U.S. patent application number 14/946224 was filed with the patent office on 2019-02-28 for genome editing in rats using zinc-finger nucleases.
The applicant listed for this patent is Sangamo BioSciences, Inc., Sigma-Aldrich Co. LLC. Invention is credited to Xiaoxia Cui, Aron M. Geurts, Fyodor Urnov.
Application Number | 20190062789 14/946224 |
Document ID | / |
Family ID | 42233523 |
Filed Date | 2019-02-28 |
![](/patent/app/20190062789/US20190062789A9-20190228-D00001.png)
![](/patent/app/20190062789/US20190062789A9-20190228-D00002.png)
![](/patent/app/20190062789/US20190062789A9-20190228-D00003.png)
![](/patent/app/20190062789/US20190062789A9-20190228-D00004.png)
![](/patent/app/20190062789/US20190062789A9-20190228-D00005.png)
![](/patent/app/20190062789/US20190062789A9-20190228-D00006.png)
![](/patent/app/20190062789/US20190062789A9-20190228-D00007.png)
![](/patent/app/20190062789/US20190062789A9-20190228-D00008.png)
![](/patent/app/20190062789/US20190062789A9-20190228-D00009.png)
![](/patent/app/20190062789/US20190062789A9-20190228-D00010.png)
![](/patent/app/20190062789/US20190062789A9-20190228-D00011.png)
View All Diagrams
United States Patent
Application |
20190062789 |
Kind Code |
A9 |
Cui; Xiaoxia ; et
al. |
February 28, 2019 |
GENOME EDITING IN RATS USING ZINC-FINGER NUCLEASES
Abstract
Disclosed herein are methods and compositions for genome editing
of one or more loci in a rat, using fusion proteins comprising a
zinc-finger protein and a cleavage domain or cleavage half-domain.
Polynucleotides encoding said fusion proteins are also provided, as
are cells comprising said polynucleotides and fusion proteins.
Inventors: |
Cui; Xiaoxia; (St. Louis,
MO) ; Geurts; Aron M.; (New Berlin, WI) ;
Urnov; Fyodor; (Richmond, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sigma-Aldrich Co. LLC
Sangamo BioSciences, Inc. |
St. Louis
Richmond |
MO
CA |
US
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20160068865 A1 |
March 10, 2016 |
|
|
Family ID: |
42233523 |
Appl. No.: |
14/946224 |
Filed: |
November 19, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12592852 |
Dec 3, 2009 |
9206404 |
|
|
14946224 |
|
|
|
|
61200985 |
Dec 4, 2008 |
|
|
|
61205970 |
Jan 26, 2009 |
|
|
|
61263904 |
Nov 24, 2009 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01K 67/0276 20130101;
C12N 15/1024 20130101; A01K 2267/03 20130101; A01K 2227/10
20130101; C12N 15/8509 20130101; C12N 2015/8527 20130101; A01K
2217/15 20130101; A01K 2267/0393 20130101; C12N 15/907 20130101;
C12N 2800/80 20130101; A01K 2217/054 20130101; A01K 67/0278
20130101; A01K 67/0275 20130101; C07K 2319/00 20130101; C07K
2319/81 20130101; A01K 2227/105 20130101; C12N 9/22 20130101 |
International
Class: |
C12N 15/90 20060101
C12N015/90; C12N 15/10 20060101 C12N015/10; C12N 15/85 20060101
C12N015/85 |
Claims
1. A method for modifying one or more endogenous cellular genes in
a rat cell, the method comprising: introducing, into the rat cell,
one or more polynucleotides encoding one or more zinc finger
nucleases (ZFNs) that bind to a target site in the one or more
genes under conditions such that the ZFN(s) is (are) expressed and
the one or more endogenous cellular genes are cleaved and
modified.
2. The method of claim 1, wherein the modification comprises
introducing an exogenous sequence into the genome of a rat cell by
homologous recombination stimulated by cleavage of the one or more
endogenous cellular genes.
3. The method of claim 2, wherein the exogenous sequence is
integrated physically into the genome.
4. The method of claim 2, wherein the exogenous sequence is
integrated into the genome by copying of the exogenous sequence
into the host cell genome via nucleic acid replication
processes.
5. The method of claim 4, wherein the nucleic acid replication
process comprises homology-directed repair of the double strand
break.
6. The method of claim 4, wherein the nucleic acid replication
process comprises non-homology dependent targeted integration.
7. The method of claim 1, wherein the modification results from
non-homologous end joining following cleavage.
8. The method of claim 7, wherein first and second zinc finger
nucleases cleave the genome at two sites and further wherein the
non-homologous end joining results in a deletion between the first
and second cleavage sites.
9. The method of claim 1, wherein the zinc finger nucleases
comprise a cleavage domain or cleavage half-domain of a Type IIS
restriction endonuclease.
10. A method for germline disruption of one or more target genes in
rat, the method comprising modifying one or more gene sequences in
the genome of one or more cells of a rat embryo the method
comprising: modifying one or more of the target genes in one or
more cells of a rat embryo according to the method of claim 1; and
allowing the rat embryo to develop, wherein that the modified gene
sequences are present in at least a portion of gametes of the
sexually mature rat.
11. The method of claim 10, wherein the modification comprises
integration of an exogenous sequence into the genome of a rat cell
by homologous recombination stimulated by cleavage of the one or
more endogenous cellular genes.
12. The method of claim 10, wherein the modification results from
non-homologous end joining following cleavage.
13. A method of creating one or more heritable mutant alleles in
rat loci of interest, the method comprising modifying one or more
loci in the genome of one or more cells of a rat embryo by the
method of claim 1; raising the rat embryo to sexual maturity; and
allowing the sexually mature rat to produce offspring; wherein at
least some of the offspring comprise the mutant alleles.
14. The method of claim 13, wherein the modification comprises
integration of an exogenous sequence into the genome of a rat cell
by homologous recombination stimulated by cleavage of the one or
more endogenous cellular genes.
15. The method of claim 13, wherein the modification results from
non-homologous end joining following cleavage.
16. A method for making a deletion in an endogenous IgM gene, the
method comprising: (i) introducing mRNA into a rat embryo, wherein
the mRNA comprises (i) mRNA encoding a first zinc finger nuclease
(ZFN) that binds to a first target site, wherein the first ZFN
comprises a cleavage domain and a zinc finger protein, wherein the
first zinc finger protein-comprises the following recognition
helices in the following order: TABLE-US-00017 (SEQ ID NO: 41)
DRSHLTR; (SEQ ID NO: 40) RSDALTQ; (SEQ ID NO: 28) DRSDLSR; (SEQ ID
NO: 39) RSDALAR; (SEQ ID NO: 38) RSDSLSA; and (SEQ ID NO: 37)
TSSNRKT;
and (ii) mRNA encoding a second ZFN that binds to a second target
site in the endogenous rat IgM gene, wherein the second ZFN
comprises a cleavage domain and a zinc finger protein comprising
the following recognition helices in the following order:
TABLE-US-00018 (SEQ ID NO: 46) NKVGLIE; (SEQ ID NO: 45) TSSDLSR;
(SEQ ID NO: 44) RSDHLSR; (SEQ ID NO: 43) RSDNLSE; and (SEQ ID NO:
42) QNAHRKT;
such that the first and second ZFNs are expressed, dimerize and
cleave the endogenous rat IgM gene and non-homologous end joining
occurs causing the cell to have a deletion in the endogenous rat
IgM gene.
17. The method of claim 16, wherein the cleavage domain is a Type
IIS restriction endonuclease cleavage domain.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 12/592,852, filed Dec. 3, 2009, which claims
the benefit of U.S. Provisional Application Nos. 61/200,985, filed
Dec. 4, 2008; 61/205,970, filed Jan. 26, 2009 and 61/263,904, filed
Nov. 24, 2009, the disclosures of which are hereby incorporated by
reference in their entireties.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] Not applicable.
TECHNICAL FIELD
[0003] The present disclosure is in the fields of genome
engineering of rats, including somatic and heritable gene
disruptions, genomic alterations, generation of alleles carrying
random mutations at specific positions of rat genes and induction
of homology-directed repair.
BACKGROUND
[0004] Rats (Rattus norvegicus) are a widely used animal model in
the fields of hypertension, cardiovascular physiology, diabetes,
metabolic disorders, behavioral studies and toxicity testing.
Michalkiewicz et al. (2007) J. Amer. Phys. Society 293:H881-H894.
The availability of these model systems, advances in rat genomics
and sequence of the rat, human and mouse genomes have greatly
accelerated the use of inbred rat models for discovery of the
genetic basis of complex diseases and provided animal models for
therapeutic drug discovery.
[0005] However, the advances in the information about the rat
genome have not been accompanied by parallel progress in genome
modification technology. Unlike mice, rat embryonic stem cell
clones for gene targeting are not readily produced. Pronuclear
injection has also proven difficult and has a poor success rate in
generating transgenic rats. Michalkiewicz et al. (2007) J. Amer.
Phys. Society 293:H881-H894 report generation of transgenic rats
using a lentiviral construct expressing an enhanced green
fluorescent protein (eGFP) reporter gene, where the eGFP transgene
was found to be present in 1-4 copies integrated at random sites
within the genome.
[0006] There remains a need for methods of modifying rat genomes in
a targeted fashion. Precisely targeted site-specific cleavage of
genomic loci offers an efficient supplement and/or alternative to
conventional homologous recombination. Creation of a double-strand
break (DSB) increases the frequency of homologous recombination at
the targeted locus more than 1000-fold. More simply, the imprecise
repair of a site-specific DSB by non-homologous end joining (NHEJ)
can also result in gene disruption. Creation of two such DSBs
results in deletion of arbitrarily large regions. The modular DNA
recognition preferences of zinc-fingers protein allows for the
rational design of site-specific multi-finger DNA binding proteins.
Fusion of the nuclease domain from the Type II restriction enzyme
Fok I to site-specific zinc-finger proteins allows for the creation
of site-specific nucleases. See, for example, United States Patent
Publications 20030232410; 20050208489; 20050026157; 20050064474;
20060188987; 20060063231; 20070134796; 2008015164; 20080131962;
2008015996 and International Publication WOs 07/014275 and
2008/133938, which all describe use of zinc-finger nucleases and
which are incorporated by reference in their entireties for all
purposes.
SUMMARY
[0007] Disclosed herein are compositions for genome editing in rat,
including, but not limited to: cleaving of one or more genes in rat
resulting in targeted alteration (insertion, deletion and/or
substitution mutations) in one or more rat genes, including the
incorporation of these targeted alterations into the germline;
targeted introduction of non-endogenous nucleic acid sequences, the
partial or complete inactivation of one or more genes in rat;
methods of inducing homology-directed repair and/or generation of
random mutations encoding novel allelic forms of rat genes.
[0008] In one aspect, described herein is a zinc-finger protein
(ZFP) that binds to target site in a region of interest in a rat
genome, wherein the ZFP comprises one or more engineered
zinc-finger binding domains. In one embodiment, the ZFP is a
zinc-finger nuclease (ZFN) that cleaves a target genomic region of
interest in rat, wherein the ZFN comprises one or more engineered
zinc-finger binding domains and a nuclease cleavage domain or
cleavage half-domain. Cleavage domains and cleavage half domains
can be obtained, for example, from various restriction
endonucleases and/or homing endonucleases. In one embodiment, the
cleavage half-domains are derived from a Type IIS restriction
endonuclease (e.g., Fok I). The ZFN may specifically cleave one
particular rat gene sequence. Alternatively, the ZFN may cleave two
or more homologous rat gene sequences.
[0009] The ZFN may bind to and/or cleave a rat gene within the
coding region of the gene or in a non-coding sequence within or
adjacent to the gene, such as, for example, a leader sequence,
trailer sequence or intron, or within a non-transcribed region,
either upstream or downstream of the coding region. In certain
embodiments, the ZFN binds to and/or cleaves a coding sequence or a
regulatory sequence of the target rat gene.
[0010] In another aspect, described herein are compositions
comprising one or more of the zinc-finger nucleases described
herein. In certain embodiments, the composition comprises one or
more zinc-finger nucleases in combination with a pharmaceutically
acceptable excipient.
[0011] In another aspect, described herein is a polynucleotide
encoding one or more ZFNs described herein. The polynucleotide may
be, for example, mRNA.
[0012] In another aspect, described herein is a ZFN expression
vector comprising a polynucleotide, encoding one or more ZFNs
described herein, operably linked to a promoter.
[0013] In another aspect, described herein is a rat host cell
comprising one or more ZFN expression vectors. The rat host cell
may be stably transformed or transiently transfected or a
combination thereof with one or more ZFP expression vectors. In one
embodiment, the rat host cell is an embryonic stem cell. In other
embodiments, the one or more ZFP expression vectors express one or
more ZFNs in the rat host cell. In another embodiment, the rat host
cell may further comprise an exogenous polynucleotide donor
sequence. In any of the embodiments, described herein, the rat host
cell can comprise an embryo cell, for example a one or more cell
embryo.
[0014] In another aspect, described herein is a method for cleaving
one or more genes in a rat cell, the method comprising: (a)
introducing, into the rat cell, one or more polynucleotides
encoding one or more ZFNs that bind to a target site in the one or
more genes under conditions such that the ZFN(s) is (are) expressed
and the one or more genes are cleaved.
[0015] In yet another aspect, described herein is a method for
introducing an exogenous sequence into the genome of a rat cell,
the method comprising the steps of: (a) introducing, into the rat
cell, one or more polynucleotides encoding one or more ZFNs that
bind to a target site in the one or more genes under conditions
such that the ZFN(s) is (are) expressed and the one or more genes
are cleaved; and (b) contacting the cell with an exogenous
polynucleotide; such that cleavage of the gene(s) stimulates
integration of the exogenous polynucleotide into the genome by
homologous recombination. In certain embodiments, the exogenous
polynucleotide is integrated physically into the genome. In other
embodiments, the exogenous polynucleotide is integrated into the
genome by copying of the exogenous sequence into the host cell
genome via nucleic acid replication processes (e.g.,
homology-directed repair of the double strand break). In yet other
embodiments, integration into the genome occurs through
non-homology dependent targeted integration (e.g. "end-capture").
In certain embodiments, the one or more nucleases are fusions
between the cleavage domain of a Type IIS restriction endonuclease
and an engineered zinc-finger binding domain.
[0016] In another embodiment, described herein is a method for
modifying one or more gene sequence(s) in the genome of a rat cell,
the method comprising (a) providing a rat cell comprising one or
more target gene sequences; and (b) expressing first and second
zinc-finger nucleases (ZFNs) in the cell, wherein the first ZFN
cleaves at a first cleavage site and the second ZFN cleaves at a
second cleavage site, wherein the gene sequence is located between
the first cleavage site and the second cleavage site, wherein
cleavage of the first and second cleavage sites results in
modification of the gene sequence by non-homologous end joining. In
certain embodiments, non-homologous end joining results in a
deletion between the first and second cleavage sites. The size of
the deletion in the gene sequence is determined by the distance
between the first and second cleavage sites. Accordingly, deletions
of any size, in any genomic region of interest, can be obtained.
Deletions of 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,
1,000 nucleotide pairs, or any integral value of nucleotide pairs
within this range, can be obtained. In addition deletions of a
sequence of any integral value of nucleotide pairs greater than
1,000 nucleotide pairs can be obtained using the methods and
compositions disclosed herein. In other embodiments, non-homologous
end joining results in an insertion between the first and second
cleavage sites. Methods of modifying the genome of a rat as
described herein can be used to create models of animal (e.g.,
human) disease, for example by inactivating (partially or fully) a
gene or by creating random mutations at defined positions of genes
that allow for the identification or selection of transgenic rats
carrying novel allelic forms of those genes, by insertion of
humanized rat genes (to study, by way of a non-limiting example,
drug metabolism) or by insertion of a mutant alleles of interest to
examine, for example, the phenotypic affect of such a mutant
allele.
[0017] In yet another aspect, described herein is a method for
germline disruption of one or more target genes in rat, the method
comprising modifying one or more gene sequences in the genome of
one or more cells of a rat embryo by any of the methods described
herein and allowing the rat embryo to develop, wherein that the
modified gene sequences are present in at least a portion of
gametes of the sexually mature rat.
[0018] In another aspect, described herein is a method of creating
one or more heritable mutant alleles in rat loci of interest, the
method comprising modifying one or more loci in the genome of one
or more cells of a rat embryo by any of the methods described
herein; raising the rat embryo to sexual maturity; and allowing the
sexually mature rat to produce offspring; wherein at least some of
the offspring comprise the mutant alleles.
[0019] In any of the methods described herein, the polynucleotide
encoding the zinc finger nuclease(s) can comprise DNA, RNA or
combinations thereof. In certain embodiments, the polynucleotide
comprises a plasmid. In other embodiments, the polynucleotide
encoding the nuclease comprises mRNA.
