12 <210> SEQ ID NO 8 <211> LENGTH: 50
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: a tracrRNA
<400> SEQUENCE: 8 aaggcuaguc cguuaucaac uugaaaaagu ggcaccgagu
cggugcuuuu 50 <210> SEQ ID NO 9 <211> LENGTH: 203
<212> TYPE: PRT <213> ORGANISM: Mus Musculus
<400> SEQUENCE: 9 Met Lys Val Leu Ala Ala Gly Ile Val Pro Leu
Leu Leu Leu Val Leu 1 5 10 15 His Trp Lys His Gly Ala Gly Ser Pro
Leu Pro Ile Thr Pro Val Asn 20 25 30 Ala Thr Cys Ala Ile Arg His
Pro Cys His Gly Asn Leu Met Asn Gln 35 40 45 Ile Lys Asn Gln Leu
Ala Gln Leu Asn Gly Ser Ala Asn Ala Leu Phe 50 55 60 Ile Ser Tyr
Tyr Thr Ala Gln Gly Glu Pro Phe Pro Asn Asn Val Glu 65 70 75 80 Lys
Leu Cys Ala Pro Asn Met Thr Asp Phe Pro Ser Phe His Gly Asn 85 90
95 Gly Thr Glu Lys Thr Lys Leu Val Glu Leu Tyr Arg Met Val Ala Tyr
100 105 110 Leu Ser Ala Ser Leu Thr Asn Ile Thr Arg Asp Gln Lys Val
Leu Asn 115 120 125 Pro Thr Ala Val Ser Leu Gln Val Lys Leu Asn Ala
Thr Ile Asp Val 130 135 140 Met Arg Gly Leu Leu Ser Asn Val Leu Cys
Arg Leu Cys Asn Lys Tyr 145 150 155 160 Arg Val Gly His Val Asp Val
Pro Pro Val Pro Asp His Ser Asp Lys 165 170 175 Glu Ala Phe Gln Arg
Lys Lys Leu Gly Cys Gln Leu Leu Gly Thr Tyr 180 185 190 Lys Gln Val
Ile Ser Val Val Val Gln Ala Phe 195 200 <210> SEQ ID NO 10
<211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: ZFN1a binding site <400> SEQUENCE: 10 caggccctga
accgc 15 <210> SEQ ID NO 11 <211> LENGTH: 6 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: ZFN1 cutting site
<400> SEQUENCE: 11 ttctgg 6 <210> SEQ ID NO 12
<211> LENGTH: 16 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: ZFN1b binding site <400> SEQUENCE: 12 gattacctgc
gctggg 16 <210> SEQ ID NO 13 <211> LENGTH: 15
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: ZF21a binding
site <400> SEQUENCE: 13 ttcaccctcc gcacc 15 <210> SEQ
ID NO 14 <211> LENGTH: 7 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: ZFN2 cutting site <400> SEQUENCE: 14
tgctgag 7 <210> SEQ ID NO 15 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: ZF21b binding
site <400> SEQUENCE: 15 tatccagatc caggggtt 18 <210>
SEQ ID NO 16 <211> LENGTH: 9 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: conserved domain of a family of
homing nucleases <400> SEQUENCE: 16 Leu Ala Gly Leu Ile Asp
Ala Asp Gly 1 5 <210> SEQ ID NO 17 <211> LENGTH: 15
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: ZFN1a binding site <400>
SEQUENCE: 17 caggccctga accgc 15 <210> SEQ ID NO 18
<211> LENGTH: 6 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: ZFN1 cutting site <400> SEQUENCE: 18 ttctgg 6
<210> SEQ ID NO 19 <211> LENGTH: 16 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: ZFN1b binding site <400>
SEQUENCE: 19 gattacctgc gctggg 16 <210> SEQ ID NO 20
<211> LENGTH: 369 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 20 Met Leu Lys Pro Ser Leu Pro
Phe Thr Ser Leu Leu Phe Leu Gln Leu 1 5 10 15 Pro Leu Leu Gly Val
Gly Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly 20 25 30 Asn Glu Asp
Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp 35 40 45 Ser
Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val 50 55
60 Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro
65 70 75 80 Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser
Asp Asn 85 90 95 Asp Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser
Glu Glu Ile Thr 100 105 110 Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile
His Leu Tyr Gln Thr Phe 115 120 125 Val Val Gln Leu Gln Asp Pro Arg
Glu Pro Arg Arg Gln Ala Thr Gln 130 135 140 Met Leu Lys Leu Gln Asn
Leu Val Ile Pro Trp Ala Pro Glu Asn Leu 145 150 155 160 Thr Leu His
Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn 165 170 175 Arg
Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp 180 185
190 Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys Phe
195 200 205 Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg
Val Arg 210 215 220 Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala Gln His
Trp Ser Glu Trp 225 230 235 240 Ser His Pro Ile His Trp Gly Ser Asn
Thr Ser Lys Glu Asn Pro Phe 245 250 255 Leu Phe Ala Leu Glu Ala Val
Val Ile Ser Val Gly Ser Met Gly Leu 260 265 270 Ile Ile Ser Leu Leu
Cys Val Tyr Phe Trp Leu Glu Arg Thr Met Pro 275 280 285 Arg Ile Pro
Thr Leu Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr His 290 295 300 Gly
Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly Leu Ala Glu Ser 305 310
315 320 Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu Val Ser Glu Ile
Pro 325 330 335 Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly Ala Ser
