U.S. patent application number 10/415440 was filed with the patent office on 2005-06-30 for methods of modifying eukaryotic cells.
Invention is credited to Economides, Aris N., Frendewey, David, Murphy, Andrew J., Valenzuela, David M., Yancopoulos, George D..
Application Number | 20050144655 10/415440 |
Document ID | / |
Family ID | 46150314 |
Filed Date | 2005-06-30 |
United States Patent
Application |
20050144655 |
Kind Code |
A1 |
Economides, Aris N. ; et
al. |
June 30, 2005 |
Methods of modifying eukaryotic cells
Abstract
A method for engineering and utilizing large DNA vectors to
target, via homologous recombination, and modify, in any desirable
fashion, endogenous genes and chromosomal loci in eukaryotic cells.
These large DNA targeting vectors for eukaryotic cells, termed
LTVECs, are derived from fragments of cloned genomic DNA larger
than those typically used by other approaches intended to perform
homologous targeting in eukaryotic cells. Also provided is a rapid
and convenient method of detecting eukaryotic cells in which the
LTVEC has correctly targeted and modified the desired endogenous
genes(s) or chromosomal locus (loci) as well as the use of these
cells to generate organisms bearing the genetic modification.
Inventors: |
Economides, Aris N.;
(Tarrytown, NY) ; Murphy, Andrew J.;
(Croton-on-Hudson, NY) ; Valenzuela, David M.;
(Yorktown Heights, NY) ; Frendewey, David; (New
York, NY) ; Yancopoulos, George D.; (Yoktown Heights,
NY) |
Correspondence
Address: |
Linda O Palladino
Regeneron Pharmaceuticals Inc
777 Old Saw Mill River Road
Tarrytown
NY
10591
US
|
Family ID: |
46150314 |
Appl. No.: |
10/415440 |
Filed: |
April 29, 2003 |
PCT Filed: |
October 31, 2001 |
PCT NO: |
PCT/US01/45375 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10415440 |
Apr 29, 2003 |
|
|
|
09732234 |
Dec 7, 2000 |
|
|
|
6586251 |
|
|
|
|
60244665 |
Oct 31, 2000 |
|
|
|
60244665 |
Oct 31, 2000 |
|
|
|
Current U.S.
Class: |
800/8 ; 435/325;
435/353; 435/354; 435/455; 435/6.16; 800/14; 800/18 |
Current CPC
Class: |
C12N 2510/00 20130101;
C12N 2810/10 20130101; C12N 15/85 20130101; C12Q 1/6827 20130101;
A01K 2217/05 20130101; C12N 5/0606 20130101; C12N 15/907 20130101;
C12N 15/79 20130101; A01K 2227/105 20130101; A01K 67/0275
20130101 |
Class at
Publication: |
800/008 ;
435/455; 435/325; 435/006; 435/353; 435/354; 800/018; 800/014 |
International
Class: |
A01K 067/027; C12Q
001/68; C12N 005/06; C12N 015/85 |
Claims
We claim:
1. A method for genetically modifying an endogenous gene or
chromosomal locus of interest in eukaryotic cells, comprising: a)
obtaining a large cloned genomic fragment containing a DNA sequence
of interest; b) using bacterial homologous recombination to
genetically modify the large cloned genomic fragment of (a) to
create a large targeting vector for use in the eukaryotic cells
(LTVEC); c) introducing the LTVEC of (b) into the eukaryotic cells
to modify the endogenous gene or chromosomal locus in the cells;
and d) using a quantitative assay to detect modification of allele
(MOA) in the eukaryotic cells of (c) to identify those eukaryotic
cells in which the endogenous gene or chromosomal locus has been
genetically modified.
2. The method of claim 1 wherein the large cloned genomic fragment
containing a DNA sequence is homologous to the endogenous gene or
chromosomal locus of interest.
3. The method of claim 1 wherein the genetic modification to the
endogenous gene or chromosomal locus comprises deletion of a coding
sequence, gene segment, or regulatory element; alteration of a
coding sequence, gene segment, or regulatory element; insertion of
a new coding sequence, gene segment, or regulatory element;
creation of a conditional allele; or replacement of a coding
sequence or gene segment from one species with an homologous or
orthologous coding sequence from the same or a different
species.
4. The method of claim 3 wherein the alteration of a coding
sequence, gene segment, or regulatory element comprises a
substitution, addition, or fusion.
5. The method of claim 4 wherein the fusion comprises an epitope
tag or bifunctional protein.
6. The method of claim 1 wherein the quantitative assay comprises
quantitative PCR, FISH, comparative genomic hybridization,
isothermic DNA amplification, quantitative hybridization to an
immobilized probe, Invader Probes.RTM., or MMP assays.RTM..
7. The method of claim 6 wherein the quantitative PCR comprises
TaqMan.RTM., Molecular Beacon, or Eclipse.TM. probe technology.
8. The method of claim 1 wherein the eukaryotic cell is a mammalian
embryonic stem cell.
9. The method of claim 8 wherein the embryonic stem cell is a
mouse, rat, or other rodent embryonic stem cell.
10. The method of claim 1 wherein the endogenous gene or
chromosomal locus is a mammalian gene or chromosomal locus.
11. The method of claim 10 wherein the endogenous gene or
chromosomal locus is a human gene or chromosomal locus.
12. The method of claim 10 wherein the endogenous gene or
chromosomal locus is a mouse, rat, or other rodent gene or
chromosomal locus.
13. The method of claim 1 wherein the LTVEC is capable of
accommodating large DNA fragments greater than 20 kb.
14. The method of claim 13 wherein the LTVEC is capable of
accommodating large DNA fragments greater than 100 kb.
15. A method for genetically modifying an endogenous gene or
chromosomal locus of interest in mouse embryonic stem cells,
comprising: a) obtaining a large cloned genomic fragment greater
than 20 kb which contains a DNA sequence of interest, wherein the
large cloned DNA fragment is homologous to the endogenous gene or
chromosomal locus; b) using bacterial homologous recombination to
genetically modify the large cloned genomic fragment of (a) to
create a large targeting vector for use in the mouse embryonic stem
cells, wherein the genetic modification is deletion of a coding
sequence, gene segment, or regulatory element; c) introducing the
large targeting vector of (b) into the mouse embryonic stem cells
to modify the endogenous gene or chromosomal locus in the cells;
and d) using a quantitative assay to detect modification of allele
(MOA) in the mouse embryonic stem cells of (c) to identify those
mouse embryonic stem cells in which the endogenous gene or
chromosomal locus has been genetically modified, wherein the
quantitative assay is quantitative PCR.
16. A genetically modified endogenous gene or chromosomal locus
produced by the method of any one of claims 1 or 15.
17. The genetically modified endogenous gene or chromosomal locus
of claim 16 wherein the genetic modification to the endogenous gene
or chromosomal locus comprises deletion of a coding sequence, gene
segment, or regulatory element; alteration of a coding sequence,
gene segment, or regulatory element; insertion of a new coding
sequence, gene segment, or regulatory element; creation of a
conditional allele; or replacement of a coding sequence or gene
segment from one species with an homologous or orthologous coding
sequence from the same or a different species.
18. The genetically modified endogenous gene or chromosomal locus
of claim 17 wherein the alteration of a coding sequence, gene
segment, or regulatory element comprises a substitution, addition,
or fusion.
19. The genetically modified endogenous gene or chromosomal locus
of claim 18 wherein the fusion comprises an epitope tag or
bifunctional protein.
20. A genetically modified eukaryotic cell produced by the method
of claim 1.
21. A genetically modified mouse embryonic stem cell produced by
the method of claim 15.
22. The genetically modified eukaryotic cell of claim 20 wherein
the genetic modification to the endogenous gene or chromosomal
locus comprises deletion of a coding sequence, gene segment, or
regulatory element; alteration of a coding sequence, gene segment,
or regulatory element; insertion of a new coding sequence, gene
segment, or regulatory element; creation of a conditional allele;
or replacement of a coding sequence or gene segment from one
species with an homologous or orthologous coding sequence from the
same or a different species.
23. The genetically modified eukaryotic cell of claim 22 wherein
the alteration of a coding sequence, gene segment, or regulatory
element comprises a substitution, addition, or fusion.
24. The genetically modified eukaryotic cell of claim 23 wherein
the fusion comprises an epitope tag or bifunctional protein.
25. A non-human organism containing a genetically modified
endogenous gene or chromosomal locus produced by the method of
claim 1.
26. A mouse containing a genetically modified endogenous gene or
chromosomal locus produced by the method of claim 15.
27. A non-human organism produced from the genetically modified
eukaryotic cell of any one of claims 20, 22, 23, or 24.
28. A mouse produced from the genetically modified mouse embryonic
stem cell of claim 21.
29. A genetically modified embryonic stem cell produced by the
method of claim 1.
30. The genetically modified embryonic stem cell of claim 29
wherein the genetic modification to the endogenous gene or
chromosomal locus comprises deletion of a coding sequence, gene
segment, or regulatory element; alteration of a coding sequence,
gene segment, or regulatory element; insertion of a new coding
sequence, gene segment, or regulatory element; creation of a
conditional allele; or replacement of a coding sequence or gene
segment from one species with an homologous or orthologous coding
sequence from a different species.
31. The genetically modified embryonic stem cell of claim 30
wherein the alteration of a coding sequence, gene segment, or
regulatory element comprises a substitution, addition, or
fusion.
32. The genetically modified embryonic stem cell of claim 31
wherein the fusion comprises an epitope tag or bifunctional
protein.
33. A non-human organism containing a genetically modified
endogenous gene or chromosomal locus of interest, produced by a
method comprising the steps of: a) obtaining a large cloned genomic
fragment containing a DNA sequence of interest; b) using bacterial
homologous recombination to genetically modify the large cloned
genomic fragment of (a) to create a large targeting vector (LTVEC)
for use in embryonic stem cells; c) introducing the LTVEC of (b)
into the embryonic stem cells to modify the endogenous gene or
chromosomal locus in the cells; d) using a quantitative assay to
detect modification of allele (MOA) in the embryonic stem cells of
(c) to identify those embryonic stem cells in which the endogenous
gene or chromosomal locus has been genetically modified; e)
introducing the embryonic stem cell of (d) into a blastocyst; and
f) introducing the blastocyst of (e) into a surrogate mother for
gestation.
34. A mouse containing a genetically modified endogenous gene or
chromosomal locus of interest, produced by a method comprising the
steps of: a) obtaining a large cloned genomic fragment greater than
20 kb which contains a DNA sequence of interest, wherein the large
cloned DNA fragment is homologous to the endogenous gene or
chromosomal locus; b) using bacterial homologous recombination to
genetically modify the large cloned genomic fragment of (a) to
create a large targeting vector for use in the mouse embryonic stem
cells, wherein the genetic modification is deletion of a coding
sequence, gene segment, or regulatory element; c) introducing the
large targeting vector of (b) into the mouse embryonic stem cells
to modify the endogenous gene or chromosomal locus in the cells;
and d) using a quantitative assay to detect modification of allele
(MOA) in the mouse embryonic stem cells of (c) to identify those
mouse embryonic stem cells in which the endogenous gene or
chromosomal locus has been genetically modified, wherein the
quantitative assay is quantitative PCR; e) introducing the mouse
embryonic stem cell of (d) into a blastocyst; and f) introducing
the blastocyst of (e) into a surrogate mother for gestation.
35. A non-human organism containing a genetically modified
endogenous gene or chromosomal locus, produced by a method
comprising the steps of: a) obtaining a large cloned genomic
fragment containing a DNA sequence of interest; b) using bacterial
homologous recombination to genetically modify the large cloned
genomic fragment of (a) to create a large targeting vector for use
in eukaryotic cells (LTVEC); c) introducing the LTVEC of (b) into
the eukaryotic cells to genetically modify the endogenous gene or
chromosomal locus in the cells; d) using a quantitative assay to
detect modification of allele (MOA) in the eukaryotic cells of (c)
to identify those eukaryotic cells in which the endogenous gene or
chromosomal locus has been genetically modified; e) removing the
nucleus from the eukaryotic cell of (d); f) introducing the nucleus
of (e) into an oocyte; and g) introducing the oocyte of (f) into a
surrogate mother for gestation.