[0020] In a still further aspect, provided herein is a method for
site specific integration of a nucleic acid sequence into a
chromosome. In certain embodiments, the method comprises: (a)
injecting an embryo with (i) at least one DNA vector, wherein the
DNA vector comprises an upstream sequence and a downstream sequence
flanking the nucleic acid sequence to be integrated, and (ii) at
least one RNA molecule encoding a zinc finger nuclease that
recognizes the chromosomal site of integration, and (b) culturing
the embryo to allow expression of the zinc finger nuclease, wherein
a double stranded break introduced into the site of integration by
the zinc finger nuclease is repaired, via homologous recombination
with the DNA vector, so as to integrate the nucleic acid sequence
into the chromosome. Suitable embryos may be derived from several
different vertebrate species, including mammalian, bird, reptile,
amphibian, and fish species. Generally speaking, a suitable embryo
is an embryo that may be collected, injected, and cultured to allow
the expression of a zinc finger nuclease. In some embodiments,
suitable embryos may include embryos from rodents, companion
animals, livestock, and primates. Non-limiting examples of rodents
may include mice, rats, hamsters, gerbils, and guinea pigs.
Non-limiting examples of companion animals may include cats, dogs,
rabbits, hedgehogs, and ferrets. Non-limiting examples of livestock
may include horses, goats, sheep, swine, llamas, alpacas, and
cattle. Non-limiting examples of primates may include capuchin
monkeys, chimpanzees, lemurs, macaques, marmosets, tamarins, spider
monkeys, squirrel monkeys, and vervet monkeys. In other
embodiments, suitable embryos may include embryos from fish,
reptiles, amphibians, or birds. Alternatively, suitable embryos may
be insect embryos, for instance, a Drosophila embryo or a mosquito
embryo.
[0021] Also provided is an embryo comprising at least one DNA
vector, wherein the DNA vector comprises an upstream sequence and a
downstream sequence flanking the nucleic acid sequence to be
integrated, and at least one RNA molecule encoding a zinc finger
nuclease that recognizes the chromosomal site of integration.
Organisms derived from any of the embryos as described herein are
also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows Surveyor.TM. nuclease ("CEL-I") assays results
of p53-specific ZFN pairs in rat C6 cells. The ZFN pair used in
each lane is shown above the lane and the percent NHEJ activity as
detected by the Surveyor mismatch assay is shown at the bottom.
[0023] FIG. 2 shows Surveyor.TM. nuclease ("CEL-I") assays results
of eGFP-targeted ZFN pairs 16834/16833, 16856/16855 and 16859/16860
in rat C6 cells carrying the eGFP gene. The ZFN pair used is shown
above each lane and the % non-homologous end joining (% NHEJ) is
indicated below each lane as appropriate.
[0024] FIG. 3 is a schematic depicting targeted modification of a
GFP transgene using ZFNs in transgenic GFP rats.
[0025] FIGS. 4A and 4B show targeted disruption of GFP by ZFNs in
rat pups born from pronuclear injection of GFP-targeted ZFNs into
embryos obtained from transgenic GFP rats. FIG. 4A shows the 5 pups
under ultra-violet light, revealing 3 GFP positive animals and 2
animals that do not express GFP (GFP-negative). FIG. 4B shows
results of PCR analysis of tail biopsies of GFP+ and GFP- pups.
[0026] FIG. 5 depicts ZFN-mediated cleavage in exon 1 of endogenous
IgM in C6 cells using ZFN pairs driven by the CMV promoter (CMV) or
the CAG promoter. "Linked" refers to ZFN pairs on the same plasmid
linked by the 2A peptide while "unlinked" refers to ZFN pairs not
linked by the 2A peptide.
[0027] FIG. 6 depicts analysis, by Surveyor.TM. nuclease, of
genomic DNA was prepared from the tails of 43 animals resulting
from live births of IgM ZFN-injected one-cell embryos. As
indicated, rats #6, 7, 8, 19, and 46 scored positive for
modification at the IgM locus. Bars with white numbers indicate
pups born from individual (numbered) mothers.
[0028] FIG. 7 depicts results of PCR analysis of the IgM modified
rats (#6, 7, 8, 19, and 46 as identified in FIG. 6) for insertion
of the ZFN plasmids into the genome.
[0029] FIGS. 8A and 8B show CEL-I and sequencing analysis of IgM
modified rat #19. The alignment of the WT, Rat 19 wild type allele
and the Rat 19 deletion allele sequences (FIG. 8B) (SEQ ID NOS:
166-168) demonstrates the sequences that have been deleted from the
Rat 19 deletion allele.
[0030] FIGS. 9A through 9C depict analysis of IgM modified rats
(#6, 7, 8, 19, and 46 as identified in FIG. 6) for activity at 8
different off-target sites. Off target sites (Site 1, Site 2 etc.)
are as delineated in Table 9.
[0031] FIG. 10 (SEQ ID NOS: 169-171) depicts sequence analysis of
ZFN mediated modification of Rab38. Shown in this Figure is an
alignment of the wildtype allele with two deletion alleles
(.DELTA.6 and .DELTA.42).
[0032] FIGS. 11A through 11C depict analysis of the pups obtained
from crossing ZFN-IgM modified rats and a wild-type rat. FIG. 11A
shows PCR and CEL-I analysis of the 5 pups (numbered 224 to 228)
from crossing rat #19 (Example 3) with a wild-type rat. FIG. 11B
(SEQ ID NOS: 172-179) shows sequencing analysis confirmation that
the 3 IgM modified pups (#225, 227 and 228 as identified in the
figure) include the same 64 basepair deletion allele at the IgM
locus as parent rat #19. FIG. 11C shows PCR and CEL-I analysis of
additions pups of rat #19 as well as pups from crosses of IgM
modified rats #46 and #8. The parental IgM-modified rat is
indicated at the top "F0" and the numbers of the pups are indicated
above each lane.
[0033] FIG. 12 is a schematic depicting the repair outcomes after a
targeted ZFN-induced double stranded break. Shaded bars represent
the donor fragment, whereas white bars depict target site for ZFN
double stranded break.
[0034] FIG. 13 is a schematic depicting the construction of RFLP
donor plasmids. Shown, are the plasmid, and left and right
PCR-amplified fragments homologous to the integration target site.
Restriction enzymes used for cloning are denoted. The left fragment
used KpnI and NotI or PmeI. The right fragment used NotI or PmeI
and SacII.
[0035] FIG. 14 is a schematic depicting the construction of
GFP-expressing donor plasmids. The GFP cassette was PCR amplified
from an existing plasmid and closed into the NotI RFLP donor using
a NotI site.
[0036] FIGS. 15A and 15B depict methods of detecting RFLP
integration. FIG. 15A is a schematic depicting methods of detecting
RFLP integration and restriction enzyme digestion. FIG. 15B is a
schematic depicting integration of the GFP expression cassette
using PCR amplification.
[0037] FIG. 16 is a photographic image of fluorescently stained PCR
fragments resolved on an agarose gel. The leftmost lane contains a
DNA ladder. Lanes 1 to 6 contain PCR fragments amplified using
mouse Mdr1a-specific primers from a whole or a fraction of a mouse
blastocyst. Lanes 1 and 2 were amplified from and 1/6 of a
blastocyst, respectively. Lane 3 was from one whole blastocyst.
Lanes 4 to 6 were from 1/2, 1/3, and 1/6 of the same blastocyst,
respective. Lane 7 contains a positive control PCR fragment
amplified using the same primers from extracted mouse toe DNA.
[0038] FIGS. 17A and 17B depict photographic images of
fluorescently stained DNA fragments resolved on an agarose gel. The
leftmost lanes contain a DNA ladder. Lanes 1 to 39 of FIG. 17A
contain PCR fragments amplified using mMdr1a-specific primers from
37 mouse embryos cultured in vitro after being microinjected with
ZFN RNA against mouse Mdr1a and RFLP donor with NotI site, along
with one positive and negative control for PCR amplification. Lanes
1 to 39 of FIG. 17B contain the PCR fragments of FIG. 17A after
performing the Surveyor.TM. mutation detection assay.
[0039] FIGS. 18A and 18B are photographic images of fluorescently
stained DNA fragments resolved on an agarose gel. The leftmost and
rightmost lanes contain a DNA ladder. Lanes contain PCR fragments
amplified using mMdr1a-specific primers from mouse embryos shown in
FIG. 17, and digested with NotI without purifying the PCR product.
FIG. 18B is a longer run of the same gel in FIG. 18A. The uncut PCR
products are around 1.8 kb, and the digested products are two bands
around 900 bp.
[0040] FIG. 19 is a photographic image of fluorescently stained DNA
fragments resolved on an agarose gel. The leftmost lane contains a
DNA ladder. Lanes 1 to 6 contain some of the PCR fragments from as
shown in FIG. 18 digested with NotI after the PCR products were
column purified so that NotI can work in its optimal buffer. Lines
7 and 8 are two of the samples digested with NotI (as in FIG. 18).
This gel shows NotI digestion in PCR reactions was complete.
[0041] FIG. 20 is a photographic image of fluorescently stained PCR
fragments resolved on an agarose gel. The leftmost lane contains a
DNA ladder. Lanes 1 to 5 contain PCR fragments amplified using
PXR-specific primers from 1, 1/2, 1/6, 1/10, 1/30 of a rat
blastocyst. Lane 6 is a positive control amplified using the same
primers from purified Sprague Dawley genomic DNA.
[0042] FIGS. 21A and 21B are photographic images of fluorescently
stained DNA fragments resolved on an agarose gel. The leftmost and
rightmost lanes contain a DNA ladder. FIG. 21A shows PCR fragments
amplified from rat embryos cultured in vitro after microinjection
of PXR ZFN mRNA and the NotI RFLP donor, using PXR-specific primers
and digested with NotI. FIG. 21B shows the same PCR fragments as in
FIG. 21A after performing the Surveyor.TM. mutation detection
assay.
[0043] FIG. 22 is a photographic image of fluorescently stained DNA
fragments resolved on an agarose gel. The first 4 lanes are PCR
amplified from 4 well developed fetus at 12.5 days post conception
from embryos injected with mMdr1a ZFN mRNA with the NotI RFLP
donor. The PCR was digested with NotI. Lane 4 is positive one.
Lanes 5-8 are 4 decidua, aborted implantations. All four were
negative.
[0044] FIGS. 23A through 23E are schematic and photographic images
of fluorescently stained DNA fragments resolved on an agarose gel.
FIG. 23A is a schematic showing the location of the primers used.
FIGS. 23B and 23C show results from primers PF and GR. FIGS. 23D
and 23E show results from primers PR+GF. Expected fragment size is
2.4 kb. Two out of forty fetuses were positive for GFP.
[0045] FIG. 24 is a photographic image of DNA fragments resolved on
an agarose gel. Lane 8 represents a 13 dpc fetus positive for the
NotI site.
DETAILED DESCRIPTION
[0046] Described herein are compositions and methods for genomic
editing in rat (e.g., cleaving of genes; alteration of genes, for
example by cleavage followed by insertion (physical insertion or
insertion by replication via homology-directed repair) of an
exogenous sequence and/or cleavage followed by non-homologous end
joining (NHEJ); partial or complete inactivation of one or more
genes; generation of alleles with random mutations to create
altered expression of endogenous genes; etc.) and alterations of
the rat genome which are carried into the germline. Also disclosed
are methods of making and using these compositions (reagents), for
example to edit (alter) one or more genes in a target rat cell.
Thus, the methods and compositions described herein provide highly
efficient methods for targeted gene alteration (e.g., knock-in)
and/or knockout (partial or complete) of one or more rat genes
and/or for randomized mutation of the sequence of any target
allele, and, therefore, allow for the generation of animal models
of human diseases.
[0047] The compositions and methods described herein provide rapid,
complete, and permanent targeted disruption of endogenous loci in
rats without the need for labor-intensive selection and/or
screening and with minimal off-target effects. Whole animal gene
knockouts can also be readily generated in a single-step by
injecting ZFN mRNA or ZFN expression cassettes.
[0048] General
[0049] Practice of the methods, as well as preparation and use of
the compositions disclosed herein employ, unless otherwise
indicated, conventional techniques in molecular biology,
biochemistry, chromatin structure and analysis, computational
chemistry, cell culture, recombinant DNA and related fields as are
within the skill of the art. These techniques are fully explained
in the literature. See, for example, Sambrook et al. MOLECULAR
CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989 and Third edition, 2001; Ausubel et al.,
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New
York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY,
Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND
FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS
IN ENZYMOLOGY, Vol. 304, "Chromatin" (P. M. Wassarman and A. P.
Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN
MOLECULAR BIOLOGY, Vol. 119, "Chromatin Protocols" (P. B. Becker,
ed.) Humana Press, Totowa, 1999.
DEFINITIONS
[0050] The terms "nucleic acid," "polynucleotide," and
"oligonucleotide" are used interchangeably and refer to a
deoxyribonucleotide or ribonucleotide polymer, in linear or
circular conformation, and in either single- or double-stranded
form. For the purposes of the present disclosure, these terms are
not to be construed as limiting with respect to the length of a
polymer. The terms can encompass known analogues of natural
nucleotides, as well as nucleotides that are modified in the base,
sugar and/or phosphate moieties (e.g., phosphorothioate backbones).
In general, an analogue of a particular nucleotide has the same
base-pairing specificity; i.e., an analogue of A will base-pair
with T.
[0051] The terms "polypeptide," "peptide" and "protein" are used
interchangeably to refer to a polymer of amino acid residues. The
term also applies to amino acid polymers in which one or more amino
acids are chemical analogues or modified derivatives of a
corresponding naturally-occurring amino acids.
[0052] "Binding" refers to a sequence-specific, non-covalent
interaction between macromolecules (e.g., between a protein and a
nucleic acid). Not all components of a binding interaction need be
sequence-specific (e.g., contacts with phosphate residues in a DNA
backbone), as long as the interaction as a whole is
sequence-specific. Such interactions are generally characterized by
a dissociation constant (K.sub.d) of 10.sup.-6 M.sup.-1 or lower.
"Affinity" refers to the strength of binding: increased binding
affinity being correlated with a lower K.sub.d.
[0053] A "binding protein" is a protein that is able to bind
non-covalently to another molecule. A binding protein can bind to,
for example, a DNA molecule (a DNA-binding protein), an RNA
molecule (an RNA-binding protein) and/or a protein molecule (a
protein-binding protein). In the case of a protein-binding protein,
it can bind to itself (to form homodimers, homotrimers, etc.)
and/or it can bind to one or more molecules of a different protein
or proteins. A binding protein can have more than one type of
binding activity. For example, zinc-finger proteins have
DNA-binding, RNA-binding and protein-binding activity.
[0054] A "zinc-finger DNA binding protein" (or binding domain) is a
protein, or a domain within a larger protein, that binds DNA in a
sequence-specific manner through one or more zinc-fingers, which
are regions of amino acid sequence within the binding domain whose
structure is stabilized through coordination of a zinc ion. The
term zinc-finger DNA binding protein is often abbreviated as
zinc-finger protein or ZFP.
[0055] Zinc-finger binding domains can be "engineered" to bind to a
predetermined nucleotide sequence. Non-limiting examples of methods
for engineering zinc-finger proteins are design and selection. A
designed zinc-finger protein is a protein not occurring in nature
whose design/composition results principally from rational
criteria. Rational criteria for design include application of
substitution rules and computerized algorithms for processing
information in a database storing information of existing ZFP
designs and binding data. See, for example, U.S. Pat. Nos.
6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO
98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.
[0056] A "selected" zinc-finger protein is a protein not found in
nature whose production results primarily from an empirical process
such as phage display, interaction trap or hybrid selection. See
e.g., U.S. Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat.
No. 6,007,988; U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,200,759; WO
95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO
01/60970 WO 01/88197 and WO 02/099084.
[0057] The term "sequence" refers to a nucleotide sequence of any
length, which can be DNA or RNA; can be linear, circular or
branched and can be either single-stranded or double stranded. The
term "donor sequence" refers to a nucleotide sequence that is
inserted into a genome. A donor sequence can be of any length, for
example between 2 and 10,000 nucleotides in length (or any integer
value therebetween or thereabove), preferably between about 100 and
1,000 nucleotides in length (or any integer therebetween), more
preferably between about 200 and 500 nucleotides in length.
[0058] A "homologous, non-identical sequence" refers to a first
sequence which shares a degree of sequence identity with a second
sequence, but whose sequence is not identical to that of the second
sequence. For example, a polynucleotide comprising the wild-type
sequence of a mutant gene is homologous and non-identical to the
sequence of the mutant gene. In certain embodiments, the degree of
homology between the two sequences is sufficient to allow
homologous recombination therebetween, utilizing normal cellular
mechanisms. Two homologous non-identical sequences can be any
length and their degree of non-homology can be as small as a single
nucleotide (e.g., for correction of a genomic point mutation by
targeted homologous recombination) or as large as 10 or more
kilobases (e.g., for insertion of a gene at a predetermined ectopic
site in a chromosome). Two polynucleotides comprising the
homologous non-identical sequences need not be the same length. For
example, an exogenous polynucleotide (i.e., donor polynucleotide)
of between 20 and 10,000 nucleotides or nucleotide pairs can be
used.