Pro Cys Asn 340 345 350 Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr
Thr Leu Lys Pro Glu 355 360 365 Thr <210> SEQ ID NO 21
<211> LENGTH: 368 <212> TYPE: PRT <213> ORGANISM:
Rattus norvegicus <400> SEQUENCE: 21 Met Leu Lys Pro Leu Leu
Pro Ser Arg Ser Phe Leu Leu Leu Gln Leu 1 5 10 15 Leu Leu Leu Arg
Val Gly Trp Ser Ser Lys Val Leu Met Ser Ser Gly 20 25 30 Asn Glu
Asp Thr Lys Ser Asp Leu Leu Leu Thr Ser Met Asp Leu Lys 35 40 45
His Leu Ser Val Pro Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val 50
55 60 Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu
Pro 65 70 75 80 Gln Pro Thr Asn Leu Thr Met His Tyr Arg Tyr Lys Gly
Ser Asp Asn 85 90 95 Asn Thr Phe Gln Glu Cys Ser His Tyr Leu Phe
Ser Lys Glu Ile Thr 100 105 110 Ser Gly Cys Gln Ile Gln Lys Glu Asp
Ile Gln Leu Tyr Gln Thr Phe 115 120 125 Val Val Gln Leu Gln Asp Pro
Gln Lys Pro Gln Arg Arg Ala Glu Gln 130 135 140 Lys Leu Asn Leu Gln
Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu 145 150 155 160 Thr Leu
Tyr Asn Leu Ser Glu Ser Gln Val Glu Leu Arg Trp Lys Ser 165 170 175
Arg Tyr Ile Glu Arg Cys Leu Gln Tyr Leu Val Gln Tyr Arg Ser Asn 180
185 190 Arg Asp Arg Ser Trp Thr Glu Gln Ile Val Asp His Glu Pro Arg
Phe 195 200 205 Ser Leu Pro Ser Val Asp Glu Gln Lys Leu Tyr Thr Phe
Arg Val Arg 210 215 220 Ser Arg Phe Asn Pro Ile Cys Gly Ser Thr Gln
Gln Trp Ser Lys Trp 225 230 235 240 Ser Gln Pro Ile His Trp Gly Ser
His Thr Ala Glu Glu Asn Pro Ser 245 250 255 Leu Phe Ala Leu Glu Ala
Val Leu Ile Pro Val Gly Thr Met Gly Leu 260 265 270 Ile Ile Thr Leu
Ile Phe Val Tyr Cys Trp Leu Glu Arg Met Pro Arg 275 280 285 Ile Pro
Ala Ile Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr His Gly 290 295 300
Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly Leu Thr Glu Ser Leu 305
310 315 320 Gln Pro Asp Tyr Ser Glu Arg Phe Cys His Val Ser Glu Ile
Pro Pro 325 330 335 Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly Gly Ser
Pro Cys Ser Leu 340 345 350 His Ser Pro Tyr Trp Pro Pro Pro Cys Tyr
Ser Leu Lys Pro Glu Ala 355 360 365 <210> SEQ ID NO 22
<211> LENGTH: 368 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: chimeric IL-2 receptor gamma comprising the rat IL-2
receptor gamma protein having the ecto domain of IL-2 gamma
receptor from human <400> SEQUENCE: 22 Met Leu Lys Pro Ser
Leu Pro Phe Thr Ser Leu Leu Phe Leu Gln Leu 1 5 10 15 Pro Leu Leu
Gly Val Gly Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly 20 25 30 Asn
Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp 35 40
45 Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val
50 55 60 Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser
Glu Pro 65 70 75 80 Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys
Asn Ser Asp Asn 85 90 95 Asp Lys Val Gln Lys Cys Ser His Tyr Leu
Phe Ser Glu Glu Ile Thr 100 105 110 Ser Gly Cys Gln Leu Gln Lys Lys
Glu Ile His Leu Tyr Gln Thr Phe 115 120 125 Val Val Gln Leu Gln Asp
Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln 130 135 140 Met Leu Lys Leu
Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu 145 150 155 160 Thr
Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn 165 170
175 Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp
180 185 190 Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His
Lys Phe 195 200 205 Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr
Phe Arg Val Arg 210 215 220 Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala
Gln His Trp Ser Glu Trp 225 230 235 240 Ser His Pro Ile His Trp Gly
Ser Asn Thr Ser Lys Glu Asn Pro Phe 245 250 255 Leu Phe Ala Leu Glu
Ala Val Leu Ile Pro Val Gly Thr Met Gly Leu 260 265 270 Ile Ile Thr
Leu Ile Phe Val Tyr Cys Trp Leu Glu Arg Met Pro Arg
275 280 285 Ile Pro Ala Ile Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr
His Gly 290 295 300 Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly Leu
Thr Glu Ser Leu 305 310 315 320 Gln Pro Asp Tyr Ser Glu Arg Phe Cys
His Val Ser Glu Ile Pro Pro 325 330 335 Lys Gly Gly Ala Leu Gly Glu
Gly Pro Gly Gly Ser Pro Cys Ser Leu 340 345 350 His Ser Pro Tyr Trp
Pro Pro Pro Cys Tyr Ser Leu Lys Pro Glu Ala 355 360 365 <210>
SEQ ID NO 23 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: a genomic target sequence that is
linked to a guide RNA (gRNA) <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (2)...(21) <223>
OTHER INFORMATION: n= A, T, C or G <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (0)...(0) <223>
OTHER INFORMATION: n can be from 1-20 nucleotides <400>
SEQUENCE: 23 gnnnnnnnnn nnnnnnnnnn ngg 23
* * * * *
References