36. A non-human organism containing a genetically modified
endogenous gene or chromosomal locus, produced by a method
comprising the steps of: a) obtaining a large cloned genomic
fragment containing a DNA sequence of interest; b) using bacterial
homologous recombination to genetically modify the large cloned
genomic fragment of (a) to create a large targeting vector for use
in eukaryotic cells (LTVEC); c) introducing the LTVEC of (b) into
the eukaryotic cells to genetically modify the endogenous gene or
chromosomal locus in the cells; d) using a quantitative assay to
detect modification of allele (MOA) in the eukaryotic cells of (c)
to identify those eukaryotic cells in which the endogenous gene or
chromosomal locus has been genetically modified; e) fusing the
eukaryotic cell of (d) with another eukaryotic cell; and f)
introducing the fused eukaryotic cell of (e) into a surrogate
mother for gestation.
37. The non-human organism of any one of claims 33, 35, or 36
wherein the large cloned genomic fragment containing a DNA sequence
is homologous to the endogenous gene or chromosomal locus of
interest.
38. The non-human organism of any one of claims 25, 33, 35, or 36
wherein the non-human organism is a mouse, rat, or other
rodent.
39. The non-human organism of claim 33 wherein the blastocyst is a
mouse, rat, or other rodent blastocyst.
40. The non-human organism of claim 35 wherein the oocyte is a
mouse, rat, or other rodent oocyte.
41. The non-human organism of any one of claims 33, 35, or 36
wherein the surrogate mother is a mouse, rat, or other rodent.
42. The non-human organism of claim 33 wherein the embryonic stem
cell is a mammalian embryonic stem cell.
43. The non-human organism of claim 42 wherein the mammalian
embryonic stem cell is a mouse, rat, or other rodent embryonic stem
cell.
44. The non-human organism of any one of claims 33, 35, or 36
wherein the genetic modification to the endogenous gene or
chromosomal locus comprises deletion of a coding sequence, gene
segment, or regulatory element; alteration of a coding sequence,
gene segment, or regulatory element; insertion of a new coding
sequence, gene segment, or regulatory element; creation of a
conditional allele; or replacement of a coding sequence or gene
segment from one species with an homologous or orthologous coding
sequence from the same or a different species.
45. The non-human-organism of claim 44 wherein the alteration of a
coding sequence, gene segment, or regulatory element comprises a
substitution, addition, or fusion.
46. The non-human organism of claim 45 wherein the fusion comprises
an epitope tag or bifunctional protein.
47. The non-human organism of any one of claims 33, 35, or 36
wherein the quantitative assay comprises quantitative PCR, FISH,
comparative genomic hybridization, isothermic DNA amplification,
quantitative hybridization to an immobilized probe, Invader
Probes.RTM., or MMP.RTM. assays.
48. The non-human organism of claim 47 wherein the quantitative PCR
comprises TaqMan.RTM., Molecular Beacon, or Eclipse.TM. probe
technology.s.
49. The use of the genetically modified eukaryotic cell of claim 20
for the production of a non-human organism.
50. The use of the genetically modified mouse embryonic stem cell
of claim 21 for the production of a mouse.
51. The use of the genetically modified embryonic stem cell of
claim 29 for the production of a non-human organism.
52. The method of claims 1 or 15, wherein about 1-5 .mu.g of the
large targeting vector of (c) is introduced to about
1.times.10.sup.7 cells.
53. The mouse of claim 34, wherein about 1-5 .mu.g of the large
targeting vector of (c) is introduced to about 1.times.10.sup.7
cells.
54. The non-human organism of claims 33, 35, or 36, wherein about
1-5 .mu.g of the large targeting vector of (c) is introduced to
about 1.times.10.sup.7 cells.
Description
[0001] This application claims priority to U.S. patent application
Ser. No. 09/732,234, filed Dec. 7, 2000 and U.S. Provisional
Application No. 60/244,665, filed Oct. 31, 2000. Throughout this
application various publications are referenced. The disclosures of
these publications in their entireties are hereby incorporated by
reference into this application.
FIELD OF THE INVENTION
[0002] The field of this invention is a method for engineering and
utilizing large DNA vectors to target, via homologous
recombination, and modify, in any desirable fashion, endogenous
genes and chromosomal loci in eukaryotic cells. These large DNA
targeting vectors for eukaryotic cells, termed LTVECs, are derived
from fragments of cloned genomic DNA larger than those typically
used by other approaches intended to perform homologous targeting
in eukaryotic cells. The field of the invention further provides
for a rapid and convenient method of detecting eukaryotic cells in
which the LTVEC has correctly targeted and modified the desired
endogenous gene(s) or chromosomal locus(loci). The field also
encompasses the use of these cells to generate organisms bearing
the genetic modification, the organisms, themselves, and methods of
use thereof.
INTRODUCTION
[0003] The use of LTVECs provides substantial advantages over
current methods. For example, since these are derived from DNA
fragments larger than those currently used to generate targeting
vectors, LTVECs can be more rapidly and conveniently generated from
available libraries of large genomic DNA fragments (such as BAC and
PAC libraries) than targeting vectors made using current
technologies. In addition, larger modifications as well as
modifications spanning larger genomic regions can be more
conveniently generated than using current technologies.
[0004] Furthermore, the present invention takes advantage of long
regions of homology to increase the targeting frequency of "hard to
target" loci, and also diminishes the benefit, if any, of using
isogenic DNA in these targeting vectors.
[0005] The present invention thus provides for a rapid, convenient,
and streamlined method for systematically modifying virtually all
the endogenous genes and chromosomal loci of a given organism.
BACKGROUND OF THE INVENTION
[0006] Gene targeting by means of homologous recombination between
homologous exogenous DNA and endogenous chromosomal sequences has
proven to be an extremely valuable way to create deletions,
insertions, design mutations, correct gene mutations, introduce
transgenes, or make other genetic modifications in mice. Current
methods involve using standard targeting vectors, with regions of
homology to endogenous DNA typically totaling less than 10-20 kb,
to introduce the desired genetic modification into mouse embryonic
stem (ES) cells, followed by the injection of the altered ES cells
into mouse embryos to transmit these engineered genetic
modifications into the mouse germline (Smithies et al., Nature,
317:230-234, 1985; Thomas et al., Cell, 51:503-512, 1987; Koller et
al., Proc Natl Acad Sci USA, 86:8927-8931, 1989; Kuhn et al.,
Science, 254:707-710, 1991; Thomas et al., Nature, 346:847-850,
1990; Schwartzberg et al., Science, 246:799-803, 1989; Doetschman
et al., Nature, 330:576-578, 1987; Thomson et al., Cell, 5:313-321;
1989; DeChiara et al., Nature, 345:78-80, 1990; U.S. Pat. No.
5,789,215, issued Aug. 4, 1998 in the name of GenPharm
International) In these current methods, detecting the rare ES
cells in which the standard targeting vectors have correctly
targeted and modified the desired endogenous gene(s) or chromosomal
locus(loci) requires sequence information outside of the homologous
targeting sequences contained within the targeting vector. Assays
for successful targeting involve standard Southern blotting or long
PCR (Cheng, et al., Nature, 369:684-5, 1994; Foord and Rose, PCR
Methods Appl, 3:S149-61, 1994; Ponce and Micol, Nucleic Acids Res,
20:623, 1992; U.S. Pat. No. 5,436,149 issued to Takara Shuzo Co.,
Ltd.) from sequences outside the targeting vector and spanning an
entire homology arm (see Definitions); thus, because of size
considerations that limit these methods, the size of the homology
arms are restricted to less than 10-20 kb in total (Joyner, The
Practical Approach Series, 293, 1999).
[0007] The ability to utilize targeting vectors with homology arms
larger than those used in current methods would be extremely
valuable. For example, such targeting vectors could be more rapidly
and conveniently generated from available libraries containing
large genomic inserts (e.g. BAC or PAC libraries) than targeting
vectors made using current technologies, in which such genomic
inserts have to be extensively characterized and trimmed prior to
use. In addition, larger modifications as well as modifications
spanning larger genomic regions could be more conveniently
generated and in fewer steps than using current technologies.
Furthermore, the use of long regions of homology could increase the
targeting frequency of "hard to target" loci in eukaryotic cells,
since the targeting of homologous recombination in eukaryotic cells
appears to be related to the total homology contained within the
targeting vector (Deng and Capecchi, Mol Cell Biol,
12:3365-71,1992). In addition, the increased targeting frequency
obtained using long homology arms could diminish any potential
benefit that can be derived from using isogenic DNA in these
targeting vectors.
[0008] The problem of engineering precise modifications into very
large genomic fragments, such as those cloned in BAC libraries, has
largely been solved through the use of homologous recombination in
bacteria (Zhang, et al., Nat Genet, 20:123-8, 1998; Yang, et al.,
Nat Biotechnol, 15:859-65, 1997; Angrand, et al., Nucleic Acids
Res, 27:e16, 1999; Muyrers, et al., Nucleic Adds Res, 27:1555-7,
1999; Narayanan, et al., Gene Ther, 6:442-7, 1999), allowing for
the construction of vectors containing large regions of homology to
eukaryotic endogenous genes or chromosomal loci. However, once
made, these vectors have not been generally useful for modifying
endogenous genes or chromosomal loci via homologous recombination
because of the difficulty in detecting rare correct targeting
events when homology arms are larger than 10-20 kb (Joyner, The
Practical Approach Series, 293, 1999). Consequently, vectors
generated using bacterial homologous recombination from BAC genomic
fragments must still be extensively trimmed prior to use as
targeting vectors (Hill et al., Genomics, 64:111-3, 2000).
Therefore, there is still a need for a rapid and convenient
methodology that makes possible the use of targeting vectors
containing large regions of homology so as to modify endogenous
genes or chromosomal loci in eukaryotic cells.
[0009] In accordance with the present invention, Applicants provide
novel methods that enables the use of targeting vectors containing
large regions of homology so as to modify endogenous genes or
chromosomal loci in eukaryotic cells via homologous recombination.
Such methods overcome the above-described limitations of current
technologies. In addition, the skilled artisan will readily
recognize that the methods of the invention are easily adapted for
use with any genomic DNA of any eukaryotic organism including, but
not limited to, animals such as mouse, rat, other rodent, or human,
as well as plants such as soy, corn and wheat.
SUMMARY OF THE INVENTION
[0010] In accordance with the present invention, Applicants have
developed a novel, rapid, streamlined, and efficient method for
creating and screening eukaryotic cells which contain modified
endogenous genes or chromosomal loci. This novel methods combine,
for the first time:
[0011] 1. Bacterial homologous recombination to precisely engineer
a desired genetic modification within a large cloned genomic
fragment, thereby creating a large targeting vector for use in
eukaryotic cells (LTVECs);
[0012] 2. Direct introduction of these LTVECs into eukaryotic cells
to modify the endogenous chromosomal locus of interest in these
cells; and
[0013] 3. An analysis to determine the rare eukaryotic cells in
which the targeted allele has been modified as desired, involving
an assay for modification of allele (MOA) of the parental allele
that does not require sequence information outside of the targeting
sequence, such as, for example, quantitative PCR.
[0014] A preferred embodiment of the invention is a method for
genetically modifying an endogenous gene or chromosomal locus in
eukaryotic cells, comprising: a) obtaining a large cloned genomic
fragment containing a DNA sequence of interest; b) using bacterial
homologous recombination to genetically modify the large cloned
genomic fragment of (a) to create a large targeting vector for use
in the eukaryotic cells (LTVEC); c) introducing the LTVEC of (b)
into the eukaryotic cells to modify the endogenous gene or
chromosomal locus in the cells; and d) using a quantitative assay
to detect modification of allele (MOA) in the eukaryotic cells of
(c) to identify those eukaryotic cells in which the endogenous gene
or chromosomal locus has been genetically modified.
[0015] Another embodiment of the invention is a method wherein the
genetic modification to the endogenous gene or chromosomal locus
comprises deletion of a coding sequence, gene segment, or
regulatory element; alteration of a coding sequence, gene segment,
or regulatory element; insertion of a new coding sequence, gene
segment, or regulatory element; creation of a conditional allele;
or replacement of a coding sequence or gene segment from one
species with an homologous or orthologous coding sequence from a
different species.
[0016] An alternative embodiment of the invention is a method
wherein the alteration of a coding sequence, gene segment, or
regulatory element comprises a substitution, addition, or fusion,
wherein the fusion comprises an epitope tag or bifunctional
protein.