[0059] Techniques for determining nucleic acid and amino acid
sequence identity are known in the art. Typically, such techniques
include determining the nucleotide sequence of the mRNA for a gene
and/or determining the amino acid sequence encoded thereby, and
comparing these sequences to a second nucleotide or amino acid
sequence. Genomic sequences can also be determined and compared in
this fashion. In general, identity refers to an exact
nucleotide-to-nucleotide or amino acid-to-amino acid correspondence
of two polynucleotides or polypeptide sequences, respectively. Two
or more sequences (polynucleotide or amino acid) can be compared by
determining their percent identity. The percent identity of two
sequences, whether nucleic acid or amino acid sequences, is the
number of exact matches between two aligned sequences divided by
the length of the shorter sequences and multiplied by 100. An
approximate alignment for nucleic acid sequences is provided by the
local homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2:482-489 (1981). This algorithm can be applied to
amino acid sequences by using the scoring matrix developed by
Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff
ed., 5 suppl. 3:353-358, National Biomedical Research Foundation,
Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res.
14(6):6745-6763 (1986). An exemplary implementation of this
algorithm to determine percent identity of a sequence is provided
by the Genetics Computer Group (Madison, Wis.) in the "BestFit"
utility application. The default parameters for this method are
described in the Wisconsin Sequence Analysis Package Program
Manual, Version 8 (1995) (available from Genetics Computer Group,
Madison, Wis.). A preferred method of establishing percent identity
in the context of the present disclosure is to use the MPSRCH
package of programs copyrighted by the University of Edinburgh,
developed by John F. Collins and Shane S. Sturrok, and distributed
by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite
of packages the Smith-Waterman algorithm can be employed where
default parameters are used for the scoring table (for example, gap
open penalty of 12, gap extension penalty of one, and a gap of
six). From the data generated the "Match" value reflects sequence
identity. Other suitable programs for calculating the percent
identity or similarity between sequences are generally known in the
art, for example, another alignment program is BLAST, used with
default parameters. For example, BLASTN and BLASTP can be used
using the following default parameters: genetic code=standard;
filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;
Descriptions=50 sequences; sort by=HIGH SCORE;
Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+Swiss protein+Spupdate+PIR. Details of these programs
can be found at the following internet address:
http://www.ncbi.nlm.gov/cgi-bin/BLAST. With respect to sequences
described herein, the range of desired degrees of sequence identity
is approximately 80% to 100% and any integer value therebetween.
Typically the percent identities between sequences are at least
70-75%, preferably 80-82%, more preferably 85-90%, even more
preferably 92%, still more preferably 95%, and most preferably 98%
sequence identity.
[0060] Alternatively, the degree of sequence similarity between
polynucleotides can be determined by hybridization of
polynucleotides under conditions that allow formation of stable
duplexes between homologous regions, followed by digestion with
single-stranded-specific nuclease(s), and size determination of the
digested fragments. Two nucleic acid, or two polypeptide sequences
are substantially homologous to each other when the sequences
exhibit at least about 70%-75%, preferably 80%-82%, more preferably
85%-90%, even more preferably 92%, still more preferably 95%, and
most preferably 98% sequence identity over a defined length of the
molecules, as determined using the methods above. As used herein,
substantially homologous also refers to sequences showing complete
identity to a specified DNA or polypeptide sequence. DNA sequences
that are substantially homologous can be identified in a Southern
hybridization experiment under, for example, stringent conditions,
as defined for that particular system. Defining appropriate
hybridization conditions is within the skill of the art. See, e.g.,
Sambrook et al., supra; Nucleic Acid Hybridization: A Practical
Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford;
Washington, D.C.; IRL Press).
[0061] Selective hybridization of two nucleic acid fragments can be
determined as follows. The degree of sequence identity between two
nucleic acid molecules affects the efficiency and strength of
hybridization events between such molecules. A partially identical
nucleic acid sequence will at least partially inhibit the
hybridization of a completely identical sequence to a target
molecule. Inhibition of hybridization of the completely identical
sequence can be assessed using hybridization assays that are well
known in the art (e.g., Southern (DNA) blot, Northern (RNA) blot,
solution hybridization, or the like, see Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold
Spring Harbor, N.Y.). Such assays can be conducted using varying
degrees of selectivity, for example, using conditions varying from
low to high stringency. If conditions of low stringency are
employed, the absence of non-specific binding can be assessed using
a secondary probe that lacks even a partial degree of sequence
identity (for example, a probe having less than about 30% sequence
identity with the target molecule), such that, in the absence of
non-specific binding events, the secondary probe will not hybridize
to the target.
[0062] When utilizing a hybridization-based detection system, a
nucleic acid probe is chosen that is complementary to a reference
nucleic acid sequence, and then by selection of appropriate
conditions the probe and the reference sequence selectively
hybridize, or bind, to each other to form a duplex molecule. A
nucleic acid molecule that is capable of hybridizing selectively to
a reference sequence under moderately stringent hybridization
conditions typically hybridizes under conditions that allow
detection of a target nucleic acid sequence of at least about 10-14
nucleotides in length having at least approximately 70% sequence
identity with the sequence of the selected nucleic acid probe.
Stringent hybridization conditions typically allow detection of
target nucleic acid sequences of at least about 10-14 nucleotides
in length having a sequence identity of greater than about 90-95%
with the sequence of the selected nucleic acid probe. Hybridization
conditions useful for probe/reference sequence hybridization, where
the probe and reference sequence have a specific degree of sequence
identity, can be determined as is known in the art (see, for
example, Nucleic Acid Hybridization: A Practical Approach, editors
B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL
Press).
[0063] Conditions for hybridization are well-known to those of
skill in the art. Hybridization stringency refers to the degree to
which hybridization conditions disfavor the formation of hybrids
containing mismatched nucleotides, with higher stringency
correlated with a lower tolerance for mismatched hybrids. Factors
that affect the stringency of hybridization are well-known to those
of skill in the art and include, but are not limited to,
temperature, pH, ionic strength, and concentration of organic
solvents such as, for example, formamide and dimethylsulfoxide. As
is known to those of skill in the art, hybridization stringency is
increased by higher temperatures, lower ionic strength and lower
solvent concentrations.
[0064] With respect to stringency conditions for hybridization, it
is well known in the art that numerous equivalent conditions can be
employed to establish a particular stringency by varying, for
example, the following factors: the length and nature of the
sequences, base composition of the various sequences,
concentrations of salts and other hybridization solution
components, the presence or absence of blocking agents in the
hybridization solutions (e.g., dextran sulfate, and polyethylene
glycol), hybridization reaction temperature and time parameters, as
well as, varying wash conditions. The selection of a particular set
of hybridization conditions is selected following standard methods
in the art (see, for example, Sambrook, et al., Molecular Cloning:
A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor,
N.Y.).
[0065] "Recombination" refers to a process of exchange of genetic
information between two polynucleotides. For the purposes of this
disclosure, "homologous recombination (HR)" refers to the
specialized form of such exchange that takes place, for example,
during repair of double-strand breaks in cells via
homology-directed repair mechanisms. This process requires
nucleotide sequence homology, uses a "donor" molecule to template
repair of a "target" molecule (i.e., the one that experienced the
double-strand break), and is variously known as "non-crossover gene
conversion" or "short tract gene conversion," because it leads to
the transfer of genetic information from the donor to the target.
Without wishing to be bound by any particular theory, such transfer
can involve mismatch correction of heteroduplex DNA that forms
between the broken target and the donor, and/or
"synthesis-dependent strand annealing," in which the donor is used
to resynthesize genetic information that will become part of the
target, and/or related processes. Such specialized HR often results
in an alteration of the sequence of the target molecule such that
part or all of the sequence of the donor polynucleotide is
incorporated into the target polynucleotide.
[0066] In the methods of the disclosure, one or more targeted
nucleases as described herein create a double-stranded break in the
target sequence (e.g., cellular chromatin) at a predetermined site,
and a "donor" polynucleotide, having homology to the nucleotide
sequence in the region of the break, can be introduced into the
cell. The presence of the double-stranded break has been shown to
facilitate integration of the donor sequence. The donor sequence
may be physically integrated or, alternatively, the donor
polynucleotide is used as a template for repair of the break via
homologous recombination, resulting in the introduction of all or
part of the nucleotide sequence as in the donor into the cellular
chromatin. Thus, a first sequence in cellular chromatin can be
altered and, in certain embodiments, can be converted into a
sequence present in a donor polynucleotide. Thus, the use of the
terms "replace" or "replacement" can be understood to represent
replacement of one nucleotide sequence by another, (i.e.,
replacement of a sequence in the informational sense), and does not
necessarily require physical or chemical replacement of one
polynucleotide by another.
[0067] In any of the methods described herein, additional pairs of
zinc-finger proteins can be used for additional double-stranded
cleavage of additional target sites within the cell.
[0068] In certain embodiments of methods for targeted recombination
and/or replacement and/or alteration of a sequence in a region of
interest in cellular chromatin, a chromosomal sequence is altered
by homologous recombination with an exogenous "donor" nucleotide
sequence. Such homologous recombination is stimulated by the
presence of a double-stranded break in cellular chromatin, if
sequences homologous to the region of the break are present.
[0069] In any of the methods described herein, the first nucleotide
sequence (the "donor sequence") can contain sequences that are
homologous, but not identical, to genomic sequences in the region
of interest, thereby stimulating homologous recombination to insert
a non-identical sequence in the region of interest. Thus, in
certain embodiments, portions of the donor sequence that are
homologous to sequences in the region of interest exhibit between
about 80 to 99% (or any integer therebetween) sequence identity to
the genomic sequence that is replaced. In other embodiments, the
homology between the donor and genomic sequence is higher than 99%,
for example if only 1 nucleotide differs as between donor and
genomic sequences of over 100 contiguous base pairs. In certain
cases, a non-homologous portion of the donor sequence can contain
sequences not present in the region of interest, such that new
sequences are introduced into the region of interest. In these
instances, the non-homologous sequence is generally flanked by
sequences of 50-1,000 base pairs (or any integral value
therebetween) or any number of base pairs greater than 1,000, that
are homologous or identical to sequences in the region of interest.
In other embodiments, the donor sequence is non-homologous to the
first sequence, and is inserted into the genome by non-homologous
recombination mechanisms.
[0070] Any of the methods described herein can be used for partial
or complete inactivation of one or more target sequences in a cell
by targeted integration of donor sequence that disrupts expression
of the gene(s) of interest. Cell lines with partially or completely
inactivated genes are also provided.
[0071] Furthermore, the methods of targeted integration as
described herein can also be used to integrate one or more
exogenous sequences. The exogenous nucleic acid sequence can
comprise, for example, one or more genes or cDNA molecules, or any
type of coding or noncoding sequence, as well as one or more
control elements (e.g., promoters). In addition, the exogenous
nucleic acid sequence may produce one or more RNA molecules (e.g.,
small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs
(miRNAs), etc.).
[0072] "Cleavage" refers to the breakage of the covalent backbone
of a DNA molecule. Cleavage can be initiated by a variety of
methods including, but not limited to, enzymatic or chemical
hydrolysis of a phosphodiester bond. Both single-stranded cleavage
and double-stranded cleavage are possible, and double-stranded
cleavage can occur as a result of two distinct single-stranded
cleavage events. DNA cleavage can result in the production of
either blunt ends or staggered ends. In certain embodiments, fusion
polypeptides are used for targeted double-stranded DNA
cleavage.
[0073] A "cleavage half-domain" is a polypeptide sequence which, in
conjunction with a second polypeptide (either identical or
different) forms a complex having cleavage activity (preferably
double-strand cleavage activity). The terms "first and second
cleavage half-domains;" "+ and - cleavage half-domains" and "right
and left cleavage half-domains" are used interchangeably to refer
to pairs of cleavage half-domains that dimerize.
[0074] An "engineered cleavage half-domain" is a cleavage
half-domain that has been modified so as to form obligate
heterodimers with another cleavage half-domain (e.g., another
engineered cleavage half-domain). See, also, U.S. Patent
Publication Nos. 2005/0064474; 2007/0218528 and 2008/0131962,
incorporated herein by reference in their entireties.
[0075] "Chromatin" is the nucleoprotein structure comprising the
cellular genome. Cellular chromatin comprises nucleic acid,
primarily DNA, and protein, including histones and non-histone
chromosomal proteins. The majority of eukaryotic cellular chromatin
exists in the form of nucleosomes, wherein a nucleosome core
comprises approximately 150 base pairs of DNA associated with an
octamer comprising two each of histones H2A, H2B, H3 and H4; and
linker DNA (of variable length depending on the organism) extends
between nucleosome cores. A molecule of histone H1 is generally
associated with the linker DNA. For the purposes of the present
disclosure, the term "chromatin" is meant to encompass all types of
cellular nucleoprotein, both prokaryotic and eukaryotic. Cellular
chromatin includes both chromosomal and episomal chromatin.
[0076] A "chromosome," is a chromatin complex comprising all or a
portion of the genome of a cell. The genome of a cell is often
characterized by its karyotype, which is the collection of all the
chromosomes that comprise the genome of the cell. The genome of a
cell can comprise one or more chromosomes.
[0077] An "episome" is a replicating nucleic acid, nucleoprotein
complex or other structure comprising a nucleic acid that is not
part of the chromosomal karyotype of a cell. Examples of episomes
include plasmids and certain viral genomes.
[0078] A "target site" or "target sequence" is a nucleic acid
sequence that defines a portion of a nucleic acid to which a
binding molecule will bind, provided sufficient conditions for
binding exist. For example, the sequence 5'-GAATTC-3' is a target
site for the Eco RI restriction endonuclease.
[0079] An "exogenous" molecule is a molecule that is not normally
present in a cell, but can be introduced into a cell by one or more
genetic, biochemical or other methods. "Normal presence in the
cell" is determined with respect to the particular developmental
stage and environmental conditions of the cell. Thus, for example,
a molecule that is present only during embryonic development of
muscle is an exogenous molecule with respect to an adult muscle
cell. Similarly, a molecule induced by heat shock is an exogenous
molecule with respect to a non-heat-shocked cell. An exogenous
molecule can comprise, for example, a functioning version of a
malfunctioning endogenous molecule or a malfunctioning version of a
normally-functioning endogenous molecule. An exogenous molecule can
also be a molecule normally found in another species, for example,
a human sequence introduced into a rat genome.
[0080] An exogenous molecule can be, among other things, a small
molecule, such as is generated by a combinatorial chemistry
process, or a macromolecule such as a protein, nucleic acid,
carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any
modified derivative of the above molecules, or any complex
comprising one or more of the above molecules. Nucleic acids
include DNA and RNA, can be single- or double-stranded; can be
linear, branched or circular; and can be of any length. Nucleic
acids include those capable of forming duplexes, as well as
triplex-forming nucleic acids. See, for example, U.S. Pat. Nos.
5,176,996 and 5,422,251. Proteins include, but are not limited to,
DNA-binding proteins, transcription factors, chromatin remodeling
factors, methylated DNA binding proteins, polymerases, methylates,
demethylases, acetylases, deacetylases, kinases, phosphatases,
integrases, recombinases, ligases, topoisomerases, gyrases and
helicases.
[0081] An exogenous molecule can be the same type of molecule as an
endogenous molecule, e.g., an exogenous protein or nucleic acid.
For example, an exogenous nucleic acid can comprise an infecting
viral genome, a plasmid or episome introduced into a cell, or a
chromosome that is not normally present in the cell. Methods for
the introduction of exogenous molecules into cells are known to
those of skill in the art and include, but are not limited to,
lipid-mediated transfer (i.e., liposomes, including neutral and
cationic lipids), electroporation, direct injection, cell fusion,
particle bombardment, calcium phosphate co-precipitation,
DEAE-dextran-mediated transfer and viral vector-mediated
transfer.
[0082] By contrast, an "endogenous" molecule is one that is
normally present in a particular cell at a particular developmental
stage under particular environmental conditions. For example, an
endogenous nucleic acid can comprise a chromosome, the genome of a
mitochondrion, chloroplast or other organelle, or a
naturally-occurring episomal nucleic acid. Additional endogenous
molecules can include proteins, for example, transcription factors
and enzymes.
[0083] A "fusion" molecule is a molecule in which two or more
subunit molecules are linked, preferably covalently. The subunit
molecules can be the same chemical type of molecule, or can be
different chemical types of molecules. Examples of the first type
of fusion molecule include, but are not limited to, fusion proteins
(for example, a fusion between a ZFP DNA-binding domain and a
cleavage domain) and fusion nucleic acids (for example, a nucleic
acid encoding the fusion protein described supra). Examples of the
second type of fusion molecule include, but are not limited to, a
fusion between a triplex-forming nucleic acid and a polypeptide,
and a fusion between a minor groove binder and a nucleic acid.
[0084] Expression of a fusion protein in a cell can result from
delivery of the fusion protein to the cell or by delivery of a
polynucleotide encoding the fusion protein to a cell, wherein the
polynucleotide is transcribed, and the transcript is translated, to
generate the fusion protein. Trans-splicing, polypeptide cleavage
and polypeptide ligation can also be involved in expression of a
protein in a cell. Methods for polynucleotide and polypeptide
delivery to cells are presented elsewhere in this disclosure.
[0085] A "gene," for the purposes of the present disclosure,
includes a DNA region encoding a gene product (see infra), as well
as all DNA regions which regulate the production of the gene
product, whether or not such regulatory sequences are adjacent to
coding and/or transcribed sequences. Accordingly, a gene includes,
but is not necessarily limited to, promoter sequences, terminators,
translational regulatory sequences such as ribosome binding sites
and internal ribosome entry sites, enhancers, silencers,
insulators, boundary elements, replication origins, matrix
attachment sites and locus control regions.
[0086] "Gene expression" refers to the conversion of the
information, contained in a gene, into a gene product. A gene
product can be the direct transcriptional product of a gene (e.g.,
mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any
other type of RNA) or a protein produced by translation of a mRNA.
Gene products also include RNAs which are modified, by processes
such as capping, polyadenylation, methylation, and editing, and
proteins modified by, for example, methylation, acetylation,
phosphorylation, ubiquitination, ADP-ribosylation, myristilation,
and glycosylation.
[0087] "Modulation" of gene expression refers to a change in the
activity of a gene. Modulation of expression can include, but is
not limited to, gene activation and gene repression. Genome editing
(e.g., cleavage, alteration, inactivation, random mutation) can be
used to modulate expression. Gene inactivation refers to any
reduction in gene expression as compared to a cell that does not
include a ZFP as described herein. Thus, gene inactivation may be
partial or complete.
[0088] A "region of interest" is any region of cellular chromatin,
such as, for example, a gene or a non-coding sequence within or
adjacent to a gene, in which it is desirable to bind an exogenous
molecule. Binding can be for the purposes of targeted DNA cleavage
and/or targeted recombination. A region of interest can be present
in a chromosome, an episome, an organellar genome (e.g.,
mitochondrial, chloroplast), or an infecting viral genome, for
example. A region of interest can be within the coding region of a
gene, within transcribed non-coding regions such as, for example,
leader sequences, trailer sequences or introns, or within
non-transcribed regions, either upstream or downstream of the
coding region. A region of interest can be as small as a single
nucleotide pair or up to 2,000 nucleotide pairs in length, or any
integral value of nucleotide pairs.
[0089] The terms "operative linkage" and "operatively linked" (or
"operably linked") are used interchangeably with reference to a
juxtaposition of two or more components (such as sequence
elements), in which the components are arranged such that both
components function normally and allow the possibility that at
least one of the components can mediate a function that is exerted
upon at least one of the other components. By way of illustration,
a transcriptional regulatory sequence, such as a promoter, is
operatively linked to a coding sequence if the transcriptional
regulatory sequence controls the level of transcription of the
coding sequence in response to the presence or absence of one or
more transcriptional regulatory factors. A transcriptional
regulatory sequence is generally operatively linked in cis with a
coding sequence, but need not be directly adjacent to it. For
example, an enhancer is a transcriptional regulatory sequence that
is operatively linked to a coding sequence, even though they are
not contiguous.
[0090] With respect to fusion polypeptides, the term "operatively
linked" can refer to the fact that each of the components performs
the same function in linkage to the other component as it would if
it were not so linked. For example, with respect to a fusion
polypeptide in which a ZFP DNA-binding domain is fused to a
cleavage domain, the ZFP DNA-binding domain and the cleavage domain
are in operative linkage if, in the fusion polypeptide, the ZFP
DNA-binding domain portion is able to bind its target site and/or
its binding site, while the cleavage domain is able to cleave DNA
in the vicinity of the target site.
[0091] A "functional fragment" of a protein, polypeptide or nucleic
acid is a protein, polypeptide or nucleic acid whose sequence is
not identical to the full-length protein, polypeptide or nucleic
acid, yet retains the same function as the full-length protein,
polypeptide or nucleic acid. A functional fragment can possess
more, fewer, or the same number of residues as the corresponding
native molecule, and/or can contain one ore more amino acid or
nucleotide substitutions. Methods for determining the function of a
nucleic acid (e.g., coding function, ability to hybridize to
another nucleic acid) are well-known in the art. Similarly, methods
for determining protein function are well-known. For example, the
DNA-binding function of a polypeptide can be determined, for
example, by filter-binding, electrophoretic mobility-shift, or
immunoprecipitation assays. DNA cleavage can be assayed by gel
electrophoresis. See Ausubel et al., supra. The ability of a
protein to interact with another protein can be determined, for
example, by co-immunoprecipitation, two-hybrid assays or
complementation, both genetic and biochemical. See, for example,
Fields et al. (1989) Nature 340:245-246; U.S. Pat. No. 5,585,245
and PCT WO 98/44350.
[0092] Zinc-Finger Nucleases
[0093] Described herein are zinc-finger nucleases (ZFNs) that can
be used for genomic editing (e.g., cleavage, alteration,
inactivation and/or random mutation) of one or more rat genes. ZFNs
comprise a zinc-finger protein (ZFP) and a nuclease (cleavage)
domain (e.g., cleavage half-domain).
[0094] A. Zinc-Finger Proteins
[0095] Zinc-finger binding domains can be engineered to bind to a
sequence of choice. See, for example, Beerli et al. (2002) Nature
Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem.
70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660;
Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al.
(2000) Curr. Opin. Struct. Biol. 10:411-416. An engineered
zinc-finger binding domain can have a novel binding specificity,
compared to a naturally-occurring zinc-finger protein. Engineering
methods include, but are not limited to, rational design and
various types of selection. Rational design includes, for example,
using databases comprising triplet (or quadruplet) nucleotide
sequences and individual zinc-finger amino acid sequences, in which
each triplet or quadruplet nucleotide sequence is associated with
one or more amino acid sequences of zinc-fingers which bind the
particular triplet or quadruplet sequence. See, for example,
co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by
reference herein in their entireties.
[0096] Exemplary selection methods, including phage display and
two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538;
5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759;
and 6,242,568; as well as WO 98/37186; WO 98/53057; WO 00/27878; WO
01/88197 and GB 2,338,237. In addition, enhancement of binding
specificity for zinc-finger binding domains has been described, for
example, in co-owned WO 02/077227.
[0097] Selection of target sites; ZFPs and methods for design and
construction of fusion proteins (and polynucleotides encoding same)
are known to those of skill in the art and described in detail in
U.S. Patent Application Publication Nos. 20050064474 and
20060188987, incorporated by reference in their entireties
herein.
[0098] In addition, as disclosed in these and other references,
zinc-finger domains and/or multi-fingered zinc-finger proteins may
be linked together using any suitable linker sequences, including
for example, linkers of 5 or more amino acids in length (e.g.,
TGEKP (SEQ ID NO:1), TGGQRP (SEQ ID NO:2), TGQKP (SEQ ID NO:3),
and/or TGSQKP (SEQ ID NO:4)). See, also, U.S. Pat. Nos. 6,479,626;
6,903,185; and 7,153,949 for exemplary linker sequences 6 or more
amino acids in length. The proteins described herein may include
any combination of suitable linkers between the individual
zinc-fingers of the protein.
[0099] As described below, in certain embodiments, a four-, five-,
or six-finger binding domain is fused to a cleavage half-domain,
such as, for example, the cleavage domain of a Type IIs restriction
endonuclease such as FokI. One or more pairs of such
zinc-finger/nuclease half-domain fusions are used for targeted
cleavage, as disclosed, for example, in U.S. Patent Publication No.
20050064474.
[0100] For targeted cleavage, the near edges of the binding sites
can separated by 5 or more nucleotide pairs, and each of the fusion
proteins can bind to an opposite strand of the DNA target. All
pairwise combinations 1 can be used for targeted cleavage of a rat
gene. Following the present disclosure, ZFNs can be targeted to any
sequence in the rat genome.
[0101] In some embodiments, the DNA binding domain is an engineered
domain from a TAL effector derived from the plant pathogen
Xanthomonas (see Boch et al, (2009) Science 29 Oct. 2009
(10.1126/science.117881) and Moscou and Bogdanove, (2009) Science
29 Oct. 2009 (10.1126/science.1178817).
[0102] B. Cleavage Domains
[0103] The ZFNs also comprise a nuclease (cleavage domain, cleavage
half-domain). The cleavage domain portion of the fusion proteins
disclosed herein can be obtained from any endonuclease or
exonuclease. Exemplary endonucleases from which a cleavage domain
can be derived include, but are not limited to, restriction
endonucleases and homing endonucleases. See, for example, 2002-2003
Catalogue, New England Biolabs, Beverly, Mass.; and Belfort et al.
(1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes which
cleave DNA are known (e.g., 51 Nuclease; mung bean nuclease;
pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease;
see also Linn et al. (eds.) Nucleases, Cold Spring Harbor
Laboratory Press, 1993). One or more of these enzymes (or
functional fragments thereof) can be used as a source of cleavage
domains and cleavage half-domains.
[0104] Similarly, a cleavage half-domain can be derived from any
nuclease or portion thereof, as set forth above, that requires
dimerization for cleavage activity. In general, two fusion proteins
are required for cleavage if the fusion proteins comprise cleavage
half-domains. Alternatively, a single protein comprising two
cleavage half-domains can be used. The two cleavage half-domains
can be derived from the same endonuclease (or functional fragments
thereof), or each cleavage half-domain can be derived from a
different endonuclease (or functional fragments thereof). In
addition, the target sites for the two fusion proteins are
preferably disposed, with respect to each other, such that binding
of the two fusion proteins to their respective target sites places
the cleavage half-domains in a spatial orientation to each other
that allows the cleavage half-domains to form a functional cleavage
domain, e.g., by dimerizing. Thus, in certain embodiments, the near
edges of the target sites are separated by 5-8 nucleotides or by
15-18 nucleotides. However any integral number of nucleotides or
nucleotide pairs can intervene between two target sites (e.g., from
2 to 50 nucleotide pairs or more). In general, the site of cleavage
lies between the target sites.
[0105] Restriction endonucleases (restriction enzymes) are present
in many species and are capable of sequence-specific binding to DNA
(at a recognition site), and cleaving DNA at or near the site of
binding. Certain restriction enzymes (e.g., Type IIS) cleave DNA at
sites removed from the recognition site and have separable binding
and cleavage domains. For example, the Type IIS enzyme Fok I
catalyzes double-stranded cleavage of DNA, at 9 nucleotides from
its recognition site on one strand and 13 nucleotides from its
recognition site on the other. See, for example, U.S. Pat. Nos.
5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992)
Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc.
Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl.
Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.
269:31,978-31,982. Thus, in one embodiment, fusion proteins
comprise the cleavage domain (or cleavage half-domain) from at
least one Type IIS restriction enzyme and one or more zinc-finger
binding domains, which may or may not be engineered.
[0106] An exemplary Type IIS restriction enzyme, whose cleavage
domain is separable from the binding domain, is Fok I. This
particular enzyme is active as a dimer. Bitinaite et al. (1998)
Proc. Natl. Acad. Sci. USA 95: 10,570-10,575. Accordingly, for the
purposes of the present disclosure, the portion of the Fok I enzyme
used in the disclosed fusion proteins is considered a cleavage
half-domain. Thus, for targeted double-stranded cleavage and/or
targeted replacement of cellular sequences using zinc-finger-Fok I
fusions, two fusion proteins, each comprising a FokI cleavage
half-domain, can be used to reconstitute a catalytically active
cleavage domain. Alternatively, a single polypeptide molecule
containing a zinc-finger binding domain and two Fok I cleavage
half-domains can also be used. Parameters for targeted cleavage and
targeted sequence alteration using zinc-finger-Fok I fusions are
provided elsewhere in this disclosure.
[0107] A cleavage domain or cleavage half-domain can be any portion
of a protein that retains cleavage activity, or that retains the
ability to multimerize (e.g., dimerize) to form a functional
cleavage domain.
[0108] Exemplary Type IIS restriction enzymes are described in
International Publication WO 07/014275, incorporated herein in its
entirety. Additional restriction enzymes also contain separable
binding and cleavage domains, and these are contemplated by the
present disclosure. See, for example, Roberts et al. (2003) Nucleic
Acids Res. 31:418-420.
[0109] In certain embodiments, the cleavage domain comprises one or
more engineered cleavage half-domain (also referred to as
dimerization domain mutants) that minimize or prevent
homodimerization, as described, for example, in U.S. Patent
Publication Nos. 20050064474; 20060188987 and 20080131962, the
disclosures of all of which are incorporated by reference in their
entireties herein. Amino acid residues at positions 446, 447, 479,
483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537,
and 538 of Fok I are all targets for influencing dimerization of
the Fok I cleavage half-domains.
[0110] Exemplary engineered cleavage half-domains of Fok I that
form obligate heterodimers include a pair in which a first cleavage
half-domain includes mutations at amino acid residues at positions
490 and 538 of Fok I and a second cleavage half-domain includes
mutations at amino acid residues 486 and 499.
[0111] Thus, in one embodiment, a mutation at 490 replaces Glu (E)
with Lys (K); the mutation at 538 replaces Iso (I) with Lys (K);
the mutation at 486 replaced Gln (Q) with Glu (E); and the mutation
at position 499 replaces Iso (I) with Lys (K). Specifically, the
engineered cleavage half-domains described herein were prepared by
mutating positions 490 (E.fwdarw.K) and 538 (I.fwdarw.K) in one
cleavage half-domain to produce an engineered cleavage half-domain
designated "E490K:I538K" and by mutating positions 486 (Q.fwdarw.E)
and 499 (I.fwdarw.L) in another cleavage half-domain to produce an
engineered cleavage half-domain designated "Q486E:I499L". The
engineered cleavage half-domains described herein are obligate
heterodimer mutants in which aberrant cleavage is minimized or
abolished. See, e.g., Example 1 of U.S. Patent Publication No.
2008/0131962, the disclosure of which is incorporated by reference
in its entirety for all purposes.
[0112] Engineered cleavage half-domains described herein can be
prepared using any suitable method, for example, by site-directed
mutagenesis of wild-type cleavage half-domains (Fok I) as described
in U.S. Patent Publication No. 20050064474 and U.S. Pat. No.
8,313,925.
[0113] C. Additional Methods for Targeted Cleavage in Rat
[0114] Any nuclease having a target site in any rat gene(s) can be
used in the methods disclosed herein. For example, homing
endonucleases and meganucleases have very long recognition
sequences, some of which are likely to be present, on a statistical
basis, once in a human-sized genome. Any such nuclease having a
target site in a rat gene can be used instead of, or in addition
to, a zinc-finger nuclease, for targeted cleavage in a rat
gene.
[0115] Exemplary homing endonucleases include I-SceI, I-CeuI,
PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI,
I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. Their recognition
sequences are known. See also U.S. Pat. No. 5,420,032; U.S. Pat.
No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res.
25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al.
(1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet.
12:224-228; Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast
et al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs
catalogue.
[0116] Although the cleavage specificity of most homing
endonucleases is not absolute with respect to their recognition
sites, the sites are of sufficient length that a single cleavage
event per mammalian-sized genome can be obtained by expressing a
homing endonuclease in a cell containing a single copy of its
recognition site. It has also been reported that the specificity of
homing endonucleases and meganucleases can be engineered to bind
non-natural target sites. See, for example, Chevalier et al. (2002)
Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res.
31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et
al. (2007) Current Gene Therapy 7:49-66.
[0117] Delivery
[0118] The ZFNs described herein may be delivered to a target rat
cell by any suitable means, including, for example, by injection of
ZFN mRNA. See, Hammerschmidt et al. (1999) Methods Cell Biol.
59:87-115
[0119] Methods of delivering proteins comprising zinc-fingers are
described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717;
6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113;
6,979,539; 7,013,219; and 7,163,824, the disclosures of all of
which are incorporated by reference herein in their entireties.
[0120] ZFNs as described herein may also be delivered using vectors
containing sequences encoding one or more of the ZFNs. Any vector
systems may be used including, but not limited to, plasmid vectors,
retroviral vectors, lentiviral vectors, adenovirus vectors,
poxvirus vectors; herpesvirus vectors and adeno-associated virus
vectors, etc. See, also, U.S. Pat. Nos. 6,534,261; 6,607,882;
6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824,
incorporated by reference herein in their entireties. Furthermore,
it will be apparent that any of these vectors may comprise one or
more ZFN encoding sequences. Thus, when one or more pairs of ZFNs
are introduced into the cell, the ZFNs may be carried on the same
vector or on different vectors. When multiple vectors are used,
each vector may comprise a sequence encoding one or multiple
ZFNs.
[0121] Conventional viral and non-viral based gene transfer methods
can be used to introduce nucleic acids encoding engineered ZFPs in
rat cells. Such methods can also be used to administer nucleic
acids encoding ZFPs to rat cells in vitro. In certain embodiments,
nucleic acids encoding ZFPs are administered for in vivo or ex vivo
uses.
[0122] Non-viral vector delivery systems include electroporation,
lipofection, microinjection, biolistics, virosomes, liposomes,
immunoliposomes, polycation or lipid:nucleic acid conjugates, naked
DNA, artificial virions, and agent-enhanced uptake of DNA.
Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can
also be used for delivery of nucleic acids. Viral vector delivery
systems include DNA and RNA viruses, which have either episomal or
integrated genomes after delivery to the cell. Additional exemplary
nucleic acid delivery systems include those provided by Amaxa
Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX
Molecular Delivery Systems (Holliston, Mass.) and Copernicus
Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).
Lipofection is described in e.g., U.S. Pat. No. 5,049,386, U.S.