[0017] Yet another embodiment of the invention is a method wherein
the quantitative assay comprises quantitative PCR, comparative
genomic hybridization, isothermal DNA amplification, quantitative
hybridization to an immobilized probe, Invader Probes.RTM., or MMP
assays.RTM., and wherein the quantitative PCR comprises TaqMan.RTM.
Molecular Beacon, or Eclipse.TM. probe technology.
[0018] Another preferred embodiment of the invention is a method
wherein the eukaryotic cell is a mammalian embryonic stem cell and
in particular wherein the embryonic stem cell is a mouse, rat, or
other rodent embryonic stem cell.
[0019] Another preferred embodiment of the invention is a method
wherein the endogenous gene or chromosomal locus is a mammalian
gene or chromosomal locus, preferably a human gene or chromosomal
locus or a mouse, rat, or other rodent gene or chromosomal
locus.
[0020] An additional preferred embodiment is one in which the LTVEC
is capable of accommodating large DNA fragments greater than 20 kb,
and in particular large DNA fragments greater than 100 kb.
[0021] Another preferred embodiment is a genetically modified
endogenous gene or chromosomal locus that is produced by the method
of the invention.
[0022] Yet another preferred embodiment is a genetically modified
eukaryotic cell that is produced by the method of the
invention.
[0023] A preferred embodiment of the invention is a non-human
organism containing the genetically modified endogenous gene or
chromosomal locus produced by the method of the invention.
[0024] Also preferred in a non-human organism produced from the
genetically modified eukaryotic cells or embryonic stem cells
produced by the method of the invention.
[0025] A preferred embodiment is a non-human organism containing a
genetically modified endogenous gene or chromosomal locus, produced
by a method comprising the steps of: a) obtaining a large cloned
genomic fragment containing a DNA sequence of interest; b) using
bacterial homologous recombination to genetically modify the large
cloned genomic fragment of (a) to create a large targeting vector
(LTVEC) for use in embryonic stem cells; c) introducing the LTVEC
of (b) into the embryonic stem cells to modify the endogenous gene
or chromosomal locus in the cells; d) using a quantitative assay to
detect modification of allele (MOA) in the embryonic stem cells of
(c) to identify those embryonic stem cells in which the endogenous
gene or chromosomal locus has been genetically modified; e)
introducing the embryonic stem cell of (d) into a blastocyst; and
f) introducing the blastocyst of (e) into a surrogate mother for
gestation.
[0026] An additional preferred embodiment of the invention is a
non-human organism containing a genetically modified endogenous
gene or chromosomal locus, produced by a method comprising the
steps of: a) obtaining a large cloned genomic fragment containing a
DNA sequence of interest; b) using bacterial homologous
recombination to genetically modify the large cloned genomic
fragment of (a) to create a large targeting vector for use in
eukaryotic cells (LTVEC); c) introducing the LTVEC of (b) into the
eukaryotic cells to genetically modify the endogenous gene or
chromosomal locus in the cells; d) using a quantitative assay to
detect modification of allele (MOA) in the eukaryotic cells of (c)
to identify those eukaryotic cells in which the endogenous gene or
chromosomal locus has been genetically modified; e) removing the
nucleus from the eukaryotic cell of (d); f) introducing the nucleus
of (e) info an oocyte; and g) introducing the oocyte of (f) into a
surrogate mother for gestation.
[0027] Yet another preferred embodiment is a non-human organism
containing a genetically modified endogenous gene or chromosomal
locus, produced by a method comprising the steps of: a) obtaining a
large cloned genomic fragment containing a DNA sequence of
interest; b) using bacterial homologous recombination to
genetically modify the large cloned genomic fragment of (a) to
create a large targeting vector for use in eukaryotic cells
(LTVEC); c) introducing the LTVEC of (b) into the eukaryotic cells
to genetically modify the endogenous gene or chromosomal locus in
the cells; d) using a quantitative assay to detect modification of
allele (MOA) in the eukaryotic cells of (c) to identify those
eukaryotic cells in which the endogenous gene or chromosomal locus
has been genetically modified; e) fusing the eukaryotic cell of (d)
with another eukaryotic cell; f) introducing the fused eukaryotic
cell of (e) into a surrogate mother for gestation.
[0028] In preferred embodiments, the non-human organism is a mouse,
rat, or other rodent; the blastocyst is a mouse, rat, or other
rodent blastocyst; the oocyte is a mouse, rat, or other rodent
oocyte; and the surrogate mother is a mouse, rat, or other
rodent.
[0029] Another preferred embodiment is one in which the embryonic
stem cell is a mammalian embryonic stem cell, preferably a mouse,
rat, or other rodent embryonic stem cell.
[0030] An additional preferred embodiment is the use of the
genetically modified eukaryotic cells of the invention for the
production of a non-human organism, and in particular, the use of
the genetically modified embryonic stem cell of the invention for
the production of a non-human organism.
[0031] A preferred embodiment of the invention is a method for
genetically modifying an endogenous gene or chromosomal locus of
interest in mouse embryonic stem cells, comprising: a) obtaining a
large cloned genomic fragment greater than 20 kb which contains a
DNA sequence of interest, wherein the large cloned DNA fragment is
homologous to the endogenous gene or chromosomal locus; b) using
bacterial homologous recombination to genetically modify the large
cloned genomic fragment of (a) to create a large targeting vector
for use in the mouse embryonic stem cells, wherein the genetic
modification is deletion of a coding sequence, gene segment, or
regulatory element; c) introducing the large targeting vector of
(b) into the mouse embryonic stem cells to modify the endogenous
gene or chromosomal locus in the cells; and d) using a quantitative
assay to detect modification of allele (MOA) in the mouse embryonic
stem cells of (c) to identify those mouse embryonic stem cells in
which the endogenous gene or chromosomal locus has been genetically
modified, wherein the quantitative assay is quantitative PCR. Also
preferred is a genetically modified mouse embryonic stem cell
produced by this method; a mouse containing a genetically modified
endogenous gene or chromosomal locus produced by this method; and a
mouse produced from the genetically modified mouse embryonic stem
cell.
[0032] Another preferred embodiment is a mouse containing a
genetically modified endogenous gene or chromosomal locus of
interest, produced by a method comprising the steps of: a)
obtaining a large cloned genomic fragment greater than 20 kb which
contains a DNA sequence of interest, wherein the large cloned DNA
fragment is homologous to the endogenous gene or chromosomal locus;
b) using bacterial homologous recombination to genetically modify
the large cloned genomic fragment of (a) to create a large
targeting vector for use in the mouse embryonic stem cells, wherein
the genetic modification is deletion of a coding sequence, gene
segment, or regulatory element; c) introducing the large targeting
vector of (b) into the mouse embryonic stem cells to modify the
endogenous gene or chromosomal locus in the cells; and d) using a
quantitative assay to detect modification of allele (MOA) in the
mouse embryonic stem cells of (c) to identify those mouse embryonic
stem cells in which the endogenous gene or chromosomal locus has
been genetically modified, wherein the quantitative assay is
quantitative PCR; e) introducing the mouse embryonic stem cell of
(d) into a blastocyst; and f) introducing the blastocyst of (e)
into a surrogate mother for gestation.
[0033] Also preferred is the use of the genetically modified mouse
embryonic stem cell described above for the production of a
mouse.
[0034] Also preferred are methods wherein about 1-5 .mu.g of large
targeting vector DNA is introduced into about 1.times.10.sup.7
eukaryotic cells.
BRIEF DESCRIPTION OF THE FIGURES
[0035] FIG. 1: Schematic diagram of the generation of a typical
LTVEC using bacterial homologous recombination. (hb1=homology box
1; hb2=homology box 2; RE=restriction enzyme site).
[0036] FIG. 2: Schematic diagram of donor fragment and LIVEC for
mouse OCR10. (hb1=homology box 1; lacZ=.beta.-galactosidase ORF;
SV40 polyA=a DNA fragment derived from Simian Virus 40, containing
a polyadenylation site and signal; PGKp=mouse phosphoglycerate
kinase (PGK) promoter; EM7=a bacterial promoter; neo=neomycin
phosphotransferase; PGK polyA=3' untranslated region derived from
the PGK gene and containing a polyadenylation site and signal;
hb2=homology box 2)
[0037] FIG. 3A-3D: Sequence of the mouse OCR10 cDNA, homology box 1
(hb1), homology box 2 (hb2), and TaqMan.RTM. probes and primers
used in a quantitative PCR assay to detect modification of allele
(MOA) in ES cells targeted using the mOCR10 LTVEC.
[0038] hb1: base pairs 1 to 211
[0039] hb2: base pairs 1586 to 1801
[0040] TaqMan.RTM. probe and corresponding PCR primer set derived
from mOCR10 exon 3:
[0041] TaqMan.RTM. probe: nucleotides 413 to 439--upper strand
[0042] Primer ex3-5': nucleotides 390 to 410--upper strand
[0043] Primer ex3-3': nucleotides 445 to 461--lower strand
[0044] TaqMan.RTM. probe and corresponding PCR primer set derived
from mOCR10 exon 4:
[0045] TaqMan.RTM. probe: nucleotides 608 to 639--upper strand
[0046] Primer ex4-5': nucleotides 586 to 605--upper strand
[0047] Primer ex4-3': nucleotides 642 to 662--lower strand
Definitions
[0048] A "targeting vector" is a DNA construct that contains
sequences "homologous" to endogenous chromosomal nucleic acid
sequences flanking a desired genetic modification(s). The flanking
homology sequences, referred to as "homology arms", direct the
targeting vector to a specific chromosomal location within the
genome by virtue of the homology that exists between the homology
arms and the corresponding endogenous sequence and introduce the
desired genetic modification by a process referred to as
"homologous recombination".
[0049] "Homologous" means two or more nucleic acid sequences that
are either identical or similar enough that they are able to
hybridize to each other or undergo intermolecular exhange.
[0050] "Gene targeting" is the modification of an endogenous
chromosomal locus by the insertion into, deletion of, or
replacement of the endogenous sequence via homologous recombination
using a targeting vector.
[0051] A "gene knockout" is a genetic modification resulting from
the disruption of the genetic information encoded in a chromosomal
locus.
[0052] A "gene knockin" is a genetic modification resulting from
the replacement of the genetic information encoded in a chromosomal
locus with a different DNA sequence.
[0053] A "knockout organism" is an organism in which a significant
proportion of the organism's cells harbor a gene knockout.
[0054] A "knockin organism" is an organism in which a significant
proportion of the organism's cells harbor a gene knockin.
[0055] A "marker" or a "selectable marker" is a selection marker
that allows for the isolation of rare transfected cells expressing
the marker from the majority of treated cells in the population.
Such marker's gene's include, but are not limited to, neomycin
phosphotransferase and hygromycin B phosphotransferase, or
fluorescing proteins such as GFP.
[0056] An "ES cell" is an embryonic stem cell. This cell is usually
derived from the inner cell mass of a blastocyst-stage embryo.
[0057] An "ES cell clone" is a subpopulation of cells derived from
a single cell of the ES cell population following introduction of
DNA and subsequent selection.
[0058] A "flanking DNA" is a segment of DNA that is collinear with
and adjacent to a particular point of reference.
[0059] "LTVECs" are large targeting vectors for eukaryotic cells
that are derived from fragments of cloned genomic DNA larger than
those typically used by other approaches intended to perform
homologous targeting in eukaryotic cells.
[0060] A "non-human organism" is an organism that is not normally
accepted by the public as being human.
[0061] "Modification of allele" (MOA) refers to the modification of
the exact DNA sequence of one allele of a gene(s) or chromosomal
locus (loci) in a genome. This modification of allele (MOA)
includes, but is not limited to, deletions, substitutions, or
insertions of as little as a single nucleotide or deletions of many
kilobases spanning a gene(s) or chromosomal locus (loci) of
interest, as well as any and all possible modifications between
these two extremes.
[0062] "Orthologous" sequence refers to a sequence from one species
that is the functional equivalent of that sequence in another
species.