Pat. No. 4,946,787; and U.S. Pat. No. 4,897,355) and lipofection
reagents are sold commercially (e.g., Transfectam.TM. and
Lipofectin.TM.). Cationic and neutral lipids that are suitable for
efficient receptor-recognition lipofection of polynucleotides
include those of Felgner, WO 91/17424, WO 91/16024. Delivery can be
to cells (ex vivo administration) or target tissues (in vivo
administration). The preparation of lipid:nucleic acid complexes,
including targeted liposomes such as immunolipid complexes, is well
known to one of skill in the art (see, e.g., Crystal, Science
270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297
(1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et
al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy
2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992);
U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975,
4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
[0123] As noted above, the disclosed methods and compositions can
be used in any type of rat cell. Progeny, variants and derivatives
of rat cells can also be used.
Applications
[0124] The disclosed methods and compositions can be used for
genomic editing of any rat gene or genes. In certain applications,
the methods and compositions can be used for inactivation of rat
genomic sequences. In other applications, the methods and
compositions allow for generation of random mutations, including
generation of novel allelic forms of genes with different
expression as compared to unedited genes or integration of
humanized rat genes, which in turn allows for the generation of
animal models. In other applications, the methods and compositions
can be used for creating random mutations at defined positions of
genes that allows for the identification or selection of animals
carrying novel allelic forms of those genes. In other applications,
the methods and compositions allow for targeted integration of an
exogenous (donor) sequence into any selected area of the rat
genome. Regulatory sequences (e.g. promoters) could be integrated
in a targeted fashion at a site of interest. By "integration" is
meant both physical insertion (e.g., into the genome of a host
cell) and, in addition, integration by copying of the donor
sequence into the host cell genome via the nucleic acid replication
processes. Donor sequences can also comprise nucleic acids such as
shRNAs, miRNAs etc. These small nucleic acid donors can be used to
study their effects on genes of interest within the rat genome.
Genomic editing (e.g., inactivation, integration and/or targeted or
random mutation) of a rat gene can be achieved, for example, by a
single cleavage event, by cleavage followed by non-homologous end
joining, by cleavage followed by homology-directed repair
mechanisms, by cleavage followed by physical integration of a donor
sequence, by cleavage at two sites followed by joining so as to
delete the sequence between the two cleavage sites, by targeted
recombination of a missense or nonsense codon into the coding
region, by targeted recombination of an irrelevant sequence (i.e.,
a "stuffer" sequence) into the gene or its regulatory region, so as
to disrupt the gene or regulatory region, or by targeting
recombination of a splice acceptor sequence into an intron to cause
mis-splicing of the transcript. See, U.S. Patent Publication Nos.
20030232410; 20050208489; 20050026157; 20050064474; 20060188987;
20060063231; and International Publication WO 07/014275, the
disclosures of which are incorporated by reference in their
entireties for all purposes.
[0125] There are a variety of applications for ZFN-mediated genomic
editing of rat. The methods and compositions described herein allow
for the generation of rat models of human diseases. For example,
editing of the p53 gene allows for the generation of a "cancer rat"
that provides an animal model for studying cancer and testing
cancer therapies.
EXAMPLES
Example 1
ZFNs Induce Targeted Disruption in Rat C6 Cells
[0126] ZFNs targeted to rat p53 were designed and incorporated into
plasmids essentially as described in Urnov et al. (2005) Nature
435(7042):646-651. The recognition helices for representative rat
p53 designs are shown below in Table 1. The target sites for these
ZFNs are shown in Table 2.
TABLE-US-00001 TABLE 1 rat p53-specific ZFN designs ZFN Name F1 F2
F3 F4 10356 RSDDLTR RSDHLSR DNPNLNR RSDDLSR (SEQ ID (SEQ ID (SEQ ID
(SEQ ID NO: 16) NO: 44) NO: 55) NO: 100) 10358 DNPNLNR RSDDLSR
NSQHLTE QSSHLSR (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 55) NO: 100)
NO: 101) NO: 102) 10359 QSGNLAR RSDDLTR NSQHLTE QSSHLSR (SEQ ID
(SEQ ID (SEQ ID (SEQ ID NO: 21) NO: 16) NO: 101) NO: 102) 10357
RSDDLTR RSDHLSR QSGNLAR RSDDLTR (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:
16) NO: 44) NO: 21) NO: 16) 10360 RSDNLAR RSDHLTT RSDNLSQ ASNDRKK
(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 103) NO: 104) NO: 105) NO: 106)
10362 RSDHLSE RSAALAR RSDHLSE RNQHRIT (SEQ ID (SEQ ID (SEQ ID (SEQ
ID NO: 87) NO: 107) NO: 87) NO: 108) 10361 RSDNLAR RSDHLTT RSDNLSE
DSRSRIN (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 103) NO: 104) NO: 43)
NO: 109) 10363 DRSHLSR RSDDLTR RSDHLSR DRSHLAR (SEQ ID (SEQ ID (SEQ
ID (SEQ ID NO: 110) NO: 16) NO: 44) NO: 12)
TABLE-US-00002 TABLE 2 rat p53-specific ZFN targets ZFN Name Target
Site (5' to 3') 10356 aaGCGGAAGGGGCGggccatagcccggg (SEQ ID NO: 111)
10358 caGGACGTGCGGAAtgcgttaagggaat (SEQ ID NO: 112) 10359
caGGACGTGCGGAAtgcgttaagggaat (SEQ ID NO: 112) 10357
aaGCGGAAGGGGCGggccatagcccggg (SEQ ID NO: 111) 10360
ctTCCCAGTGGGAGgtgacagaaccctg (SEQ ID NO: 113) 10362
acCGGCGGGTGCGGgeggactgcactta (SEQ ID NO: 114) 10361
ctTCCCAGTGGGAGgtgacagaaccctg (SEQ ID NO: 113) 10363
ccGGCGGGtGCGGGCggactgcacttag (SEQ ID NO: 115)
[0127] ZFN-encoding plasmids were transfected into rat C6 cells. To
determine the ZFN activity at the p53 locus, CEL-I mismatch assays
were performed essentially as per the manufacturer's instructions
(Trangenomic SURVEYOR.TM.). Cells were harvested and chromosomal
DNA prepared using a Quickextract.TM. Kit according to
manufacturer's directions (Epicentre.RTM.). The appropriate region
of the p53 locus was PCR amplified using Accuprime.TM.
High-fidelity DNA polymerase (Invitrogen). PCR reactions were
heated to 94.degree. C., and gradually cooled to room temperature.
Approximately 200 ng of the annealed DNA was mixed with 0.334 CEL-I
enzyme and incubated for 20 minutes at 42.degree. C. Reaction
products were analyzed by polyacrylamide gel electrophoresis in
1.times. Tris-borate-EDTA buffer.
[0128] Results are shown in FIG. 1 where various pairs of
p53-specific ZFNs descried in Tables 1 and 2 were tested in
combination. Percent mismatch, a measure of NHEJ activity are shown
at the bottom of each lane. The results indicate that these ZFNs
are active against this rat locus.
[0129] ZFNs targeted to GFP were designed and incorporated into
plasmids essentially as described in Urnov et al. (2005) Nature
435(7042):646-651. ZFN pairs were screened for activity in a
yeast-based chromosomal system as described in U.S. Ser. No.
12/284,887, entitled "Rapid in vivo Identification of Biologically
Active Nucleases." Briefly, galactose-inducible ZFNs were
transformed into a yeast strain containing an integrated Single
Strand Annealing (ySSA) reporter, which consisted of the full eGFP
sequence inserted between two overlapping segments of the MEL1 gene
driven by the PGK promoter. The expression of the ZFNs was induced
for 6 hours, then repressed for 18 hours, after which time a
standard colorometric assay was used to quantify the amount of MEL1
protein in the supernatant.
[0130] The recognition helices for representative GFP zinc-finger
designs are shown below in Table 3.
TABLE-US-00003 GFP Zinc-finger Designs ZFN Name F1 F2 F3 F4 F5 F6
16833 RSAHLSR TSANLSR RSDNLSV DRSNLTR ''33'' (SEQ ID (SEQ ID (SEQ
ID (SEQ ID NO: 5) NO: 6) NO: 7) NO: 8) 16834 RSDTLSQ QRDHRIK
DRSNLSR DRSHLAR DRSNLTR ''34'' (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ
ID NO: 9) NO: 10) NO: 11) NO: 12) NO: 8) 16855 RSDHLSA DSSTRKT
TSGSLSR RSDDLTR TSANLSR ''55'' (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ
ID NO: 13) NO: 14) NO: 15) NO: 16) NO: 6) 16856 RSDNLST DSSSRIK
RSAVLSE TNSNRIT RSAHLSR QSGNLAR ''56'' (SEQ ID (SEQ ID (SEQ ID (SEQ
ID (SEQ ID (SEQ ID NO: 17) NO: 18) NO: 19) NO: 20) NO: 5) NO: 21)
16859 TSGSLSR QSGSLTR TSGSLSR QSSDLRR RSDALSR TSGSLTR ''59'' (SEQ
ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 15) NO: 22) NO: 15)
NO: 23) NO: 24) NO: 25) 16860 RSANLSV DRANLSR DRSDLSR RSDSLSV
DSSARKK ''60'' (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 30) NO:
29) NO: 28) NO: 27) NO: 26)
[0131] Target sites of the GFP zinc-finger designs are shown below
in Table 4. Nucleotides in the target site that are contacted by
the ZFP recognition helices are indicated in uppercase letters;
non-contacted nucleotides indicated in lowercase.
TABLE-US-00004 TABLE 4 Target Sites of GFP Zinc-fingers ZFN Name
Target Site (5' to 3') 16833 GACCAGGATGGG (SEQ ID NO: 31) 16834
GACGGCGACgTAAACG (SEQ ID NO: 32) 16855 GATGCGGTTcACCAGG (SEQ ID NO:
33) 16856 GAAGGGCATCGAcTTCAAG (SEQ ID NO: 34) 16859
GTTGTGGCTGTTGTAGTT (SEQ ID NO: 35) 16860 ATCATGGCCGACAAG (SEQ ID
NO: 36)
[0132] Active GFP-targeted ZFN expression constructs were
transfected into rat C6 cells containing a GFP expression
construct.
[0133] As shown in FIG. 2, all ZFN pairs tested cleaved the GFP
gene in the target cells.
Example 2
ZFNs Induce Targeted Disruption in Transgenic Rats
[0134] GFP-specific ZFNs as described in Example 1 were also
introduced by pronuclear injection (PNI) or cytoplasmic injection
(of ZFN mRNA) at varying concentrations into one-cell embryos
obtained from transgenic rats expressing GFP described in
Michalkiewicz et al. (2007) J. Amer. Phys. Society 293:H881-H894.
See, FIG. 3.
[0135] The injected embryos were cultured for 2-3 days until they
reached the 2-4 cell stage. Some of the 2-4 cell embryos were then
transferred to pseudo-pregnant females. DNA was extracted from both
cultured embryos and transferred embryos and cleavage of the GFP
gene assessed.
[0136] Results of the different mode of injection and concentration
of ZFNs injected into the embryos injected using ZFN pair
16859/16860 are shown in the Table 5 below.
TABLE-US-00005 TABLE 5 Method ZFN of conc. Embryos Sur- Divided
Trans- injection (ng/.mu.L) injected vived 2-cells % ferred Born
PNI 1 40 23 19 83 2 39 29 19 66 1.5 36 25 25 5 cyto- 5 40 26 16 62
plasmic 10 39 32 16 50 20 38 31 24 77 10 256 138 138
[0137] GFP imaging of cytoplasmic injections of ZFN mRNA showed
that many more ZFN-containing embryos failed to express GFP than
uninjected embryos, indicating that no mosaicism was present in the
cells in which ZFNs were active.
[0138] Five pups were born from pronuclear injection (PNI) of ZFNs
into embryos that were transferred into pseudo-pregnant females.
See, Table 5. As shown in FIG. 4A, 3 of the five pups expressed GFP
while 2 pups did not.
[0139] Genomic DNA was prepared from the tails of the two
GFP-negative animals and screened for modification via PCR. When
compared to the wild-type eGFP locus, the regions bordering the
site targeted by the ZFN 59/60 pair were significantly reduced,
suggesting deletions of approximately 150 bp for both GFP-negative
animals. Again, no mosaicism was evident in the tail biopsy as
indicated by the absence of a wild-type eGFP band. These deletions
were then directly analyzed by sequencing, which revealed deletions
of 162 nt and 156 nt, resulting in the smaller bands evident in
FIG. 4B. Furthermore, as shown in FIG. 4B, no mosaicism was evident
in GFP negative pups since no wild-type eGFP band was detected.
[0140] Thus, ZFNs successfully modified the chromosomal GFP
transgene.
Example 3
ZFNs Cleave Endogenous Rat Loci
[0141] ZFNs were designed to cleave endogenous loci as described
below.
A. IgM
[0142] In one experiment, ZFNs were designed to cleave the
endogenous rat IgM gene, as described above and were tested for
cleavage activity in rat C6 cells. Exemplary rat IgM-targeted ZFPs
are shown below in Table 6 below.
TABLE-US-00006 TABLE 6 IgM Zinc-finger Designs ZFN Name F1 F2 F3 F4
F5 F6 17747 DRSHLTR RSDALTQ DRSDLSR RSDALAR RSDSLSA TSSNRKT (SEQ ID
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 41) NO: 40) NO: 28) NO:
39) NO: 38) NO: 37) 17749 NKVGLIE TSSDLSR RSDHLSR RSDNLSE QNAHRKT
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 46) NO: 45) NO: 44) NO:
43) NO: 42) 17759 DRSALSR TSGHLSR RSDNLST HNATRIN DRSALSR QSGNLAR
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 51) NO: 52) NO:
53) NO: 54) NO: 51) NO: 21) 17756 RSANLAR RSDNLRE TSGSLSR QSGSLTR
RSDVLSE TSGSLTR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:
56) NO: 57) NO: 58) NO: 59) NO: 60) NO: 25) 17767 QSSDLSR RSDALAR
TSGHLSR RSDALSR DRSDLSR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:
61) NO: 39) NO: 52) NO: 39) NO: 28) 17764 RSDALAR RSDHLST HSNARKN
DRSDLSR TSGHLSR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 39) NO:
62) NO: 63) NO: 28) NO: 52) 17782 RSANLSV DRANLSR RSDALAR DRSDLSR
RSDDLTR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 30) NO: 29) NO:
39) NO: 28) NO: 16) 17778 RSAHLSR QSGDLTR RSDALAR RSDTLSV DNSTRIK
(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 5) NO: 64) NO: 39) NO:
65) NO: 66)
[0143] Target sites of the rat IgM-targeted zinc-finger designs are
shown below in Table 7. Nucleotides in the target site that are
contacted by the ZFP recognition helices are indicated in uppercase
letters; non-contacted nucleotides indicated in lowercase.
TABLE-US-00007 TABLE 7 Target Sites of IgM Zinc-fingers ZFN Name
Target Site (5' to 3') 17747 AATTTGGTGGCCATGGGC (SEQ ID NO: 47)
17749 AGACAGGGGGCTCTC (SEQ ID NO: 48) 17759
ctGAAGTCATGCAGGGTGTCagaacctt (SEQ ID NO: 67) 17756
ttGTTCTGGTAGTTcCAGGAGaaggaaa (SEQ ID NO: 68) 17767
gtGCTGTGGGTGTGGCTagtgtttgtat (SEQ ID NO: 69) 17764
aaGGTGCCATTGGGGTGactttccatga (SEQ ID NO: 70) 17782
gaGAGGACcGTGGACAAGtccactggta (SEQ ID NO: 71) 17778
tcACCATGtGTGGCAGGGcctcgtggcc (SEQ ID NO: 72)
[0144] All IgM-targeted ZFNs contained the EL/KK Fok I mutations as
described in U.S. Patent Publication No. 2008/0131962. ZFN
expression was driven by either the CAG or the CMV promoter. ZFN (1
.mu.g each) were transfected into 200,000 C6 cells via Amaxa
nucleofection using the solution SF and the Amaxa Shuttle 96-well
nucleofector. The IgM locus was PCR amplified using GJC153F
(5'-ggaggcaagaagatggattc-3') and GJC154R
(5'-gaatcggcacatgcagatct-3') and ZFN cleavage was assayed with the
Surveyor.TM. nuclease as described, for example, in U.S. Patent
Publication Nos. 20080015164; 20080131962 and 20080159996.
[0145] In C6 cells, ZFN pair 17747/17749 cleaved 3% of chromosomes
when the CMV promoter was used and approximately 1% when the CAG
promoter was used (FIG. 4). This ZFN pair cleaved the rat IgM gene
in the coding region of exon 1. Rat oocytes were injected with 10
ng/uL of a plasmid encoding ZFN pair 17747/17749 under the control
of the CAG promoter using standard techniques. Oocytes were
fertilized and implanted into pseudo-pregnant females. Out of 430
oocytes injected and implanted, 43 live births resulted. Genomic
DNA was prepared from the tails of these 43 animals and screened
for modification using the Surveyor.TM. nuclease.
[0146] As shown in FIG. 7, five of the 43 animals (rats #6, 7, 8,
19, and 46) scored positive for modification at the IgM locus. The
patterns of Surveyor.TM. nuclease digestion were identical both
with and without the addition of wild-type rat genomic DNA,
suggesting that none of the rats has a homozygous mutation.
[0147] GJC153F/GJC154R PCR products from the positive rats were
cloned and sequenced. A description of the mutated alleles is in
Table 8.
TABLE-US-00008 TABLE 8 Approx. Rat Allele Count NHEJ % Notes 6
Wild-type 8 49 6 .DELTA.9 2 in frame deletion of DEN 7 Wild-type 5
31 7 .DELTA.5 1 out of frame 7 .DELTA.13 1 out of frame 7 .DELTA.15
3 in frame deletion of SDENL (SEQ ID NO: 182) 7 .DELTA.18 1 in
frame deletion of DENLA (SEQ ID NO: 183) 7 .DELTA.39 1 in frame
del. of SCESPLSDENLVA (SEQ ID NO: 184) 8 Wild-type 7 25 8 .DELTA.3,
7b bp mut. 3 in frame deletion of D, E->P 8 .DELTA.23 2 out of
frame 19 Wild-type 7 70 19 .DELTA.64 17 largest deletion, out of
frame 46 Wild-type 9 47 46 .DELTA.5 2 out of frame Count refers to
the number of time a particular sequence was isolated. NHEJ, % is
the approximate percentage of chromosomes modified in the tail
DNA
[0148] Sequencing of the IgM locus in these rats confirmed the
results of the Surveyor.TM. nuclease assay. All deletions overlap
the ZFN binding sites. The spectrum of small deletions seen here is
typical of NHEJ-mediated mutation. Rats 7 and 8 have more than one
mutated allele and are therefore mosaics for the IgM mutation.
Although sequencing of rats 6, 19, and 46 gave only one mutated
allele, they may be mosaic for IgM modification in other
tissues.
[0149] Thus, ZFNs successfully modified the endogenous rat IgM
locus.
[0150] To determine whether the ZFN plasmid itself integrated into
the rat genome, a PCR-based assay was developed to test for ZFN
plasmid integration. Briefly, rat genomic DNA and the ZFN plasmid
were mixed so as to mimic a plasmid insertion frequency of once per
genome. Performing 35 cycles of PCR amplification of this mixture
with one oligo in the CAG promoter (5'-GCT AAC CAT GTT CAT GCC
TTC-3') (SEQ ID NO:49) and another oligo in the 2A region of the
plasmid (5'-CAT CCT AGG GCC GGG ATT CTC-3') (SEQ ID NO:50) gave a
band of 1338 bp (FIG. 7, lane 3). When genomic DNA from wild-type
and the five ZFN-modified rats was analyzed, no PCR product was
detectable; indicating that insertion of the plasmid into the rat
genome is not a high-frequency event.
[0151] In addition, IgM modified rat #19 was further analyzed by
CEL-I assay and sequencing. As shown in FIGS. 7A and 7B, IgM-ZFNs
produced a 64 base pair deletion in this rat in the IgM locus.
[0152] Finally, ZFNs cleavage at off-target sites was also
evaluated. A computer algorithm was used to predict the location of
the most likely off-target sites (Doyon et al (2008) Nature
Biotechnology 26(6):702-708). All likely off-target sites were
assayed for ZFN modification using the Surveyor.TM. nuclease assay
as described above. The results of this analysis are shown in Table
9 and FIGS. 9A-C.
TABLE-US-00009 TABLE 9 Site Score Sequence Mm Gene PCR Frag. A
Frag. B Hit 1 8.18E-17 AGtcAGCttCCTGTCTAGAAGA 8 320 221 99 No
GAAcTgGGTGtCtATGGGCC (SEQ ID NO: 73) 2 2.90E-18
CaAatGCCaCCTGTCTGAATG 9 325 222 103 No GttTaTGcTGGCaATGGGCT (SEQ ID
NO: 74) 3 1.67E-18 GGtGAGaCCCCTGTCTTAACA 9 379 239 40 No
AAAgaTGGgGGggtTGGGaA (SEQ ID NO: 75) 4 7.75E-19
GatCCAaGGCCACCAAcTgGA 8 322 218 104 No GTTTAAGACAaaGGGCTCTgC (SEQ
ID NO: 76) 5 6.44E-19 TGtCCATGGCCtCCtccTcTTTG 9 Pde4d 396 200 196
No CTAGAgcGGtGGCTCTCA (SEQ ID NO: 77) 6 1.49E-19
GGAttGCCCCCTGTCaGTCAC 8 LOC499 342 200 142 No AGcATaTGGTGGCCATaGatG
913 (SEQ ID NO: 78) 7 1.14E-19 GGAGAagCCCaTGTgTACTCT 9 567 317 250
No TtAgTTGGTGGCtcTGGGaG (SEQ ID NO: 79) 8 1.07E-19
GcCCataGGCCAaCAAcTcTCA 9 Actn1 354 255 99 No GGCTAGACAacGGGCTCTCA
(SEQ ID NO: 80) Mm: mismatches relative to the intended target site
Frag. A, B: Expected sizes of Surveyor .TM. nuclease cleavage
products Hit: Rats showing correct Surveyor .TM. nuclease cleavage
products
[0153] As shown, no off-target sites tested showed evidence of
modification. As shown, no off-target sites tested showed evidence
of modification. Sequencing analysis of CEL-I positive rat #19
shown in FIG. 9A and five of its offspring shown in FIG. 11 at Site
1 revealed that the CEL-I positive signal was due to a SNP near the
potential off-target site. The mismatch occurs because the rats are
heterozygous for this SNP which was also found in non-treated rats
(data not shown). Although present in 50% of chromosomes in
CEL-I-positive animals, the SNP is poorly recognized by the CEL-I
enzyme resulting in unexpectedly lower-intensity cleavage
products.
B. Rab38
[0154] ZFNs were also designed to target the endogenous Rab38 locus
in rats, particularly exon 1 of the rat Rab38 gene. Exemplary Rab38
zinc-finger designs are shown in Table 10 below.
TABLE-US-00010 TABLE 10 Rab38 zinc-finger designs ZFN Name F1 F2 F3
F4 F5 F6 18160 DRSNLSS RSHSLLR RSDSLSA TSGSLTR QSGNLAR QSGHLSR (SEQ
ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 81) NO: 82) NO: 38)
NO: 25) NO: 21) NO: 83) 18181 TSGHLSR HKWQRNK DRSVLRR DSSTRKK
RSDHLSE DKSNRKK (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:
52) NO: 84) NO: 85) NO: 86) NO: 87) NO: 88) 16897 RSDTLSE QKRNRTK
RSDSLSA TSGSLTR QSGNLAR QSGHLSR (SEQ ID (SEQ ID (SEQ ID (SEQ ID
(SEQ ID (SEQ ID NO: 89) NO: 90) NO: 38) NO: 25) NO: 21) NO: 83)
16898 RSDHLSK HNDSRTN DRSDLSR RSDHLSE DKSNRKK N/A (SEQ ID (SEQ ID
(SEQ ID (SEQ ID (SEQ ID NO: 91) NO: 92) NO: 28) NO: 87) NO: 88)
18173 RSDYLPR QSNDLNS DRSDLSR RSDHLSE DKSNRKK N/A (SEQ ID (SEQ ID
(SEQ ID (SEQ ID (SEQ ID NO: 93) NO: 94) NO: 28) NO: 87) NO: 88)
18174 RSDYLPR QRVTRDA DRSDLSR RSDHLSE DKSNRKK N/A (SEQ ID (SEQ ID
(SEQ ID (SEQ ID (SEQ ID NO: 93) NO: 95) NO: 28) NO: 87) NO: 88)
18175 HSNARKT ASKTRTN DRSDLSR RSDHLSE DKSNRKK N/A (SEQ ID (SEQ ID
(SEQ ID (SEQ ID (SEQ ID NO: 96) NO: 97) NO: 28) NO: 87) NO: 88)
18161 RSHSLLR RSDSLSA TSGSLTR QSGNLAR QSGHLSR N/A (SEQ ID (SEQ ID
(SEQ ID (SEQ ID (SEQ ID NO: 82) NO: 38) NO: 25) NO: 21) NO: 83)
18183 RSHSLLR RSDYLPR DRSVLRR DSSTRKK RSDHLSE DKSNRKK (SEQ ID (SEQ
ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 82) NO: 93) NO: 85) NO: 86)
NO: 87) NO: 88)
[0155] Target sites of the rat Rab38-targeted zinc-finger designs
are shown below in Table 11. Nucleotides in the target site that
are contacted by the ZFP recognition helices are indicated in
uppercase letters; non-contacted nucleotides indicated in
lowercase.
TABLE-US-00011 TABLE 11 Target Sites of Rab38 Zinc-fingers ZFN Name
Target Site (5' to 3') 18161 gaGGAGAAGTTTTGGTGCACgtagcgct (SEQ ID
NO: 98) 18181 acTACCGGGCCACCATTGGTgtggactt (SEQ ID NO: 99) 16897
gaGGAGAAGTTTTGgTGCACGtagcgct (SEQ ID NO: 98) 16898
acTACCGGGCCacCATTGGtgtggactt (SEQ ID NO: 99) 18173
acTACCGGGCCaCCATTGgtgtggactt (SEQ ID NO: 99) 18174
acTACCGGGCCaCCATTGgtgtggactt (SEQ ID NO: 99) 18175
acTACCGGGCCACCATTggtgtggactt (SEQ ID NO: 99) 18160
gaGGAGAAGTTTTGGTGcacgtagcgct (SEQ ID NO: 98) 18183
acTACCGGGCCACCaTTGGTGtggactt (SEQ ID NO: 99)
[0156] All Rab38-targeted ZFNs contained the EL/KK Fok I mutations
as described in U.S. Patent Publication No. 2008/0131962. ZFN
expression was driven by either the CAG or the CMV promoter. ZFN (1
.mu.g each) were transfected into 200,000 C6 cells via Amaxa
nucleofection using the solution SF and the Amaxa Shuttle 96-well
nucleofector. Cleavage was assayed with the CEL-I Surveyor.TM.
nuclease as described, for example, in U.S. Patent Publication Nos.
20080015164; 20080131962 and 20080159996.
[0157] Rab38 ZFN-encoding expression plasmids were linearized with
XbaI, phenol chloroform extracted and precipitated. Messenger RNA
was in vitro transcribed using the MessageMax.TM. T7 ARCA-Capped
Message Transcription Kit (Epicentre Biotechnologies). The
resulting synthesis was purified using the MegaClear Kit.TM.
(Ambion) before resuspension in RNAse-free 0.1.times.TE (1 mM
Tris-Cl pH 8.0, 0.1 mM EDTA), quantitated using a NanoDrop-1000
(Thermo Scientific) and stored at -80.degree. C. until use.
Messenger RNAs encoding Rab38 ZFNs were mixed to a final total
concentration of 5 ng/.mu.L in 0.1.times.TE. Embryos were injected
with Rab38 ZFNs under constant time and pressure (Pi=65, Pc=20,
ti=1.5 s) into the cytoplasm and incubated at 37.5.degree. C. and
5% CO2 in KSOM (Millipore) overnight as previously described in
Filipiak et al. (2006) Transgenic Res 15:673-686 for molecular
analysis.
[0158] A mutation-enrichment strategy as described in Lloyd et al.
((2005) Proc Natl Acad Sci USA 102:2232-2237) was used to detect
alterations of the Rab38 target exon in chromosomes of DNA
extracted from embryos cultured for 48 hours post injection.
[0159] As shown in FIG. 10, and as demonstrated for both the GFP
and IgM loci above, multiple mutant Rab38 alleles could be detected
in the genomes of as few as 16 two-cell embryos and sequencing
revealed deletions at the target site.
[0160] Thus, these data confirm that multiple genomic loci are
suitable targets for ZFN-mediated genome editing.
Example 4
ZFN Mediated Germline Modifications
[0161] IgM-modified rats #19, #46 and #8 as described in Example 3
were mated to a wild-type rat and tail biopsies were taken, genomic
DNA isolated and then CEL-I and PCR assays were performed on the
nucleic acid purified from the pups.
[0162] As shown in FIGS. 11A and 11C, pups (numbered 225, 227, 228,
229, 230, 231, 234 and 235) resulting from a cross between rat #19
and a wild type rat carried the 64 base pair deletion of IgM
modified parental rat #19, as determined by PCR and CEL-I assay. In
addition, sequencing analysis confirmed that 3 pups of rat #19
(pups #225, 227 and 228) were modified at IgM locus. See, FIG. 11B.
Furthermore, as shown in FIG. 11C, a pup resulting from mating rat
#46 to a wild-type rat carried the same IgM modification as
parental rat #46 (see, pup number 236 of FIG. 11C).
[0163] These data demonstrate that ZFN-mediated disruption of a rat
locus is transmitted in the germline.
Example 5
Construction of Restriction Fragment Length Polymorphism (RFLP)
Donor Nucleic Acid for Targeted Integration into the PXR Nucleic
Acid Region of the Rat Genome
[0164] There are two possible DNA repair outcomes after a targeted,
ZFN-induced double-stranded break (FIG. 12). The break may be
repaired by non-homologous end joining (NHEJ), leading to mutations
containing base deletions or additions or, in the presence of a
donor DNA, the donor DNA can be used as a template to repair the
double stranded break by homologous recombination (HR). If the
donor DNA encodes specific sequence changes, these deliberate
mutations will be incorporated into the genome of the organism at
the target site.
[0165] To test targeted integration in the rat genome using
pronuclear injection, constructs were designed and prepared for
targeted integration into the PXR gene region of the rat genome.
Constructs were assembled to introduce either a NotI or PmeI
restriction fragment length polymorphism (RFLP) site into the PXR
gene region (FIG. 13). The constructs were designed with either
200, 800 or 2000 base pairs of sequence homology to the PXR gene
target site flanking the RFLP sites to be introduced. The three
sizes of regions of homology were used to determine the size of
homology required for efficient targeting and homologous
recombination.
[0166] The clones were assembled using PCR amplification to
introduce convenient restriction sites for cloning, and the RFLP
site at the extremities of the PXR homology regions (FIG. 12). PCR
primers used for amplifying the P.times.R region of homology are
described in Table 12. Accuprime HF DNA polymerase was used for PCR
reaction amplification. A 30 s extension was used for the 200 bp
fragments, a 1.5 min extension was used for the 800 bp fragments,
and a 4 min extension was used for the 2 Kbp fragments. PCR
fragments were then digested with the appropriate restriction
enzymes and cloned into pBluescript using three-way ligation to
produce six plasmids listed in Table 13.
TABLE-US-00012 TABLE 12 Primer sequences Name Sequence PXR 200 bp
5'-aaaaggtacctctgtgtttttccgttctagt F KpnI ccag (SEQ ID NO: 116) PXR
200 bp 5'-aaaaccgcggctgaagtatacgtggctctct R SacII tgga (SEQ ID NO:
117) PXR target 5'-gtgtagcggccgcgacaaggccaatggctat F NotI cac (SEQ
ID NO: 118) PXR target 5'-gtgtagtttaaacgacaaggccaatggctat F PmeI
cac (SEQ ID NO: 119) PXR target 5'-ttgtcgcggccgctacacggcagatttgaag
R NotI acc tc (SEQ ID NO: 120) PXR target
5'-ttgtcgtttaaactacacggcagatttgaag R PmeI acctc (SEQ ID NO: 121)
PXR 800 bp 5'-aaaaggtacctcagactggtccagattttag F KpnI amaagggg (SEQ
ID NO: 122) PXR 800 bp 5'-aaaaccgcggataaatctactggttcgccaa R SacII
gctag (SEQ ID NO: 123) PXR 2Kb 5'-aaaaggtaccgaggtagtaggaaatgcacttc
F KpnI (SEQ ID NO: 124) PXR 2Kb 5'-aaaaccgcgggaagagaattattgctgacagt
R SacII c (SEQ ID NO: 125) PXR 50 bp 5'-gagcctatcaacgtagatgagg F
(SEQ ID NO: 126) PXR 50 bp 5'-cttacatccttcacaggtcatgac R (SEQ ID
NO: 127)
TABLE-US-00013 TABLE 13 Plasmids constructed RFLP Length of region
of introduced homology NotI 200 bp NotI 800 bp NotI 2 Kbp PmeI 200
bp PmeI 800 bp PmeI 2 Kbp
Example 6
Construction of Restriction Fragment Length Polymorphism (RFLP)
Donor Nucleic Acid for Targeted Integration into the rRosa26
Nucleic Acid Region of the Rat Genome
[0167] Plasmids were also constructed to target integration of NotI
and PmeI RFLP sites into the rRosa26 nucleic acid region of the rat
genome. Design and construction of the plasmids was as described in
Example 5 above. The PCR primer pairs used for amplifying the
rRosa26 region of homology are described in Table 14.