[0063] The description and examples presented infra are provided to
illustrate the subject invention. One of skill in the art will
recognize that these examples are provided by way of illustration
only and are not included for the purpose of limiting the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0064] Applicants have developed a novel, rapid, streamlined, and
efficient method for creating and screening eukaryotic cells which
contain modified endogenous genes or chromosomal loci. In these
cells, the modification may be gene(s) knockouts, knockins, point
mutations, or large genomic insertions or deletions or other
modifications. By way of non-limiting example, these cells may be
embryonic stem cells which are useful for creating knockout or
knockin organisms and in particular, knockout or knockin mice, for
the purpose of determining the function of the gene(s) that have
been altered, deleted and/or inserted.
[0065] The novel methods described herein combine, for the first
time:
[0066] 1. Bacterial homologous recombination to precisely engineer
a desired genetic modification within a large cloned genomic DNA
fragment, thereby creating a large targeting vector for use in
eukaryotic cells (LTVECs);
[0067] 2. Direct introduction of these LTVECs into eukaryotic cells
to modify the corresponding endogenous gene(s) or chromosomal
locus(loci) of interest in these cells; and
[0068] 3. An analysis to determine the rare eukaryotic cells in
which the targeted allele has been modified as desired, involving a
quantitative assay for modification of allele (MOA) of the parental
allele.
[0069] It should be emphasized that previous methods to detect
successful homologous recombination in eukaryotic cells cannot be
utilized in conjunction with the LTVECs of Applicants' invention
because of the long homology arms present in the LTVECs. Utilizing
a LTVEC to deliberately modify endogenous genes or chromosomal loci
in eukaryotic cells via homologous recombination is made possible
by the novel application of an assay to determine the rare
eukaryotic cells in which the targeted allele has been modified as
desired, such assay involving a quantitative assay for modification
of allele (MOA) of a parental allele, by employing, for example,
quantitative PCR or other suitable quantitative assays for MOA.
[0070] The ability to utilize targeting vectors with homology arms
larger than those used in current methods is extremely valuable for
the following reasons:
[0071] 1. Targeting vectors are more rapidly and conveniently
generated from available libraries containing large genomic inserts
(e.g. BAC or PAC libraries) than targeting vectors made using
previous technologies, in which the genomic inserts have to be
extensively characterized and "trimmed" prior to use (explained in
detail below). In addition, minimal sequence information needs to
be known about the locus of interest, i.e. it is only necessary to
know the approximately 80-100 nucleotides that are required to
generate the homology boxes (described in detail below) and to
generate probes that can be used in quantitative assays for MOA
(described in detail below).
[0072] 2. Larger modifications as well as modifications spanning
larger genomic regions are more conveniently generated and in fewer
steps than using previous technologies. For example, the method of
the invention makes possible the precise modification of large loci
that cannot be accommodated by traditional plasmid-based targeting
vectors because of their size limitations. It also makes possible
the modification of any given locus at multiple points (e.g. the
introduction of specific mutations at different exons of a
multi-exon gene) in one step, alleviating the need to engineer
multiple targeting vectors and to perform multiple rounds of
targeting and screening for homologous recombination in ES
cells.
[0073] 3. The use of long regions of homology (long homology arms)
increase the targeting frequency of "hard to target" loci in
eukaryotic cells, consistent with previous findings that targeting
of homologous recombination in eukaryotic cells appears to be
related to the total homology contained within the targeting
vector.
[0074] 4. The increased targeting frequency obtained using long
homology arms apparently diminishes the benefit, if any, from using
isogenic DNA in these targeting vectors.
[0075] 5. The application of quantitative MOA assays for screening
eukaryotic cells for homologous recombination not only empowers the
use of LTVECs as targeting vectors (advantages outlined above) but
also reduces the time for identifying correctly modified eukaryotic
cells from the typical several days to a few hours. In addition,
the application of quantitative MOA does not require the use of
probes located outside the endogenous gene(s) or chromosomal
locus(loci) that is being modified, thus obviating the need to know
the sequence flanking the modified gene(s) or locus(loci). This is
a significant improvement in the way the screening has been
performed in the past and makes it a much less labor-intensive and
much more cost-effective approach to screening for homologous
recombination events in eukaryotic cells.
Methods
[0076] Many of the techniques used to construct DNA vectors
described herein are standard molecular biology techniques well
known to the skilled artisan (see e.g., Sambrook, J., E. F. Fritsch
And T. Maniatis. Molecular Cloning: A Laboratory Manual, Second
Edition, Vols 1, 2, and 3, 1989; Current Protocols in Molecular
Biology, Eds. Ausubel et al., Greene Publ. Assoc., Wiley
Interscience, NY). All DNA sequencing is done by standard
techniques using an ABI 373A DNA sequencer and Taq Dideoxy
Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster
City, Calif.).
[0077] Step 1. Obtain a large genomic DNA clone containing the
gene(s) or chromosomal locus (loci) of interest.
[0078] A Gene(s) or locus(loci) of interest can be selected based
on specific criteria, such as detailed structural or functional
data, or it can be selected in the absence of such detailed
information as potential genes or gene fragments become predicted
through the efforts of the various genome sequencing projects.
[0079] Importantly, it should be noted that it is not necessary to
know the complete sequence and gene structure of a gene(s) of
interest to apply the method of the subject invention to produce
LTVECs. In fact, the only sequence information that is required is
approximately 80-100 nucleotides so as to obtain the genomic clone
of interest as well as to generate the homology boxes used in
making the LTVEC (described in detail below) and to make probes for
use in quantitative MOA assays.
[0080] Once a gene(s) or locus(loci) of interest has been selected,
a large genomic clone(s) containing this gene(s) or locus(loci) is
obtained. This clone(s) can be obtained in any one of several ways
including, but not limited to, screening suitable DNA libraries
(e.g. BAC, PAC, YAC, or cosmid) by standard hybridization or PCR
techniques, or by any other methods familiar to the skilled
artisan.
[0081] Step 2. Append homology boxes 1 and 2 to a modification
cassette and generation of LTVEC.
[0082] Homology boxes mark the sites of bacterial homologous
recombination that are used to generate LTVECs from large cloned
genomic fragments (FIG. 1). Homology boxes are short segments of
DNA, generally double-stranded and at least 40 nucleotides in
length, that are homologous to regions within the large cloned
genomic fragment flanking the "region to be modified". The homology
boxes are appended to the modification cassette, so that following
homologous recombination in bacteria, the modification cassette
replaces the region to be modified (FIG. 1). The technique of
creating a targeting vector using bacterial homologous
recombination can be performed in a variety of systems (Yang et
al., Nat Biotechnol, 15:859-65, 1997; Muyrers et al., Nucleic Acids
Res, 27:1555-7, 1999; Angrand et al., Nucleic Acids Res, 27:e16,
1999; Narayanan et al., Gene Ther, 6:442-7, 1999; Yu, et al., Proc
Natl Acad Sci USA, 97:5978-83, 2000). One example of a favored
technology currently in use is ET cloning (Zhang et al., Nat Genet,
20:123-8, 1998; Narayanan et al., Gene Ther, 6:442-7, 1999) and
variations of this technology (Yu, et al., Proc Natl Acad Sci USA,
97:5978-83, 2000). ET refers to the recE (Hall and Kolodner, Proc
Natl Acad Sci USA, 91:3205-9, 1994) and recT proteins (Kusano et
al., Gene, 138:17-25, 1994) that carry out the homologous
recombination reaction. RecE is an exonuclease that trims one
strand of linear double-stranded DNA (essentially the donor DNA
fragment described infra) 5' to 3', thus leaving behind a linear
double-stranded fragment with a 3' single-stranded overhang. This
single-stranded overhang is coated by recT protein, which has
single-stranded DNA (ssDNA) binding activity (Kovall and Matthews,
Science, 277:1824-7, 1997). ET cloning is performed using E. coli
that transiently express the E. coli gene products of recE and recT
(Hall and Kolodner, Proc Natl Acad Sci USA, 91:3205-9,1994; Clark
et al., Cold Spring Harb Symp Quant Biol, 49:453-62, 1984; Noirot
and Kolodner, J Biol Chem, 273:12274-80, 1998; Thresher et al., J
Mol Biol, 254:364-71, 1995; Kolodner et al., Mol Microbiol,
11:23-30, 1994; Hall et al., J Bacteriol, 175:277-87, 1993) and the
bacteriophage lambda (.lambda.) protein .lambda.gam (Murphy, J
Bacteriol, 173:5808-21, 1991; Poteete et al., J Bacteriol,
170:2012-21, 1988). The .lambda.gam protein is required for
protecting the donor DNA fragment from degradation by the recBC
exonuclease system (Myers and Stahl, Annu Rev Genet, 28:49-70,
1994) and it is required for efficient ET-cloning in recBC.sup.+
hosts such as the frequently used E. coli strain DH10b.
[0083] The region to be modified and replaced using bacterial
homologous recombination can range from zero nucleotides in length
(creating an insertion into the original locus) to many tens of
kilobases (creating a deletion and/or a replacement of the original
locus). Depending on the modification cassette, the modification
can result in the following:
[0084] (a) deletion of coding sequences, gene segments, or
regulatory elements;
[0085] (b) alteration(s) of coding sequence, gene segments, or
regulatory elements including substitutions, additions, and fusions
(e.g. epitope tags or creation of bifunctional proteins such as
those with GFP);
[0086] (c) insertion of new coding regions, gene segments, or
regulatory elements, such as those for selectable marker genes or
reporter genes or putting new genes under endogenous
transcriptional control;
[0087] (d) creation of conditional alleles, e.g. by introduction of
loxP sites flanking the region to be excised by Cre recombinase
(Abremski and Hoess, J Biol Chem, 259:1509-14, 1984), or FRT sites
flanking the region to be excised by Flp recombinase (Andrews et
al., Cell, 40:795-803, 1985; Meyer-Leon et al., Cold Spring Harb
Symp Quant Biol, 49:797-804,1984; Cox, Proc Natl Acad Sci USA,
80:4223-7, 1983); or
[0088] (e) replacement of coding sequences or gene segments from
one species with orthologous coding sequences from a different
species, e.g. replacing a murine genetic locus with the orthologous
human genetic locus to engineer a mouse where that particular locus
has been `humanized`.
[0089] Any or all of these modifications can be incorporated into a
LTVEC. A specific, non-limiting example in which an endogenous
coding sequence is entirely deleted and simultaneously replaced
with both a reporter gene as well as a selectable marker is
provided below in Example 1, as are the advantages of the method of
the invention as compared to previous technologies.
[0090] Step 3 (optional). Verify that each LTVEC has been
engineered correctly.
[0091] Verify that each LTVEC has been engineered correctly by:
[0092] a. Diagnostic PCR to verify the novel junctions created by
the introduction of the donor fragment into the gene(s) or
chromosomal locus(loci) of interest. The PCR fragments thus
obtained can be sequenced to further verify the novel junctions
created by the introduction of the donor fragment into the gene(s)
or chromosomal locus(loci) of interest.
[0093] b. Diagnostic restriction enzyme digestion to make sure that
only the desired modifications have been introduced into the LTVEC
during the bacterial homologous recombination process.
[0094] c. Direct sequencing of the LTVEC, particularly the regions
spanning the site of the modification to verify the novel junctions
created by the introduction of the donor fragment into the gene(s)
or chromosomal locus(loci) of interest.
[0095] Step 4. Purification, preparation, and linearization of
LTVEC DNA for introduction into eukaryotic cells.
[0096] a. Preparation of LTVEC DNA:
[0097] Prepare miniprep DNA (Sambrook, J., E. F. Fritsch And T.
Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition,
Vols 1, 2, and 3, 1989; Tillett and Neilan, Biotechniques,
24:568-70, 572, 1998;
http://www.qiagen.com/literature/handbooks/plkmini/plm.sub.--399.pdf)
of the selected LTVEC and re-transform the miniprep LTVEC DNA into
E. coli using electroporation (Sambrook, J., E. F. Fritsch and T.
Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition,
Vols 1, 2, and 3, 1989). This step is necessary to get rid of the
plasmid encoding the recombinogenic proteins that are utilized for
the bacterial homologous recombination step (Zhang et al., Nat
Genet, 20:123-8, 1998; Narayanan et al., Gene Ther, 6:442-7, 1999).