TABLE-US-00014 TABLE 14 Primer sequences Name Sequence rRosa26 200
bp aaaaggtaccgggagtggatgaaggagttg F KpnI (SEQ ID NO: 128) rRosa26
200 bp aaaaccgcggcggatcacaagcaataat R SacII (SEQ ID NO: 129)
rRose26 target cttcgcggccgcgatctgcaactggagtctttc F NotI (SEQ ID NO:
130) rRosa26 target cttcgtttaaacgatctgcaactggagtctttc F PmeI (SEQ
ID NO: 131) rRosa26 target gatcgcggccgcgaagaagggggaagggaatc F NotI
(SEQ ID NO: 132) rRosa26 target gatcgtttaaacgaagaagggggaagggaatc R
PmeI (SEQ ID NO: 133) rRosa26 800 bp aaaaggtaccgcgtgtgaaaacacaaatgg
F KpnI (SEQ ID NO: 134) rRosa26 800 bp
aaaaccgcggaaggaaagaggcattcatgg R SacII (SEQ ID NO: 135) rRosa26 2Kb
aaaaggtaccattatggaggggaggactgg F KpnI (SEQ ID NO: 136) rRosa26 2Kb
aaaaccgcggacatgtggcaaacaggaga R SacII (SEQ ID NO: 137) rRosa26 50
bp F tgtcttctgaggaccgccc (SEQ ID NO: 138) rRosa26 50 bp R
ctgcccagaagactcccgc (SEQ ID NO: 139)
Example 7
Construction of Restriction Fragment Length Polymorphism (RFLP)
Donor Nucleic Acid for Targeted Integration into the Mdr1a Nucleic
Acid Region of the Mouse or Rat Genome
[0168] Plasmids were constructed to target integration of NotI and
PmeI RFLP sites into the mMdr1a nucleic acid region of the mouse
genome or the rMdr1a nucleic acid region of the rat genome. Design
and construction of the plasmids was as described in Example 5
above. The PCR primer pairs used for amplifying the Mdr1a region of
homology are described in Tables 15 and 16. "m" stands for mouse
and "r" stands for rat.
TABLE-US-00015 TABLE 15 Name Sequence mMdr1a 200 bp
aaaaggraccaacaacactaggctcaggag F KpnI (SEQ ID NO: 140) mMdr1a 200
bp aaaaccgcggcacatggctaagcacagcatg R SacII (SEQ ID NO: 141) mMdr1a
target cctgcggccgcggactgtcagctggtatttg F NotI (SEQ ID NO: 142)
mMdr1a target cctgtttaaacggactgtcagctggtatttg F PmeI (SEQ ID NO:
143) mMdr1a target gtccgcggccgcagggctgatggccaaaatc R NotI (SEQ ID
NO: 144) mMdr1a target gtccgtttaaacagggctgatggccaaaatc R PmeI (SEQ
ID NO: 145) mMdr1a 800 bp aaaaggtaccatgctgtgaagcagatacc F KpnI (SEQ
ID NO: 146) mMdr1a 800 bp aaaaccgcggctgaaaactgaatgagacatttgc R
SacII (SEQ ID NO: 147) mMdr1a 2KB aaaaggtaccgtaatgttccaattgcatcttcc
F KpnI (SEQ ID NO: 148) mMdr1a 2KB aaaaccgcggctctcagttctctgctgttg R
SacII (SEQ ID NO: 149) mMdr1a 50 bp F gatttacccgtggctggaag (SEQ ID
NO: 150) mMdr1a 50 bp R ctggactcatggacttcacc (SEQ ID NO: 151)
TABLE-US-00016 TABLE 15 Name Sequence rMdr1a 200 bp
aaaaggtacctggctcaggagaaaaattgtg F KpnI (SEQ ID NO: 152) rMdr1a 200
bp aaaaccgcggcacggctaaagacagcatga R SacII (SEQ ID NO: 153) rMdr1a
target ccctgcggccgcggactgtcagctggtatttg F NotI (SEQ ID NO: 154)
rMdr1a target ccctgtttaaacggactgtcagctggtatttg F PmeI (SEQ ID NO:
155) rMdr1a target gtccgcggccgcagggctgatggccaaaatc R NotI (SEQ ID
NO: 156) rMdr1a target gtccgtttaaacagggctgatggccaaaatc R PmeI (SEQ
ID NO: 157) rMdr1a 800 bp aaaaggtaccggagataggctggtttgacg F KpnI
(SEQ ID NO: 158) rMdr1a 700 bp aaaaccgcggatggtggtagttcggatgg R
SacII (SEQ ID NO: 159) rMdr1a 2Kb aaaaaggtaccaggttgttcttggagatgtgc
F KpnI (SEQ ID NO: 160) rMdr1a 2Kb aaaaccgcggtcctcttggctggtgagttt T
SacII (SEQ ID NO: 161) rMdr1a 50 bp F gatttactcgcggctggaag (SEQ ID
NO: 162) rMdr1a 50 bp R ctggactcacgggcttcac (SEQ ID NO: 163)
Example 8
Construction of GFP Expression Integration Cassette
[0169] To test targeted integration of nucleic acid fragments
larger than RFLPs, constructs were designed and prepared for
targeted integration of a GFP expression cassette into the PXR and
rRosa26 nucleic acid genomic regions of the rat and the mMdr1a
nucleic acid genomic regions of the mouse. Briefly, a GFP
expression cassette containing the human PGK promoter, the GFP open
reading frame, and a polyadenylation signal was amplified using PCR
to introduce NotI restriction sites at the extremities (FIG. 14)
using the following primers: PGKGFP-F NotI
(5'-aaagcggccgcttggggttgcgccttttcc) (SEQ ID NO:164) and PGKGFP-R
NotI (5'-aaaagcggccgccatagagcccaccgcatc) (SEQ ID NO:165). The PCR
fragment was then cloned into the NotI-containing plasmids
constructed in Examples 5-7.
Example 9
Preparation of Zinc Finger mRNAs for Targeted Integration
[0170] A pair of zinc finger nucleases were designed for each
targeted integration site and cloned as described on the Sigma web
site. For more information, see Science (2009) 325:433, herein
incorporated by reference. ZFN expressing mRNAs were then produced
in vitro by first digesting 20 .mu.g of each maxiprepped ZFN
expression plasmid DNA in 100 .mu.l reactions containing 10 .mu.l
buffer 2 (NEB, #B7002S), 10 .mu.l 10.times.BSA (diluted from
100.times.BSA, NEB, #B90015), 8 .mu.l XbaI (NEB, #R0145S), at
37.degree. C. for 2 h. The reactions were extracted with 100 .mu.l
of phenol/chloroform (Sigma, P2069), centrifuged at over
20,000.times.g for 10 min. The aqueous supernatant was precipitated
with 10 .mu.l 3M NaOAc (Sigma, 57899) and 250 .mu.l 100% ethanol
and centrifuged at top speed for 25 min at room temperature. The
resulting pellet was washed by adding 300 .mu.l 70% ethanol
filtered through a 0.02 .mu.M filter. The pellet was air dried and
resuspended in 20 .mu.l of 0.02 .mu.M filtered 0.1.times.TE.
[0171] The purified digested DNA was then used to produce ZFN
transcripts using in vitro transcription with MessageMax T7 Capped
Message Transcription Kit (#MMA60710) from Epicentre
Biotechnologies as described. In short, kit components are
prewarmed to room temperature, and reaction components for a 20
.mu.l reaction were combined at room temperature in the following
order: 5 .mu.l of 0.02 um filtered RNase-free water, 1 .mu.l
prepared template, 2 .mu.l lox transcription buffer, 8 .mu.l 2-way
Cap/NTP premix, 2 .mu.l 100 mM DTT and 2 .mu.l MessageMax T7 Enzyme
Solution. The reactions were then incubated in a 37.degree. C.
incubator for 30 min.
[0172] The capped RNA was then tailed with polyA using the A-Plus
Poly (A) Polymerase tailing kit (Epicentre, #PAP5 104H) as
described. Reaction components were combined at room temperature in
the following given order: 55.5 .mu.l 0.02 um filtered RNase-free
water, 10 .mu.l 10.times.A-Plus Reaction Buffer, 10 ul 10 mM ATP,
2.5 .mu.l ScriptGuard RNase Inhibitor (40 unit/.mu.l), 20 .mu.l In
vitro transcription capping reaction, 2 .mu.l A-plus poly A
polymerase. The reaction was then incubated at 37.degree. C. for 30
min. The resulting capped polyA-tailed mRNA was purified by
precipitation with an equal volume of 5M NH.sub.4Oac twice. The
mRNA pellet was then air dried, and resuspended in 30 .mu.l of
filtered injection buffer (1 mM Tris, pH7.4, 0.25 mM EDTA), and RNA
concentration was measured using a Nanodrop spectrophotometer.
Example 10
Targeted Integration into Embryos
[0173] To integrate nucleic acids into the rat or mouse genome,
zinc finger nuclease mRNA was mixed with the maxiprepped target DNA
filtered with 0.02 um filters. The nucleic acid mixture consisted
of one part ZFN mRNAs to one part donor DNA. The nucleic acid
mixture was then microinjected into the pronucleus of a one-celled
embryo using known methods. The injected embryos were either
incubated in vitro, or transferred to pseudo moms. The resulting
embryos/fetus, or the toe/tail of clip live born animals were
harvested for DNA extraction and analysis.
[0174] To extract DNA, tissue was lysed in 100 .mu.l Epicentre's
QuickExtract at 50.degree. C. for 30 min, followed by incubation at
65.degree. C. for 10 min, and 98.degree. C. for 3 min. To determine
if targeted integration occurred, PCR was used to amplify the
target region using appropriate primers. For experiments where RFLP
was integrated into the genome of the animal, the PCR products were
digested with the introduced RFLP enzyme to detect integration
(FIG. 15A). In addition, a Cel-I endonuclease assay using wild type
PCR fragments and PCR fragments derived from injected embryos was
used demonstrate ZFN mRNA was functional in the embryos by
detecting NHEJ, which is independent of targeted integration. For
experiments where GFP was integrated into the genome of the animal,
a shift in size of the PCR fragment is indicative of the
integration (FIG. 15B). Alternatively, amplification of the
integration junction, where one primer lands only on the GFP
cassette was used to assess integration of the donor nucleic
acid.
Example 11
Testing of DNA Extraction and PCR Amplification of the mMdr1a
Target Site in the Mouse Genome
[0175] PCR conditions to amplify target nucleic acid extracted from
tissue were tested using embryos with 1-64 cells extracted as
described in Example 10. A 900 bp fragment containing the mouse
mMdr1a target region was amplified using 36 amplification cycles
with 4 min extension at 60.degree. C. in reactions containing up to
5 .mu.l Epicentre's QuickExtract solution in 50 .mu.l reactions
(FIG. 16). These results show that QuickExtract does not interfere
with PCR amplification, and that DNA can be amplified from sample
extracted from only 1-10 cells. To enhance sensitivity, the number
of PCR cycles may be increased, or nested PCR reactions may be
performed.
Example 12
Integration of NotI Donor RFLP into the Rat PXR Genomic Region
[0176] A donor plasmid (with an 800 bp arm) for integrating a NotI
RFLP site into the P.times.R region of the rat genome was injected
into rat embryos with ZFN mRNAs as described above. PCR, followed
by NotI restriction enzyme analysis and Cel-I endonuclease analysis
were performed using DNA extracted from a number of embryos. PCR
amplification was successful with a number of embryos (FIG. 17A),
and Cel-I endonuclease analysis revealed that most of the fragments
had nucleic acid sequence changes at the desired target (FIG.
17B).
Example 13
Integration of NotI Donor RFLP into the Mouse mMdr1a Genomic
Region
[0177] The targeted integration of the NotI RFLP into the mouse
mMdr1a region was repeated as described in Example 8. The mMdr1a
region was amplified using PCR and digested with NotI. PCR
amplification was successful with a number of embryos (FIG. 18),
and digestion with NotI revealed that a number of embryos comprised
the integrated RFLP site (see e.g. lanes 13, 17, 19, 20 and 23). In
all, targeted integration in 7 out of the 32 embryos for which data
was generated.
[0178] These results were confirmed by repeating the NotI digestion
reaction after further cleaning the PCR reaction product (FIG.
19).
Example 14
Testing DNA Extraction and PCR Amplification of the PXR Target Site
in the Rat Genome
[0179] PCR amplification of the P.times.R region from blastocysts
was tested to determine the level of sensitivity. The PCR reaction
contained 5 .mu.l template, 5 .mu.l PCR buffer, 5 .mu.l of each
primer, 0.5 .mu.l of Taq polymerase enzyme, and 33.5 .mu.l water
for a 50 .mu.l reaction. The template consisted of undiluted DNA
extracted from rat blastocysts or DNA diluted at a ratio of 1:2,
1:6, 1:10, and 1:30 (FIG. 20).
Example 15
Integration of NotI Donor RFLP into the Rat PXR Genomic Region
[0180] A donor plasmid (with 800 bp homology arms) for integrating
a NotI RFLP site into the P.times.R region of the rat genome was
injected into rat embryos with ZFN mRNAs as described above. A
total of 123 embryos were injected, and 106 survived. Decreasing
concentrations of nucleic acids were injected to test for toxicity.
Of the 51 embryos injected with 5 ng of nucleic acids, 17 survived
and divided to two cell embryos on day two. Of the 23 embryos
injected with 2 ng of nucleic acids, 14 survived and divided to two
cell embryos on day two. Of the 29 embryos injected with 10 ng of
nucleic acids, 12 survived and divided to two cell embryos on day
two. Of the ten uninjected control embryos, all survived and
divided to two cell embryos on day two.
[0181] PCR amplification of the P.times.R region, followed by NotI
and Cel-I endonuclease analysis were performed using DNA extracted
from a number of embryos. PCR amplification was successful with a
number of embryos, and NotI and Cel-I endonuclease analysis
revealed that 18 out of 47 embryos had nucleic acid sequence
changes at the desired target (FIG. 21).
Example 16
Targeted Integration of RFLP into the mMdr1a Target Region of the
Mouse Genome in Fetus
[0182] A donor plasmid (with 800 bp homology arms) for introducing
NotI into the mMdr1a region of the mouse genome was injected into
mouse embryos with ZFN mRNAs as described above. One out of four
well-developed fetuses at 12.5 dpc were positive for the NotI site.
All four deciduas were negative. (FIG. 22).
Example 17
Targeted Integration of GFP into the mMdr1a Locus of a Fetus
[0183] A donor plasmid (with 800 bp homology arms) for introducing
GFP cassette into the mMdr1a region of the mouse genome was
injected into mouse embryos with ZFN mRNAs as described above. Two
out of forty fetuses at 12.5 dpc were positive for the GFP cassette
(FIG. 23).
Example 18
Targeted Integration of RFLP into the PXR Target Region of the Rat
Genome in a Fetus
[0184] A donor plasmid (with 800 bp homology arms) for introducing
NotI into the P.times.R region of the rat genome was injected into
mouse embryos with ZFN mRNAs as described above. One out of eight
fetuses at 13 dpc were positive for the NotI site (FIG. 24).
[0185] All patents, patent applications and publications mentioned
herein are hereby incorporated by reference in their entirety.
[0186] Although disclosure has been provided in some detail by way
of illustration and example for the purposes of clarity of
understanding, it will be apparent to those skilled in the art that
various changes and modifications can be practiced without
departing from the spirit or scope of the disclosure. Accordingly,
the foregoing descriptions and examples should not be construed as
limiting.