It is useful to get rid of this plasmid (a) because it is a high
copy number plasmid and may reduce the yields obtained in the large
scale LTVEC preps; (b) to eliminate the possibility of inducing
expression of the recombinogenic proteins; and (c) because it may
obscure physical mapping of the LTVEC. Before introducing the LTVEC
into eukaryotic cells, larger amounts of LTVEC DNA are prepared by
standard methodology
(http://www.qiagen.com/literature/handbooks/plk/plkl- ow.pdf;
Sambrook, J., E. F. Fritsch And T. Maniatis. Molecular Cloning: A
Laboratory Manual, Second Edition, Vols 1, 2, and 3, 1989; Tillett
and Neilan, Biotechniques, 24:568-70, 572, 1998). However, this
step can be bypassed if a bacterial homologous recombination method
that utilizes a recombinogenic prophage is used, i.e. where the
genes encoding the recombinogenic proteins are integrated into the
bacterial chromosome (Yu, et al., Proc Natl Acad Sci USA,
97:5978-83, 2000), is used.
[0098] b. Linearizing the LTVEC DNA:
[0099] To prepare the LTVEC for introduction into eukaryotic cells,
the LTVEC is preferably linearized in a manner that leaves the
modified endogenous gene(s) or chromosomal locus(loci) DNA flanked
with long homology arms. This can be accomplished by linearizing
the LTVEC, preferably in the vector backbone, with any suitable
restriction enzyme that digests only rarely. Examples of suitable
restriction enzymes include NotI, PacI, SfiI, SrfI, SwaI, FseI,
etc. The choice of restriction enzyme may be determined
experimentally (i.e. by testing several different candidate rare
cutters) or, if the sequence of the LTVEC is known, by analyzing
the sequence and choosing a suitable restriction enzyme based on
the analysis. In situations where the LTVEC has a vector backbone
containing rare sites such as CosN sites, then it can be cleaved
with enzymes recognizing such sites, for example .lambda. terminase
(Shizuya et al., Proc Natl Acad Sci USA, 89:8794-7, 1992; Becker
and Gold, Proc Natl Acad Sci USA, 75:4199-203, 1978; Rackwitz et
al., Gene, 40:259-66, 1985).
[0100] Step 5. Introduction of LTVEC into eukaryotic cells and
selection of cells where successful introduction of the LTVEC has
taken place.
[0101] LTVEC DNA can be introduced into eukaryotic cells using
standard methodology, such as transfection mediated by calcium
phosphate, lipids, or electroporation (Sambrook, J., E. F. Fritsch
And T. Maniatis. Molecular Cloning: A Laboratory Manual, Second
Edition, Vols 1, 2, and 3, 1989). The cells where the LTVEC has
been introduced successfully can be selected by exposure to
selection agents, depending on the selectable marker gene that has
been engineered into the LTVEC. As a non-limiting example, if the
selectable marker is the neomycin phosphotransferase (neo) gene
(Beck, et al., Gene, 19:327-36, 1982), then cells that have taken
up the LTVEC can be selected in G418-containing media; cells that
do not have the LTVEC will die whereas cells that have taken up the
LTVEC will survive (Santerre, et al., Gene, 30:147-56,1984). Other
suitable selectable markers include any drug that has activity in
eukaryotic cells (Joyner, The Practical Approach Series, 293,
1999), such as hygromycin B (Santerre, et al., Gene,
30:147-56,1984; Bernard, et al., Exp Cell Res, 158:237-43, 1985;
Giordano and McAllister, Gene, 88:285-8, 1990), Blasticidin S
(Izumi, et al., Exp Cell Res, 197:229-33, 1991), and other which
are familiar to those skilled in the art.
[0102] Step 6. Screen for homologous recombination events in
eukaryotic cells using quantitative assay for modification of
allele (MOA).
[0103] Eukaryotic cells that have been successfully modified by
targeting the LTVEC into the locus of interest can be identified
using a variety of approaches that can detect modification of
allele within the locus of interest and that do not depend on
assays spanning the entire homology arm or arms. Such approaches
can include but are not limited to:
[0104] (a) quantitative PCR using TaqMan.RTM. (Lie and Petropoulos,
Curr Opin Biotechnol, 9:43-8, 1998);
[0105] (b) quantitative MOA assay using molecular beacons (Tan, et
al., Chemistry, 6:1107-11,2000)
[0106] (c) fluorescence in situ hybridization FISH (Laan, et al.,
Hum Genet, 96:275-80, 1995) or comparative genomic hybridization
(CGH) (Forozan, et al., Trends Genet, 13:405-9, 1997; Thompson and
Gray, J Cell Biochem Suppl, 139-43, 1993; Houldsworth and Chaganti,
Am J Pathol, 145:1253-60, 1994);
[0107] (d) isothermic DNA amplification (Lizardi, et al., Nat
Genet, 19:225-32, 1998; Mitra and Church, Nucleic Acids Res,
27:e34, 1999);
[0108] (e) quantitative hybridization to an immobilized probe(s)
(Southern, J. Mol. Biol. 98: 503, 1975; Kafatos FC; Jones CW;
Efstratiadis A, Nucleic Acids Res 7(6):1541-52, 1979);
[0109] (f) Invader Probes.RTM. (Third Wave Technologies);
[0110] (g) Eclipse.TM. and Molecular Beacon probes (Synthetic
Genetics); and
[0111] (h) MMP assays (High Throughput Genomics)
[0112] Applicants provide herein an example in which TaqMan.RTM.
quantitative PCR is used to screen for successfully targeted
eukaryotic cells. In this non limiting example, TaqMan.RTM. is used
to identify eukaryotic cells which have undergone homologous
recombination wherein a portion of one of two endogenous alleles in
a diploid genome has been replaced by another sequence. In contrast
to traditional methods, in which a difference in restriction
fragment length spanning the entire homology arm or arms indicates
the modification of one of two alleles, the quantitative
TaqMan.RTM. method will detect the modification of one allele by
measuring the reduction in copy number (by half) of the unmodified
allele. Specifically, the probe detects the unmodified allele and
not the modified allele. Therefore, the method is independent of
the exact nature of the modification and not limited to the
sequence replacement described in this example. TaqMan is used to
quantify the number of copies of a DNA template in a genomic DNA
sample, especially by comparison to a reference gene (Lie and
Petropoulos, Curr Opin Biotechnol, 9:43-8, 1998). The reference
gene is quantitated in the same genomic DNA as the target gene(s)
or locus(loci). Therefore, two TaqMan.RTM. amplifications (each
with its respective probe) are performed. One TaqMan.RTM. probe
determines the "Ct" (Threshold Cycle) of the reference gene, while
the other probe determines the Ct of the region of the targeted
gene(s) or locus(loci) which is replaced by successful targeting.
The Ct is a quantity that reflects the amount of starting DNA for
each of the TaqMan.RTM. probes, i.e. a less abundant sequence
requires more cycles of PCR to reach the threshold cycle.
Decreasing by half the number of copies of the template sequence
for a TaqMan.RTM. reaction will result in an increase of about one
Ct unit. TaqMan.RTM. reactions in cells where one allele of the
target gene(s) or locus(loci) has been replaced by homologous
recombination will result in an increase of one Ct for the target
TaqMan.RTM. reaction without an increase in the Ct for the
reference gene when compared to DNA from non-targeted cells. This
allows for ready detection of the modification of one allele of the
gene(s) of interest in eukaryotic cells using LTVECs.
[0113] As stated above, modification of allele (MOA) screening is
the use of any method that detects the modification of one allele
to identify cells which have undergone homologous recombination. It
is not a requirement that the targeted alleles be identical
(homologous) to each other, and in fact, they may contain
polymorphisms, as is the case in progeny resulting from crossing
two different strains of mice. In addition, one special situation
that is also covered by MOA screening is targeting of genes which
are normally present as a single copy in cells, such as some of the
located on the sex chromosomes and in particular, on the Y
chromosome. In this case, methods that will detect the modification
of the single targeted allele, such as quantitative PCR, Southern
blottings, etc., can be used to detect the targeting event. It is
clear that the method of the invention can be used to generate
modified eukaryotic cells even when alleles are polymorphic or when
they are present in a single copy in the targeted cells.
[0114] Step 8. Uses of genetically modified eukaryotic cells.
[0115] (a) The genetically modified eukaryotic cells generated by
the methods described in steps 1 through 7 can be employed in any
in vitro or in vivo assay, where changing the phenotype of the cell
is desirable.
[0116] (b) The genetically modified eukaryotic cell generated by
the methods described in steps 1 through 7 can also be used to
generate an organism carrying the genetic modification. The
genetically modified organisms can be generated by several
different techniques including but not limited to:
[0117] 1. Modified embryonic stem (ES) cells such as the frequently
used rat and mouse ES cells. ES cells can be used to create
genetically modified rats or mice by standard blastocyst injection
technology or aggregation techniques (Robertson, Practical Approach
Series, 254, 1987; Wood, et al., Nature, 365:87-9, 1993; Joyner,
The Practical Approach Series, 293, 1999), tetraploid blastocyst
injection (Wang, et al., Mech Dev, 62:137-45, 1997), or nuclear
transfer and cloning (Wakayama, et al., Proc Natl Acad Sci USA,
96:14984-9, 1999). ES cells derived from other organisms such as
rabbits (Wang, et al., Mech Dev, 62:137-45, 1997; Schoonjans, et
al., Mol Reprod Dev, 45:439-43, 1996) or chickens (Pain, et al.,
Development, 122:2339-48, 1996) or other species should also be
amenable to genetic modification(s) using the methods of the
invention.
[0118] 2. Modified protoplasts can be used to generate genetically
modified plants (for example see U.S. Pat. No. 5,350,689 "Zea mays
plants and transgenic Zea mays plants regenerated from protoplasts
or protoplast-derived cells", and U.S. Pat. No. 5,508,189
"Regeneration of plants from cultured guard cell protoplasts" and
references therein).
[0119] 3. Nuclear transfer from modified eukaryotic cells to
oocytes to generate cloned organisms with modified allele
(Wakayama, et al., Proc Natl Acad Sci U S A, 96:14984-9, 1999;
Baguisi, et al., Nat Biotechnol, 17:456-61, 1999; Wilmut, et al.,
Reprod Fertil Dev, 10:639-43, 1998; Wilmut, et al., Nature,
385:810-3, 1997; Wakayama, et al., Nat Genet, 24:108-9, 2000;
Wakayama, et al., Nature, 394:369-74, 1998; Rideout, et al., Nat
Genet, 24:109-10,2000; Campbell, et al., Nature, 380:64-6,
1996).
[0120] 4. Cell-fusion to transfer the modified allele to another
cell, including transfer of engineered chromosome(s), and uses of
such cell(s) to generate organisms carrying the modified allele or
engineered chromosome(s) (Kuroiwa, et al., Nat Biotechnol,
18:1086-1090, 2000).
[0121] 5. The method of the invention are also amenable to any
other approaches that have been used or yet to be discovered.
[0122] While many of the techniques used in practicing the
individual steps of the methods of the invention are familiar to
the skilled artisan, Applicants contend that the novelty of the
method of the invention lies in the unique combination of those
steps and techniques coupled with the never-before-described method
of introducing a LTVEC directly into eukaryotic cells to modify a
chromosomal locus, and the use of quantitative MOA assays to
identify eukaryotic cells which have been appropriately modified.
This novel combination represents a significant improvement over
previous technologies for creating organisms possessing
modifications of endogenous genes or chromosomal loci.
EXAMPLES
Example 1
Engineering Mouse ES Cells Bearing a Deletion of the OCR10 Gene
[0123] a. Selection of a Large Genomic DNA Clone Containing
mOCR10.
[0124] A Bacterial Artificial Chromosome (BAC) clone carrying a
large genomic DNA fragment that contained the coding sequence of
the mouse OCR10 (mOCR10) gene was obtained by screening an arrayed
mouse genomic DNA BAC library (Incyte Genomics) using PCR. The
primers employed to screen this library were derived from the
mOCR10 gene cDNA sequence.
[0125] Two primer pairs where used:
1 (a) OCR10.RAA (5'-AGCTACCAGCTGCAGATGCGGGCAG-3') and OCR10.PVIrc
(5'-CTCCCCAGCCTGGGTCTGAAAGATGACG-3') which amplifies a 102 bp DNA;
and (b) OCR10.TDY (5'-GACCTCACTTGCTACACTGACTAC-3') and OCR10.QETrc
(5'-ACTTGTGTAGGCTGCAGAAGGTCTCTTG-3') which amplifies a 1500 bp
DNA.