Sequence CWU 1
1
18415PRTArtificial SequenceDescription of Artificial Sequence
Synthetic linker peptide 1Thr Gly Glu Lys Pro 1 5 26PRTArtificial
SequenceDescription of Artificial Sequence Synthetic linker peptide
2Thr Gly Gly Gln Arg Pro 1 5 35PRTArtificial SequenceDescription of
Artificial Sequence Synthetic linker peptide 3Thr Gly Gln Lys Pro 1
5 46PRTArtificial SequenceDescription of Artificial Sequence
Synthetic linker peptide 4Thr Gly Ser Gln Lys Pro 1 5
57PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger targeted to rat
IgM and GFP 5Arg Ser Ala His Leu Ser Arg 1 5 67PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized GFP zinc finger 6Thr Ser Ala Asn Leu
Ser Arg 1 5 77PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideChemically synthesized GFP zinc finger
7Arg Ser Asp Asn Leu Ser Val 1 5 87PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized GFP zinc finger 8Asp Arg Ser Asn Leu
Thr Arg 1 5 97PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideChemically synthesized GFP zinc finger
9Arg Ser Asp Thr Leu Ser Gln 1 5 107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized GFP zinc finger 10Gln Arg Asp His Arg
Ile Lys 1 5 117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideChemically synthesized GFP zinc finger
11Asp Arg Ser Asn Leu Ser Arg 1 5 127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat p53 and
GFP 12Asp Arg Ser His Leu Ala Arg 1 5 137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized GFP zinc finger 13Arg Ser Asp His Leu
Ser Ala 1 5 147PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideChemically synthesized GFP zinc finger
14Asp Ser Ser Thr Arg Lys Thr 1 5 157PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized GFP zinc finger 15Thr Ser Gly Ser Leu
Ser Arg 1 5 167PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideChemically synthesized zinc finger
targeted to rat IgM, GFP and p53 16Arg Ser Asp Asp Leu Thr Arg 1 5
177PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized GFP zinc finger 17Arg Ser
Asp Asn Leu Ser Thr 1 5 187PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideChemically synthesized GFP
zinc finger 18Asp Ser Ser Ser Arg Ile Lys 1 5 197PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized GFP zinc finger 19Arg Ser Ala Val Leu
Ser Glu 1 5 207PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideChemically synthesized GFP zinc finger
20Thr Asn Ser Asn Arg Ile Thr 1 5 217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat p53,
Rab38, IgM and GFP 21Gln Ser Gly Asn Leu Ala Arg 1 5
227PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized GFP zinc finger 22Gln Ser
Gly Ser Leu Thr Arg 1 5 237PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideChemically synthesized GFP
zinc finger 23Gln Ser Ser Asp Leu Arg Arg 1 5 247PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized GFP zinc finger 24Arg Ser Asp Ala Leu
Ser Arg 1 5 257PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideChemically synthesized zinc finger
targeted to rat Rab38, IgM and GFP 25Thr Ser Gly Ser Leu Thr Arg 1
5 267PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized GFP zinc finger 26Asp Ser
Ser Ala Arg Lys Lys 1 5 277PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideChemically synthesized GFP
zinc finger 27Arg Ser Asp Ser Leu Ser Val 1 5 287PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38,
IgM and GFP 28Asp Arg Ser Asp Leu Ser Arg 1 5 297PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat IgM and
GFP 29Asp Arg Ala Asn Leu Ser Arg 1 5 307PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat IgM and
GFP 30Arg Ser Ala Asn Leu Ser Val 1 5 3112DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized target site of GFP zinc
finger 31gaccaggatg gg 123216DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotideChemically synthesized
target site of GFP zinc finger 32gacggcgacg taaacg
163316DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideChemically synthesized target site of GFP
zinc finger 33gatgcggttc accagg 163419DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized target site of GFP zinc
finger 34gaagggcatc gacttcaag 193518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized target site of GFP zinc
finger 35gttgtggctg ttgtagtt 183615DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized target site of GFP zinc
finger 36atcatggccg acaag 15377PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideChemically synthesized zinc
finger protein targeted to rat IgM 37Thr Ser Ser Asn Arg Lys Thr 1
5 387PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger targeted to rat
Rab38 and IgM gene 38Arg Ser Asp Ser Leu Ser Ala 1 5
397PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 39Arg Ser Asp Ala Leu Ala Arg 1 5
407PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 40Arg Ser Asp Ala Leu Thr Gln 1 5
417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 41Asp Arg Ser His Leu Thr Arg 1 5
427PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 42Gln Asn Ala His Arg Lys Thr 1 5
437PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger targeted to rat
p53 and IgM 43Arg Ser Asp Asn Leu Ser Glu 1 5 447PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat IgM and
p53 44Arg Ser Asp His Leu Ser Arg 1 5 457PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger protein targeted to rat
IgM 45Thr Ser Ser Asp Leu Ser Arg 1 5 467PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger protein targeted to rat
IgM 46Asn Lys Val Gly Leu Ile Glu 1 5 4718DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized target site of IgM zinc
finger 47aatttggtgg ccatgggc 184815DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized target site of IgM zinc
finger 48agacaggggg ctctc 154921DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotideSynthetic
oligonucleotide 49gctaaccatg ttcatgcctt c 215021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideSynthetic oligonucleotide 50catcctaggg ccgggattct c
21517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 51Asp Arg Ser Ala Leu Ser Arg 1 5
527PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger targeted to rat
IgM and Rab38 52Thr Ser Gly His Leu Ser Arg 1 5 537PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger protein targeted to rat
IgM 53Arg Ser Asp Asn Leu Ser Thr 1 5 547PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger protein targeted to rat
IgM 54His Asn Ala Thr Arg Ile Asn 1 5 557PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat p53 55Asp
Asn Pro Asn Leu Asn Arg 1 5 567PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideChemically synthesized zinc
finger protein targeted to rat IgM 56Arg Ser Ala Asn Leu Ala Arg 1
5 577PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 57Arg Ser Asp Asn Leu Arg Glu 1 5
587PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 58Thr Ser Gly Ser Leu Ser Arg 1 5
597PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 59Gln Ser Gly Ser Leu Thr Arg 1 5
607PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 60Arg Ser Asp Val Leu Ser Glu 1 5
617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 61Gln Ser Ser Asp Leu Ser Arg 1 5
627PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 62Arg Ser Asp His Leu Ser Thr 1 5
637PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 63His Ser Asn Ala Arg Lys Asn 1 5
647PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 64Gln Ser Gly Asp Leu Thr Arg 1 5
657PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 65Arg Ser Asp Thr Leu Ser Val 1 5
667PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger protein
targeted to rat IgM 66Asp Asn Ser Thr Arg Ile Lys 1 5
6728DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideChemically synthesized target site of IgM
zinc finger 67ctgaagtcat gcagggtgtc agaacctt 286828DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized target site of IgM zinc
finger 68ttgttctggt agttccagga gaaggaaa 286928DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized target site of IgM zinc
finger 69gtgctgtggg tgtggctagt gtttgtat 287028DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized target site of IgM zinc
finger 70aaggtgccat tggggtgact ttccatga 287128DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized target site of IgM zinc
finger 71gagaggaccg tggacaagtc cactggta 287228DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized target site of IgM zinc
finger 72tcaccatgtg tggcagggcc tcgtggcc 287342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 73agtcagcttc ctgtctagaa gagaactggg tgtctatggg cc
427441DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 74caaatgccac ctgtctgaat ggtttatgct
ggcaatgggc t 417541DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 75ggtgagaccc ctgtcttaac
aaaagatggg ggggttggga a 417642DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 76gatccaaggc
caccaactgg agtttaagac aaagggctct gc 427741DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 77tgtccatggc ctcctcctct ttgctagagc ggtggctctc a
417842DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 78ggattgcccc ctgtcagtca cagcatatgg
tggccataga tg 427941DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 79ggagaagccc atgtgtactc
tttagttggt ggctctggga g 418042DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 80gcccataggc
caacaactct caggctagac aacgggctct ca 42817PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 81Asp Arg Ser Asn Leu Ser Ser 1 5 827PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 82Arg Ser His Ser Leu Leu Arg 1 5 837PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 83Gln Ser Gly His Leu Ser Arg 1 5 847PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 84His Lys Trp Gln Arg Asn Lys 1 5 857PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 85Asp Arg Ser Val Leu Arg Arg 1 5 867PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 86Asp Ser Ser Thr Arg Lys Lys 1 5 877PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38 and
p53 87Arg Ser Asp His Leu Ser Glu 1 5 887PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 88Asp Lys Ser Asn Arg Lys Lys 1 5 897PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 89Arg Ser Asp Thr Leu Ser Glu 1 5
907PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideChemically synthesized zinc finger targeted to rat
Rab38 gene 90Gln Lys Arg Asn Arg Thr Lys 1 5 917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 91Arg Ser Asp His Leu Ser Lys 1 5 927PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 92His Asn Asp Ser Arg Thr Asn 1 5 937PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 93Arg Ser Asp Tyr Leu Pro Arg 1 5 947PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 94Gln Ser Asn Asp Leu Asn Ser 1 5 957PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 95Gln Arg Val Thr Arg Asp Ala 1 5 967PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 96His Ser Asn Ala Arg Lys Thr 1 5 977PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat Rab38
gene 97Ala Ser Lys Thr Arg Thr Asn 1 5 9828DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized target site of rat Rab38 zinc
finger 98gaggagaagt tttggtgcac gtagcgct 289928DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized target site of rat Rab38 zinc
finger 99actaccgggc caccattggt gtggactt 281007PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat p53
100Arg Ser Asp Asp Leu Ser Arg 1 5 1017PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat p53
101Asn Ser Gln His Leu Thr Glu 1 5 1027PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat p53
102Gln Ser Ser His Leu Ser Arg 1 5 1037PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat p53
103Arg Ser Asp Asn Leu Ala Arg 1 5 1047PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat p53
104Arg Ser Asp His Leu Thr Thr 1 5 1057PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat p53
105Arg Ser Asp Asn Leu Ser Gln 1 5 1067PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat p53
106Ala Ser Asn Asp Arg Lys Lys 1 5 1077PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat p53
107Arg Ser Ala Ala Leu Ala Arg 1 5 1087PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat p53
108Arg Asn Gln His Arg Ile Thr 1 5 1097PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat p53
109Asp Ser Arg Ser Arg Ile Asn 1 5 1107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideChemically synthesized zinc finger targeted to rat p53
110Asp Arg Ser His Leu Ser Arg 1 5 11128DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized zinc finger targeted to rat
p53 111aagcggaagg ggcgggccat agcccggg 2811228DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized zinc finger targeted to rat
p53 112caggacgtgc ggaatgcgtt aagggaat 2811328DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized zinc finger targeted to rat
p53 113cttcccagtg ggaggtgaca gaaccctg 2811428DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized zinc finger targeted to rat
p53 114accggcgggt gcgggcggac tgcactta 2811528DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideChemically synthesized zinc finger targeted to rat
p53 115ccggcgggtg cgggcggact gcacttag 2811635DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer PXR 200
bp F Kpnl 116aaaaggtacc tctgtgtttt tccgttctag tccag
3511735DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer PXR 200 bp R Sacll 117aaaaccgcgg ctgaagtata
cgtggctctc ttgga 3511834DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer PXR target F Notl
118gtgtagcggc cgcgacaagg ccaatggcta tcac 3411934DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer PXR
target F Pmel 119gtgtagttta aacgacaagg ccaatggcta tcac
3412036DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer PXR target R Notl 120ttgtcgcggc cgctacacgg
cagatttgaa gacctc 3612136DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer PXR target R Pmel
121ttgtcgttta aactacacgg cagatttgaa gacctc 3612239DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer PXR 800
bp F Kpnl 122aaaaggtacc tcagactggt ccagatttta gamaagggg
3912336DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer PXR 800 bp R Sacll 123aaaaccgcgg ataaatctac
tggttcgcca agctag 3612432DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer PXR 2Kb F Kpnl 124aaaaggtacc
gaggtagtag gaaatgcact tc 3212533DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer PXR 2Kb R Sacll
125aaaaccgcgg gaagagaatt attgctgaca gtc 3312622DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer PXR 50
bp F 126gagcctatca acgtagatga gg 2212724DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer PXR 50
bp R 127cttacatcct tcacaggtca tgac 2412830DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer rRosa26
200 bp F Kpnl 128aaaaggtacc gggagtggat gaaggagttg
3012928DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer rRosa26 200 bp R Sacll 129aaaaccgcgg cggatcacaa
gcaataat 2813033DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer rRose26 target F Notl 130cttcgcggcc
gcgatctgca actggagtct ttc 3313133DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer rRosa26 target F Pmel
131cttcgtttaa acgatctgca actggagtct ttc 3313232DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer rRosa26
target F Notl 132gatcgcggcc gcgaagaagg gggaagggaa tc
3213332DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer rRosa26 target R Pmel 133gatcgtttaa acgaagaagg
gggaagggaa tc 3213430DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer rRosa26 800 bp F Kpnl
134aaaaggtacc gcgtgtgaaa acacaaatgg 3013530DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer rRosa26
800 bp R Sacll 135aaaaccgcgg aaggaaagag gcattcatgg
3013630DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer rRosa26 2Kb F Kpnl 136aaaaggtacc attatggagg
ggaggactgg 3013729DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer rRosa26 2Kb R Sacll 137aaaaccgcgg
acatgtggca aacaggaga 2913819DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer rRosa26 50 bp F 138tgtcttctga
ggaccgccc 1913919DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer rRosa26 50 bp R 139ctgcccagaa gactcccgc
1914030DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer mMdr1a 200 bp F Kpnl 140aaaaggracc aacaacacta
ggctcaggag 3014131DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer mMdr1a 200 bp R Sacll 141aaaaccgcgg
cacatggcta agcacagcat g 3114231DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer mMdr1a target F Notl
142cctgcggccg cggactgtca gctggtattt g 3114331DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer mMdr1a
target F Pmel 143cctgtttaaa cggactgtca gctggtattt g
3114431DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer mMdr1a target R Notl 144gtccgcggcc gcagggctga
tggccaaaat c 3114531DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer mMdr1a target R Pmel 145gtccgtttaa
acagggctga tggccaaaat c 3114629DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer mMdr1a 800 bp F Kpnl
146aaaaggtacc atgctgtgaa gcagatacc 2914734DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer mMdr1a
800 bp R Sacll 147aaaaccgcgg ctgaaaactg aatgagacat ttgc
3414833DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer mMdr1a 2KB F Kpnl 148aaaaggtacc gtaatgttcc
aattgcatct tcc 3314930DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer mMdr1a 2KB R Sacll
149aaaaccgcgg ctctcagttc tctgctgttg 3015020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer mMdr1a
50 bp F 150gatttacccg tggctggaag 2015120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer mMdr1a
50 bp R 151ctggactcat ggacttcacc 2015231DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer rMdr1a
200 bp F Kpnl 152aaaaggtacc tggctcagga gaaaaattgt g
3115330DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer rMdr1a 200 bp R Sacll 153aaaaccgcgg cacggctaaa
gacagcatga 3015432DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer rMdr1a target F Notl 154ccctgcggcc
gcggactgtc agctggtatt tg 3215532DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer rMdr1a target F Pmel
155ccctgtttaa acggactgtc agctggtatt tg 3215631DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer rMdr1a
target R Notl 156gtccgcggcc gcagggctga tggccaaaat c
3115731DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer rMdr1a target R Pmel 157gtccgtttaa acagggctga
tggccaaaat c 3115830DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer rMdr1a 800 bp F Kpnl 158aaaaggtacc
ggagataggc tggtttgacg 3015929DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer rMdr1a 700 bp R Sacll
159aaaaccgcgg atggtggtag ttcggatgg 2916032DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer rMdr1a
2Kb F Kpnl 160aaaaaggtac caggttgttc ttggagatgt gc
3216130DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer rMdr1a 2Kb T Sacll 161aaaaccgcgg tcctcttggc
tggtgagttt 3016220DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer rMdr1a 50 bp F 162gatttactcg cggctggaag
2016319DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer rMdr1a 50 bp R 163ctggactcac gggcttcac
1916430DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer PGKGFP-F NotI 164aaagcggccg cttggggttg cgccttttcc
3016530DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer PGKGFP-R NotI 165aaaagcggcc gccatagagc ccaccgcatc
30166111DNARattus norvegicus 166cctgcgagag ccccctgtct gatgagaatt
tggtggccat gggctgcctg gcccgggact 60tcctgcccag ctccatttcc ttctcctgga
actaccagaa caacactgaa g 111167111DNARattus norvegicus 167cctgcgagag
ccccctgtct gatgagaatt tggtggccat gggctgcctg gcccgggact 60tcctgcccag
ctccatttcc ttctcctgga actaccagaa caacactgaa g 11116847DNARattus
norvegicus 168cctgcgagag ccccctgtct cctggaacta ccagaacaac actgaag
4716975DNARattus norvegicus 169atcatcaagc gctacgtgca ccaaaacttc
tcctcccact accgggccac cattggtgtg 60gacttcgcgc tgaag
7517069DNARattus norvegicus 170atcatcaagc gctacgtgca ccaaaacttc
tcctaccggg ccaccattgg tgtggacttc 60gcgctgaag 6917133DNARattus
norvegicus 171atcatcaagc gctacgtgga cttcgcgctg aag 3317242DNARattus
norvegicus 172ctgcgagagc cccctgtctc ctggaactac cagaacaaca ct
42173106DNARattus norvegicus 173ctgcgagagc cccctgtctg atgagaattt
ggtggccatg ggctgcctgg cccgggactt 60cctgcccagc tccatttcct tctcctggaa
ctaccagaac aacact 106174106DNARattus norvegicus 174ctgcgagagc
cccctgtctg atgagaattt ggtggccatg ggctgcctgg cccgggactt 60cctgcccagc
tccatttcct tctcctggaa ctaccagaac aacact 10617542DNARattus
norvegicus 175ctgcgagagc cccctgtctc ctggaactac cagaacaaca ct
42176106DNARattus norvegicus 176ctgcgagagc cccctgtctg atgagaattt
ggtggccatg ggctgcctgg cccgggactt 60cctgcccagc tccatttcct tctcctggaa
ctaccagaac aacact 10617742DNARattus norvegicus 177ctgcgagagc
cccctgtctc ctggaactac cagaacaaca ct 42178106DNARattus norvegicus
178ctgcgagagc cccctgtctg atgagaattt ggtggccatg ggctgcctgg
cccgggactt 60cctgcccagc tccatttcct tctcctggaa ctaccagaac aacact
10617942DNARattus norvegicus 179ctgcgagagc cccctgtctc ctggaactac
cagaacaaca ct 4218020DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 180ggaggcaaga agatggattc
2018120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 181gaatcggcac atgcagatct 201825PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 182Ser
Asp Glu Asn Leu 1 5 1835PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 183Asp Glu Asn Leu Ala 1 5
18413PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 184Ser Cys Glu Ser Pro Leu Ser Asp Glu Asn Leu
Val Ala 1 5 10
* * * * *
References