[0126] This mOCR10 BAC contained approximately 180 kb of genomic
DNA including the complete mOCR10 coding sequence. This BAC clone
was used to generate an LTVEC which was subsequently used to delete
a portion of the coding region of mOCR10 while simultaneously
introducing a reporter gene whose initiation codon precisely
replaced the initiation codon of OCR10, as well as insertion of a
selectable marker gene useful for selection both in E. coli and
mammalian cells following the reporter gene (FIG. 2). The reporter
gene (in this non-limiting example LacZ, the sequence of which is
readily available to the skilled artisan), encodes the E. coli
.beta.-galactosidase enzyme. Because of the position of insertion
of LacZ (its initiating codon is at the same position as the
initiation codon of mOCR16) the expression of lacZ should mimic
that of mOCR10, as has been observed in other examples where
similar replacements with LacZ were performed using previous
technologies (see "Gene trap strategies in ES cells", by W Wurst
and A. Gossler, in Joyner, The Practical Approach Series, 293,
1999) The LacZ gene allows for a simple and standard enzymatic
assay to be performed that can reveal its expression patterns in
situ, thus providing a surrogate assay that reflects the normal
expression patterns of the replaced gene(s) or chromosomal
locus(loci).
[0127] b. Construction of Donor Fragment and Generation of
LTVEC.
[0128] The modification cassette used in the construction of the
mOCR10 LTVEC is the lacZ-SV40 polyA-PGKp-EM7-neo-PGK polyA cassette
wherein lacZ is a marker gene as described above, SV40 polyA is a
fragment derived from Simian Virus 40 (Subramanian, et al., Prog
Nucleic Acid Res Mol Biol, 19:157-64, 1976; Thimmappaya, et al., J
Biol Chem, 253:1613-8, 1978; Dhar, et al., Proc Natl Acad Sci USA,
71:371-5, 1974; Reddy, et al., Science, 200:494-502, 1978) and
containing a polyadenylation site and signal (Subramanian, et al.,
Prog Nucleic Acid Res Mol Biol, 19:157-64, 1976; Thimmappaya, et
al., J Biol Chem, 253:1613-8, 1978; Dhar, et al., Proc Natl Acad
Sci USA, 71:371-5,1974; Reddy, et al., Science, 200:494-502, 1978),
PGKp is the mouse phosphoglycerate kinase (PGK) promoter (Adra, et
al., Gene, 60:65-74, 1987) (which has been used extensively to
drive expression of drug resistance genes in mammalian cells), EM7
is a strong bacterial promoter that has the advantage of allowing
for positive selection in bacteria of the completed LTVEC construct
by driving expression of the neomycin phosphotransferase (neo)
gene, neo is a selectable marker that confers Kanamycin resistance
in prokaryotic cells and G418 resistance in eukaryotic cells (Beck,
et al., Gene, 19:327-36, 1982), and PGK polyA is a 3' untranslated
region derived from the PGK gene and containing a polyadenylation
site and signal (Boer, et al., Biochem Genet, 28:299-308,
1990).
[0129] To construct the mOCR10 LTVEC, first a donor fragment was
generated consisting of a mOCR10 homology box 1 (hb1) attached
upstream from the LacZ gene in the modification cassette and a
mOCR10 homology box 2 (hb2) attached downstream of the neo-PGK
polyA sequence in the modification cassette (FIG. 2), using
standard recombinant genetic engineering technology. Homology box 1
(hb1) consists of 211 bp of untranslated sequence immediately
upstream of the initiating methionine of the mOCR10 open reading
frame (mOCR10 ORF) (FIG. 3A-3D). Homology box 2 (hb2) consists of
last 216 bp of the mOCR10 ORF, ending at the stop codon (FIG.
3A-3D).
[0130] Subsequently, using bacterial homologous recombination
(Zhang, et al., Nat Genet, 20:123-8, 1998; Angrand, et al., Nucleic
Acids Res, 27:e16, 1999; Muyrers, et al., Nucleic Acids Res,
27:1555-7, 1999; Narayanan, et al., Gene Ther, 6:442-7, 1999; Yu,
et al., Proc Natl Acad Sci USA, 97:5978-83, 2000), this donor
fragment was used to precisely replace the mOCR10 coding region
(from initiation methionine to stop codon) with the insertion
cassette, resulting in construction of the mOCR10 LTVEC (FIG. 2).
Thus, in this mOCR10 LTVEC, the mOCR10 coding sequence was replaced
by the insertion cassette creating an approximately 20 kb deletion
in the mOCR10 locus while leaving approximately 130 kb of upstream
homology (upstream homology arm) and 32 kb of downstream homology
(downstream homology arm).
[0131] It is important to note that LTVECs can be more rapidly and
conveniently generated from available BAC libraries than targeting
vectors made using previous technologies because only a single
bacterial homologous recombination step is required and the only
sequence information required is that needed to generate the
homology boxes. In contrast, previous approaches for generating
targeting vectors using bacterial homologous recombination require
that large targeting vectors be "trimmed" prior to their
introduction in ES cells (Hill et al., Genomics, 64:111-3, 2000).
This trimming is necessary because of the need to generate homology
arms short enough to accommodate the screening methods utilized by
previous approaches. One major disadvantage of the method of Hill
et al. is that two additional homologous recombination steps are
required simply for trimming (one to trim the region upstream of
the modified locus and one to trim the region downstream of the
modified locus). To do this, substantially more sequence
information is needed, including sequence information spanning the
sites of trimming.
[0132] In addition, another obvious advantage, illustrated by the
above example, is that a very large deletion spanning the mOCR10
gene (approximately 20 kb) can be easily generated in a single
step. In contrast, using previous technologies, to accomplish the
same task may require several steps and may involve marking the
regions upstream and downstream of the coding sequences with loxP
sites in order to use the Cre recombinase to remove the sequence
flanked by these sites after introduction of the modified locus in
eukaryotic cells. This may be unattainable in one step, and thus
may require the construction of two targeting vectors using
different selection markers and two sequential targeting events in
ES cells, one to introduce the loxP site at the region upstream of
the coding sequence and another to introduce the loxP site at the
region downstream of the coding sequence. It should be further
noted that the creation of large deletions often occurs with low
efficiency using the previous targeting technologies in eukaryotic
cells, because the frequency of achieving homologous recombination
may be low when using targeting vectors containing large deletion
flanked by relatively short homology arms. The high efficiency
obtained using the method of the invention (see below) is due to
the very long homology arms present in the LTVEC that increase the
rate of homologous recombination in eukaryotic cells.
[0133] c. Verification, Preparation, and Introduction of mOCR10
LTVEC DNA into ES Cells.
[0134] The sequence surrounding the junction of the insertion
cassette and the homology sequence was verified by DNA sequencing.
The size of the mOCR10 LTVEC was verified by restriction analysis
followed by pulsed field gel electrophoresis (PFGE) (Cantor, et
al., Annu Rev Biophys Biophys Chem, 17:287-304, 1988; Schwartz and
Cantor, Cell, 37:67-75, 1984). A standard large-scale plasmid
preparation of the mOCR10 LTVEC was done, the plasmid DNA was
digested with the restriction enzyme NotI, which cuts in the vector
backbone of the mOCR10 LTVEC, to generate linear DNA. Subsequently
the linearized DNA was introduced into mouse ES cells by
electroporation (Robertson, Practical Approach Series, 254, 1987;
Joyner, The Practical Approach Series, 293, 1999; Sambrook, et al.,
Sambrook, J., E. F. Fritsch and T. Maniatis. Molecular Cloning: A
Laboratory Manual, Second Edition, Vols 1, 2, and 3, 1989). ES
cells successfully transfected with the mOCR10 LTVEC were selected
for in G418-containing media using standard selection methods
(Robertson, Practical Approach Series, 254, 1987; Joyner, The
Practical Approach Series, 293, 1999).
[0135] d. Identification of Targeted ES Cells Clones Using a
Quantitative Modification of Allele (MOA) Assay.
[0136] To identify ES cells in which one of the two endogenous
mOCR10 genes had been replaced by the modification cassette
sequence, DNA from individual ES cell clones was analyzed by
quantitative PCR using standard TaqMan.RTM. methodology as
described (Applied Biosystems, TaqMan.RTM. Universal PCR Master
Mix, catalog number P/N 4304437; see also
http://www.pebiodocs.com/pebiodocs/04304449.pdf). The primers and
TaqMan.RTM. probes used are as described in FIG. 3A-3D. A total of
69 independent ES cells clones where screened and 3 were identified
as positive, i.e. as clones in which one of the endogenous mOCR10
coding sequence had been replaced by the modification cassette
described above.
[0137] Several advantages of the MOA approach are apparent:
[0138] (i) It does not require the use of a probe outside the locus
being modified, thus obviating the need to know the sequence
flanking the modified locus.
[0139] (ii) It requires very little time to perform compared to
conventional Southern blot methodology which has been the previous
method of choice (Robertson, Practical Approach Series, 254, 1987,
Joyner, The Practical Approach Series, 293, 1999), thus reducing
the time for identifying correctly modified cells from the typical
several days to just a few hours.
[0140] This is a significant improvement in the way screening has
been performed in the past and makes it a much less labor-intensive
and more cost-effective approach to screening for homologous
recombination events in eukaryotic cells.
[0141] Yet another advantage of the method of the invention is that
it is also superior to previous technologies because of its ability
to target difficult loci. Using previous technologies, it has been
shown that for certain loci the frequency of successful targeting
may by as low as 1 in 2000 integration events, perhaps even lower.
Using the method of the invention, Applicants have demonstrated
that such difficult loci can be targeted much more efficiently
using LTVECs that contain long homology arms (i.e. greater than
those allowed by previous technologies). As the non-limiting
example described above demonstrates, the Applicants have targeted
the OCR10 locus, a locus that has previously proven recalcitrant to
targeting using conventional technology. Using the method of the
invention, Applicants have shown that they have obtained successful
targeting in 3 out of 69 ES cells clones in which the mOCR10 LTVEC
(containing more than 160 kb of homology arms, and introducing a 20
kb deletion) had integrated, whereas using previous technology for
ES cell targeting (Joyner, The Practical Approach Series, 293,
1999) using a plasmid-based vector with homology arms shorter than
10-20 kb while also introducing a deletion of less than 15 kb, no
targeted events were identified among more than 600 integrants of
the vector. These data clearly demonstrate the superiority of the
method of the invention over previous technologies.
Example 2
Increased Targeting Frequency and Abrogation of the Need to Use
Isogenic DNA when LTVECs are Used as the Targeting Vectors
[0142] As noted above, the increased targeting frequency obtained
using long homology arms should diminish the benefit, if any,
derived from using genomic DNA in constructing LTVECs that is
isogenic with (i.e. identical in sequence to) the DNA of the
eukaryotic cell being targeted. To test this hypothesis, Applicants
have constructed several LTVECs using genomic DNA derived from the
same mouse substrain as the eukaryotic cell to be targeted
(presumably isogenic), and a large number of other LTVECs using
genomic DNA derived from mouse substrains differing from that of
the eukaryotic cell to be targeted (presumably non-isogenic). The
non-isogenic LTVECs exhibited an average targeting frequency of 6%
(ranging from 1-20%, Table 1), while the isogenic LTVECs exhibited
as average targeting frequency of 3% (ranging from 2-5%),
indicating that the rate of successful targeting using LTVECs does
not depend on isogenicity.
2 TABLE 1 Approximate Size (kb) Target Gene Description DNA Origin
ES Cell BAC Size Arm 1 Arm 2 Deletion Positive Clones % Targeting
NON-ISOGENIC OGH LacZ-ATG fusion SvJ CJ7 148 50 90 5 4 4 OCR10(A)
LacZ-ATG fusion SvJ CJ7 165 135 8 20 1 1.4 OCR10(B) LacZ-ATG fusion
SvJ CJ7 160 130 32 20 3 4.3 MA61 LacZ-ATG fusion SvJ CJ7 95 N/D N/D
30 3 4.6 MA16 LacZ-ATG fusion SvJ CJ7 120 N/D N/D 8 8 13 AGRP
LacZ-ATG fusion SvJ CJ7 189 147 32 8 1 1.1 SHIP-2 LacZ-ATG fusion
SvJ CJ7 136 30 90 11 7 15 Sm22 LacZ-ATG fusion SvJ CJ7 70 35 35 0.9
18 20 LGR7L LacZ-ATG fusion SvJ CJ7 200 N/D N/D 1 3 3.2 C5aR
LacZ-ATG fusion SvJ CJ7 160 80 25 1 4 4.2 IL18 LacZ-ATG fusion SvJ
CJ7 120 50 65 10 7 7.3 PLGF LacZ-ATG fusion SvJ CJ7 130 40 20 8 1 1
NaDC-1 LacZ-ATG fusion SvJ CJ7 180 30 45 25 4 2.1 ISOGENIC ROR1
Intracell-LacZ fusion CJ7 CJ7 55 14 14 20 5 5 ROR1 Intracell-3xmyc
fusion CJ7 CJ7 55 14 14 20 2 2 ROR2 Brachydactyly mutation CJ7 CJ7
45 11 24 0.5 2 2 and Myc tag
Example 3
Detailed Description of the TagMan.RTM.-Based MOA for
Identification of Targeted ES Clones
[0143] ES cell clones that have taken up the LTVEC and incorporated
it into the genome at the targeted locus by homologous
recombination are identified by a modification of allele (MOA)
assay that uses real-time quantitative PCR to discern the
difference between targeted ES cell clones, in which one of the two
targeted alleles is modified, and non-targeted ES cell clones, in
which both alleles remain unmodified. The MOA assay consists of a
primary and a secondary screen. The primary screen contains the
following steps: (1) growth of LTVEC-transfected ES cell clones on
gelatin-coated 96-well plates; (2) isolation of genomic DNA from
each ES cell clone; (3) use of each genomic DNA sample as a
template in 8 separate quantitative PCRs on two 384-well plates in
which 2 of the PCRs employ a target-locus-specific primer set that
hybridyzes to DNA sequences at one end of the genomic fragment
targeted for deletion (`upstream PCR`), 2 of the PCRs employ a
target-locus-specific primer set that hybridyzes to DNA sequences
at the other end of the genomic fragment targeted for deletion
(`downstream PCR`), 4 of the PCRs employ primer sets that recognize
four non-targeted reference loci (`reference PCRs`), and each PCR
includes a fluorescent probe (for example a TaqMan.RTM. [ABI],
Eclipse.TM., or Molecular Beacon probe [Synthetic Genetics]) that
recognizes the amplified sequence and whose fluorescence signal is
directly proportional to the amount of PCR product; (4) running the
PCRs in a device that combines a thermocycler with a fluorescence
detector (for example the ABI 7900HT) that quantifies the
accumulation of amplification products during the PCR and
determines the threshold cycle (C.sub.T), the point in the PCR at
which the fluorescence signal is detectable above background noise;
(5) for each ES cell clone DNA sample, calculation of the
difference in the C.sub.T values (.DELTA.C.sub.T) between the
upstream PCRs and each of the four reference PCRs and between the
downstream PCRs and each of the four reference PCRs to create 8
tables of 96 .DELTA.C.sub.T values; (6) normalization of the
.DELTA.C.sub.T values to positive values; (7) calculation of the
median .DELTA.C.sub.T value for each target-reference comparison
table; (8) determination of a confidence score by use of a computer
program that examines the eight .DELTA.C.sub.T tables and
calculates the number of times a given ES cell clone DNA sample
produces a .DELTA.C.sub.T value within the tolerance ranges 0.5 to
1.5, 0.25 to 1.5, 0.5 to 2.0, 0.25 to 2.0, 0.5 to 3.0 and 0.25 to
3.0 cycles greater than the median .DELTA.C.sub.T (examples of
computer programming languages suitable for creating or writing
such a program include visual basics, Java, or any other computer
programming language familiar to the skilled artisan); (9) plotting
the values and their medians for each of the eight .DELTA.C.sub.T
tables as histograms; and (10) identification of correctly targeted
ES cell clone candidates from an inspection of the confidence
scores and the .DELTA.C.sub.T histograms. In a preferred example,
the .DELTA.C.sub.T value for the candidate targeted clone falls
within 0.5 to 1.5 cycles greater than the median in 8 out of 8
reference comparisons.
[0144] Candidate clones identified by the MOA assay primary screen
are confirmed or rejected in a secondary screen, which contains the
following steps: (1) use of the genomic DNA from each of the
positive candidate ES cell clones, from a larger number of negative
clones, and from genomic DNA copy-number standards from mice that
carry one or two copies of the LTVEC LacZ-Neo cassette per diploid
genome as templates in 8 separate quantitative PCRs on two 384-well
plates in which 1 reaction is an upstream PCR (as in the primary
screen), one reaction is a downstream PCR (as in the primary
screen), 4 reactions are reference PCRs with two reference loci
that are different from those used in the primary screen, one
reaction is a PCR with primers and a probe that are specific for
the LacZ gene of the LTVEC, and one reaction is a PCR with primers
and a probe that are specific for the Neo gene of the LTVEC; (2)
running the PCRs in a quantitative PCR device, as in the primary
screen; (3) calculation, as in the primary screen, of the
.DELTA.C.sub.T values between the upstream PCR and each of the two
reference PCRs, between the downstream PCRs and each of the two
reference PCRs, between the LacZ PCR and each of the two reference
PCRs, and between the Neo PCR and each of the two reference PCRs to
create eight .DELTA.C.sub.T tables; (4) normalization of the
.DELTA.C.sub.T values to positive values; (5) calculation of the
median value for each .DELTA.C.sub.T table; (6) calculation of
confidence scores as in the primary screen; and (7) plotting the
values and their medians for each of the eight .DELTA.C.sub.T
tables as histograms.
[0145] From an inspection of the confidence scores and the
.DELTA.C.sub.T histograms for both the primary and secondary
screens, correctly targeted ES clone candidates are either
confirmed or rejected. In a preferred example, the .DELTA.C.sub.T
value for the candidate targeted clone falls within 0.5 to 1.5
cycles greater than the median in 12 out of 12 reference
comparisons from the combined primary and secondary screens.
[0146] To score the number of copies of the LTVEC per diploid
genome in the confirmed, correctly targeted ES clones, their
.DELTA.C.sub.T values from the comparisons of the LacZ and Neo PCRs
with the two reference PCRs are compared with the .DELTA.C.sub.T
values for the LacZ-Neo copy number standards. Each ES cell clone
is scored as having 1, 2 or greater than 2 copies of the LTVEC. For
each modified allele project, ES cell clones are screened in groups
of 96 (usually fewer than 288 total clones) until 3 clones that
score positive in the MOA assay and have a single copy of the
LacZ-Neo cassette are identified.
Example 4
Use of FISH to Identify Correctly Targeted LTVECs in ES Cells
[0147] Using the LTVEC technology described herein, Applicants
knocked out the SM22alpha gene in ES cells. SM22alpha is a 22-kDa
smooth muscle cell (SMC) lineage-restricted protein that physically
associates with cytoskeletal actin filament bundles in contractile
SMCs. The targeted ES cells were then subjected to standard
fluorescence in situ hybridization (FISH) on metaphase chromosomal
spreads to verify that the gene was appropriately targeted. The
experiment was performed with two probes: 1) an SM22alpha gene
probe consisting of the unmodified SM2alpha BAC clone used to
generate the LTVEC and 2) a LacZ and Neomycin DNA probe which
detects only the gene modification made by the targeting event
(insertion of LacZ and Neo gene cassettes). Metaphase chromosomal
spreads were prepared from cells and hybridization was performed
simultaneously with both probes which were labeled with different
colored fluorophores to allow detection of hybridization of each
probe within the same spread. A non-targeted ES cell line was
analyzed in parallel as a control. As expected, in the control
spreads, two alleles of SM22alpha were detected on homologous
chromosomal arms, but there was no hybridization of the LacZ-Neo
probe. As in controls, in targeted ES cell spreads two alleles were
also detected at the same chromosomal location and on homologous
chromosomes, but double-labeling with the LacZ-Neo probe was
apparent on one of the two chromosomes indicating co-localization
of the SM22alpha and LacZ-Neo DNA sequences at that allele of
SM22alpha. Importantly, no SM22alpha or LacZ-Neo gene sequences
were detected at inappropriate locations in the spreads. Lack of
extra integration of SM22alpha gene sequences and co-localization
of LacZ-Neo with SM22alpha in one chromosome of a homologous pair
strongly suggests that correct targeting of LacZ-Neo to one of the
SM22alpha alleles via homologous recombination had occurred.
Example 5
Lowering the Amount of DNA Used to Electroporate ES Cells Improves
Targeting Efficiency
[0148] Standard methods for targeted modification of genes in mouse
embryonic stem (ES) cells typically employ 20 to 40 .mu.g of
targeting vector in the electroporation procedure. Applicants have
discovered that with LTVECs, electroporation with much lower
amounts of DNA--in the range of about 1 to 5 .mu.g per
1.times.10.sup.7 cells--doubles the frequency of correctly targeted
homologous recombination events while greatly reducing the number
of secondary, non-homologous insertion events. This clear
improvement in targeting efficiency is important because it
significantly reduces the number of ES cells clones that need to be
screened to find several positive clones with a correctly targeted,
single-copy modification. The associated benefits are reduced cost
and increased throughput.
Example 6
Use of the Method of the Invention to Create MA61 Knockout Mice to
Study Muscle Atrophy
[0149] MA61, also called MAFbx, is a recently discovered ubiquitin
ligase that is up-regulated in various conditions of muscle atrophy
(See U.S. Provisional Application No. 60/264,926, filed Jan. 30,
2001, U.S. Provisional Application No. 60/311,697, filed Aug. 10,
2001, and U.S. Provisional Application (serial number not yet
known), filed Oct. 22, 2001, all assigned to Regeneron
Pharmaceuticals, Inc., each of which is incorporated herein in its
entirety by reference). To further study the biological
significance of this gene in muscle atrophy, knockout mice were
created using the method of the invention as follows.
[0150] First, to obtain a large cloned genomic fragment containing
the MA61 gene, a Bacterial Artificial Chromosome (BAC) library was
screened with primers derived from the MA61 cDNA sequence. The BAC
clone thus obtained was then used to create a Large Targeting
Vector for Eukaryotic Cells (LTVEC) as follows. A modification
cassette containing a 5' homology box/lacZ gene/polyA/PGK
promoter/neo/polyA/3' homology box was engineered. The homology
boxes were appended to mark the sites of bacterial homologous
recombination during the generation of the LTVEC. The LacZ is a
reporter gene that was positioned such that its initiating codon
was at the same position as the initiating codon of MA61. Following
homologous recombination in bacteria, the modification cassette
replaced the MA61 gene. Thus, a MA61 LTVEC was created wherein the
MA61 coding sequences in the BAC clone was replaced by the
modification cassette engineered as described supra. LTVEC DNA was
then prepared, purified, and linearized for introduction into
eukaryotic cells as described infra.
[0151] A MA61 LTVEC DNA miniprep was prepared (Sambrook, J., E. F.
Fritsch And T. Maniatis. Molecular Cloning: A Laboratory Manual,
Second Edition, Vols 1, 2, and 3, 1989; Tillett and Neilan,
Biotechniques, 24:568-70, 572, 1998;
http://www.qiagen.com/literature/handbooks/plkmini/plm.sub.--3-
99.pdf) and re-transformed into E. coli using electroporation
(Sambrook, J., E. F. Fritsch and T. Maniatis, Molecular Cloning: A
Laboratory Manual, Second Edition, Vols 1, 2, and 3, 1989) in order
to get rid of the plasmid encoding the recombinogenic proteins that
are utilized for the bacterial homologous recombination step (Zhang
et al., Nat Genet, 20:123-8, 1998; Narayanan et al., Gene Ther,
6:442-7, 1999). Before introducing the MA61 LTVEC into eukaryotic
cells, larger amounts of MA61 LTVEC were prepared by standard
methodology (http://www.qiagen.com/litera-
ture/handbooks/plk/plklow.pdf; Sambrook, J., E. F. Fritsch And T.
Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition,
Vols 1, 2, and 3, 1989; Tillett and Neilan, Biotechniques,
24:568-70, 572, 1998).
[0152] Next, to prepare the MA61 LTVEC for introduction into
eukaryotic cells, the MA61 LTVEC was linearized. This was
accomplished by digesting with the restriction enzyme NotI which
leaves the modified endogenous gene(s) or chromosomal locus(loci)
DNA flanked with long homology arms.
[0153] The MA61 LTVEC was then introduced into eukaryotic cells
using standard electroporation methodology (Sambrook, J., E. F.
Fritsch And T. Maniatis. Molecular Cloning: A Laboratory Manual,
Second Edition, Vols 1, 2, and 3, 1989)). The cells in which the
MA61 LTVEC was introduced successfully were selected by exposure to
a selection agent. Because the selectable marker used in the
modification cassette was the neomycin phosphotransferase (neo)
gene (Beck, et al., Gene, 19:327-36, 1982), the cells that had
taken up the MA61 LTVEC were selected in a medium containing G418;
cells that do not have the MA61 LTVEC died whereas cells that have
taken up the MA61 LTVEC survived (Santerre, et al., Gene,
30:147-56, 1984).
[0154] Eukaryotic cells that have been successfully modified by
targeting the MA61 LTVEC into the MA61 locus were identified with
the quantitative PCR method TaqMan.RTM. (Lie and Petropoulos, Curr
Opin Biotechnol, 9:43-8,1998).
[0155] Finally, the genetically modified ES cells were used to
create genetically modified, in this case knock out, mice by
standard blastocyst injection technology. Thus created were the
MA61 knock-outs, mice in which the MA61 gene had been deleted.
[0156] Both these knock out mice and wild-type (WT) mice were
exposed to atrophy-inducing conditions, created by denervating the
mice, and levels of atrophy compared. First, the sciatic nerve was
isolated in the mid-thigh region of the right hind limb and
transected in mice. Transection of the sciatic nerve leads to
denervation and, over a fourteen daay period, to atrophy in the
muscles of the lower limb, specifically the tibialis anterior and
gastrocnemius muscles, over a 14-day period. At 7 and 14 days
following the denervation, animals were sacrificed by carbon
dioxide inhalation. Then the tibialis anterior (TA) and
gastrocnemius complex (GA) were removed from the right (denervated)
and left (intact) hind limbs, weighed, and frozen at a fixed length
in liquid nitrogen cooled isopentane. The amount of atrophy was
assessed by comparing the weight of the muscles from the denervated
limb with the weight of the muscles from the non-denervated
limb.
[0157] Muscle atrophy was assessed 7 and 14 days following
transection of the right sciatic nerve. The wet weights of the
right, denervated muscles were compared to the wet weights of the
left, non-denervated muscles. The right:left comparisons are given
in Table 2.
3 Gastrocnemius Complex Tibialis Anterior Sample siz Mean SE Sample
siz Mean SE 7 days Genotype WT 7 0.76 0.016 11 0.68 0.033 KO 6 0.84
0.022 11 0.80 0.015 14 days Genotype WT 5 0.55 0.024 5 0.62 0.023
KO 5 0.80 0.019 5 0.80 0.012
[0158] At 7 and 14 days, the muscles from the knock mice showed
significantly (p<0.001) less atrophy than the muscles from the
wild type mice. The difference between the knock out and wild type
mice was greater at 14 days than at 7 days. While the wild type
mice continued to atrophy between 7 and 14 days, the knock out mice
showed no additional atrophy.
[0159] In summary, the approach of creating LTVECs and directly
using them as targeting vectors combined with MOA screening for
homologous recombination events in ES cells creates a novel method
for engineering genetically modified loci that is rapid,
inexpensive and represents a significant improvement over the
tedious, time-consuming methods previously in use. It thus opens
the possibility of a rapid large scale in vivo functional genomics
analysis of essentially any and all genes in an organism's genome
in a fraction of the time and cost necessitated by previous
methodologies.
[0160] Although the foregoing invention has been described in some
detail by way of illustration and examples, it will be readily
apparent to those of ordinary skill in the art that certain changes
and modifications may be made to the teachings of the invention
without departing from the spirit or scope of the appended claims.
Sequence CWU 1
1
6 1 25 DNA Artificial Sequence Mouse OCR10 gene primer 1 agctaccagc
tgcagatgcg ggcag 25 2 28 DNA Artificial Sequence Mouse OCR10 gene
primer 2 ctccccagcc tgggtctgaa agatgacg 28 3 24 DNA Artificial
Sequence Mouse OCR10 gene primer 3 gacctcactt gctacactga ctac 24 4
28 DNA Artificial Sequence Mouse OCR10 gene primer 4 acttgtgtag
gctgcagaag gtctcttg 28 5 1799 DNA Artificial Sequence Mouse OCR10
cDNA 5 ccccgggctt cctgttctaa taagaatacc tcctaggtcc cccatgggct
aacctcatct 60 ttggtactca acaggggtct tctttatgag cttcggacca
gctcttttga tgtggcaggg 120 actgaccctg ggtggggaag ccactcagtg
catgacccca gctggttcac cacatatacc 180 acatactttt cttgcaggtc
tgggacacag catgccccgg ggcccagtgg ctgccttact 240 cctgctgatt
ctccatggag cttggagctg cctggacctc acttgctaca ctgactacct 300
ctggaccatc acctgtgtcc tggagacacg gagccccaac cccagcatac tcagtctcac
360 ctggcaagat gaatatgagg aacttcagga ccaagagacc ttctgcagcc
tacacaagtc 420 tggccacaac accacacata tatggtacac gtgccatatg
cgcttgtctc aattcctgtc 480 cgatgaagtt ttcattgtca acgtgacgga
ccagtctggc aacaactccc aagagtgtgg 540 cagctttgtc ctggctgaga
gcatcaagcc agctcccccc ttgaacgtga ctgtggcctt 600 ctcaggacgc
tatgatatct cctgggactc agcttatgac gaaccctcca actacgtgct 660
gagaggcaag ctacaatatg agctgcagta tcggaacctc agagacccct atgctgtgag
720 gccggtgacc aagctgatct cagtggactc aagaaacgtc tctcctccct
gaagagttcc 780 acaaagattc tagctaccag ctgcagatgc gggcagcgcc
tcagccaggc acttcattca 840 gggggacctg gagtgagtgg agtgaccccg
tcatctttca gacccaggct ggggagcccg 900 aggcaggctg ggaccctcac
atgctgctgc tcctggctgt cttgatcatt gtcctggttt 960 tcatgggtct
gaagatccac ctgccttgga ggctatggaa aaagatatgg gcaccagtgc 1020
ccacccctga gagtttcttc cagcccctgt acagggagca cagcgggaac ttcaagaaat
1080 gggttaatac ccctttcacg gcctccagca tagagttggt gccacagagt
tccacaacaa 1140 catcagcctt acatctgtca ttgtatccag ccaaggagaa
gaagttcccg gggctgccgg 1200 gtctggaaga gcaactggag tgtgatggaa
tgtctgagcc tggtcactgg tgcataatcc 1260 ccttggcagc tggccaagcg
gtctcagcct acagtgagga gagagaccgg ccatatggtc 1320 tggtgtccat
tgacacagtg actgtgggag atgcagaggg cctgtgtgtc tggccctgta 1380
gctgtgagga tgatggctat ccagccatga acctggatgc tggcagagag tctggtccta
1440 attcagagga tctgctcttg gtcacagacc ctgcttttct gtcttgtggc
tgtgtctcag 1500 gtagtggtct caggcttggg ggctccccag gcagcctact
ggacaggttg aggctgtcat 1560 ttgcaaagga aggggactgg acagcagacc
caacctggag aactgggtcc ccaggagggg 1620 gctctgagag tgaagcaggt
tccccccctg gtctggacat ggacacattt gacagtggct 1680 ttgcaggttc
agactgtggc agccccgtgg agactgatga aggaccccct cgaagctatc 1740
tccgccagtg ggtggtcagg acccctccac ctgtggacag tggagcccag agcagctag
1799 6 529 PRT Artificial Sequence Mouse OCR10 protein 6 Met Pro
Arg Gly Pro Val Ala Ala Leu Leu Leu Leu Ile Leu His Gly 1 5 10 15
Ala Trp Ser Cys Leu Asp Leu Thr Cys Tyr Thr Asp Tyr Leu Trp Thr 20
25 30 Ile Thr Cys Val Leu Glu Thr Arg Ser Pro Asn Pro Ser Ile Leu
Ser 35 40 45 Leu Thr Trp Gln Asp Glu Tyr Glu Glu Leu Gln Asp Gln
Glu Thr Phe 50 55 60 Cys Ser Leu His Lys Ser Gly His Asn Thr Thr
His Ile Trp Tyr Thr 65 70 75 80 Cys His Met Arg Leu Ser Gln Phe Leu
Ser Asp Glu Val Phe Ile Val 85 90 95 Asn Val Thr Asp Gln Ser Gly
Asn Asn Ser Gln Glu Cys Gly Ser Phe 100 105 110 Val Leu Ala Glu Ser
Ile Lys Pro Ala Pro Pro Leu Asn Val Thr Val 115 120 125 Ala Phe Ser
Gly Arg Tyr Asp Ile Ser Trp Asp Ser Ala Tyr Asp Glu 130 135 140 Pro
Ser Asn Tyr Val Leu Arg Gly Lys Leu Gln Tyr Glu Leu Gln Tyr 145 150
155 160 Arg Asn Leu Arg Asp Pro Tyr Ala Val Arg Pro Val Thr Lys Leu
Ile 165 170 175 Ser Val Asp Ser Arg Asn Val Ser Leu Leu Pro Glu Glu
Phe His Lys 180 185 190 Asp Ser Ser Tyr Gln Leu Gln Met Arg Ala Ala
Pro Gln Pro Gly Thr 195 200 205 Ser Phe Arg Gly Thr Trp Ser Glu Trp
Ser Asp Pro Val Ile Phe Gln 210 215 220 Thr Gln Ala Gly Glu Pro Glu
Ala Gly Trp Asp Pro His Met Leu Leu 225 230 235 240 Leu Leu Ala Val
Leu Ile Ile Val Leu Val Phe Met Gly Leu Lys Ile 245 250 255 His Leu
Pro Trp Arg Leu Trp Lys Lys Ile Trp Ala Pro Val Pro Thr 260 265 270
Pro Glu Ser Phe Phe Gln Pro Leu Tyr Arg Glu His Ser Gly Asn Phe 275
280 285 Lys Lys Trp Val Asn Thr Pro Phe Thr Ala Ser Ser Ile Glu Leu
Val 290 295 300 Pro Gln Ser Ser Thr Thr Thr Ser Ala Leu His Leu Ser
Leu Tyr Pro 305 310 315 320 Ala Lys Glu Lys Lys Phe Pro Gly Leu Pro
Gly Leu Glu Glu Gln Leu 325 330 335 Glu Cys Asp Gly Met Ser Glu Pro
Gly His Trp Cys Ile Ile Pro Leu 340 345 350 Ala Ala Gly Gln Ala Val
Ser Ala Tyr Ser Glu Glu Arg Asp Arg Pro 355 360 365 Tyr Gly Leu Val
Ser Ile Asp Thr Val Thr Val Gly Asp Ala Glu Gly 370 375 380 Leu Cys
Val Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro Ala Met 385 390 395
400 Asn Leu Asp Ala Gly Arg Glu Ser Gly Pro Asn Ser Glu Asp Leu Leu
405 410 415 Leu Val Thr Asp Pro Ala Phe Leu Ser Cys Gly Cys Val Ser
Gly Ser 420 425 430 Gly Leu Arg Leu Gly Gly Ser Pro Gly Ser Leu Leu
Asp Arg Leu Arg 435 440 445 Leu Ser Phe Ala Lys Glu Gly Asp Trp Thr
Ala Asp Pro Thr Trp Arg 450 455 460 Thr Gly Ser Pro Gly Gly Gly Ser
Glu Ser Glu Ala Gly Ser Pro Pro 465 470 475 480 Gly Leu Asp Met Asp
Thr Phe Asp Ser Gly Phe Ala Gly Ser Asp Cys 485 490 495 Gly Ser Pro
Val Glu Thr Asp Glu Gly Pro Pro Arg Ser Tyr Leu Arg 500 505 510 Gln
Trp Val Val Arg Thr Pro Pro Pro Val Asp Ser Gly Ala Gln Ser 515 520
525 Ser
* * * * *
References