U.S. patent application number 09/957659 was filed with the patent office on 2002-06-20 for method of producing non-human mammals.
This patent application is currently assigned to Whitehead Institute for Biomedical Research. Invention is credited to Eggan, Kevin C., Jaenisch, Rudolf.
Application Number | 20020078470 09/957659 |
Document ID | / |
Family ID | 27398562 |
Filed Date | 2002-06-20 |
United States Patent
Application |
20020078470 |
Kind Code |
A1 |
Eggan, Kevin C. ; et
al. |
June 20, 2002 |
Method of producing non-human mammals
Abstract
A method of producing mutant/targeted non-human mammals, such as
mutant mice that does not require production of chimera and permits
the introduction of multiple mutations in embryos and, thus, avoids
the necessity of breeding to combine all of the desired mutations
in a single animal. The method is efficient in producing ES
mice.
Inventors: |
Eggan, Kevin C.; (Cambridge,
MA) ; Jaenisch, Rudolf; (Brookline, MA) |
Correspondence
Address: |
Anne J. Collins, Esq.
HAMILTON, BROOK,
SMITH & REYNOLDS, P.C.
Two Militia Drive
Lexington
MA
02421-4799
US
|
Assignee: |
Whitehead Institute for Biomedical
Research
Cambridge
MA
|
Family ID: |
27398562 |
Appl. No.: |
09/957659 |
Filed: |
September 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09957659 |
Sep 20, 2001 |
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09755003 |
Jan 5, 2001 |
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60234378 |
Sep 20, 2000 |
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60255970 |
Dec 15, 2000 |
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Current U.S.
Class: |
800/18 ;
800/21 |
Current CPC
Class: |
C12N 2517/02 20130101;
C12N 2510/00 20130101; A01K 2217/05 20130101; A01K 2217/075
20130101; C12N 15/873 20130101; A01K 67/027 20130101 |
Class at
Publication: |
800/18 ;
800/21 |
International
Class: |
A01K 067/027 |
Goverment Interests
[0002] Work described herein was supported, in whole or in part, by
National Institutes of Health Grants No. 5-R35-CA44339 and
RO1-CA84198. The United States government has certain rights in the
invention.
Claims
What is claimed is:
1. A method of producing a non-human mammal, referred to as an ES
non-human mammal, wherein pluripotent cells are introduced into
tetraploid blastocysts of the same mammalian species under
conditions that result in production of an embryo and the resulting
embryo is transferred into a foster mother which is maintained
under conditions that result in development of live offspring,
wherein the pluripotent cells are non-inbred pluripotent cells.
2. The method of claim 1, wherein the non-human mammal is a
mouse.
3. The method of claim 2, wherein the pluripotent cells are
embryonic stem cells and are introduced into tetraploid blastocysts
by injection.
4. The method of claim 3, wherein injection is piezo
microinjection.
5. A method of producing a non-human mammalian embryo comprising
injecting non-human non-inbred ES cells into non-human tetraploid
blastocysts and maintaining the resulting tetraploid blastocysts
under conditions that result in formation of embryos, thereby
producing a non-human mammalian embryo.
6. The method of claim 5, wherein the non-human non-inbred ES cells
are mouse cells and the non-human mammalian embryo is a mouse.
7. The method of claim 6, wherein mutant mouse non-inbred ES cells
are injected into non-human tetraploid blastocysts by piezo
microinjection.
8. A non-human mammal produced by the method of claim 1.
9. A mouse produced by the method of claim 2.
10. A mouse produced by the method of claim 3.
11. A non-human mammalian embryo produced by the method of claim
5.
12. A mouse embryo produced by the method of claim 6.
13. A mouse embryo produced by the method of claim 7.
14. A method of producing a mutant non-human mammal, wherein
pluripotent cells comprising at least one mutation in genomic DNA
are introduced into tetraploid blastocysts of the same mammalian
species under conditions that result in production of an embryo and
the resulting embryo is transferred into a foster mother which is
maintained under conditions that result in development of live
offspring, thereby producing a mutant non-human mammal, wherein the
pluripotent cells are non-inbred pluripotent cells.
15. The method of claim 14, wherein the non-human mammal is a
mouse.
16. The method of claim 15, wherein the pluripotent cells are
embryonic stem cells and are introduced into tetraploid blastocysts
by injection.
17. The method of claim 16, wherein injection is piezo
microinjection.
18. A method of producing a mutant non-human mammalian embryo
comprising injecting mutant non-human non-inbred ES cells into
non-human tetraploid blastocysts and maintaining the resulting
tetraploid blastocysts under conditions that result in formation of
embryos, thereby producing a mutant non-human mammalian embryo.
19. The method of claim 18, wherein the mutant non-human non-inbred
ES cells are mouse cells and the mutant non-human mammal is a
mouse.
20. The method of claim 19, wherein mutant mouse non-inbred ES
cells are injected into non-human tetraploid blastocysts by piezo
microinjection.
21. A mutant non-human mammal produced by the method of claim
14.
22. A mutant mouse produced by the method of claim 15.
23. A mutant mouse produced by the method of claim 16.
24. A mutant mouse embryo produced by the method of claim 17.
25. A mutant mouse embryo produced by the method of claim 19.
26. A mutant mouse embryo produced by the method of claim 20.
27. A method of producing a mutant mouse, comprising: (a)
introducing mouse non-inbred ES cells comprising at least one
mutation in genomic DNA into mouse tetraploid blastocysts, thereby
producing mouse blastocysts containing mouse non-inbred ES cells;
(b) maintaining the product of (a) under conditions that result in
production of embryos; (c) introducing an embryo into a
pseudopregnant female: and (d) maintaining the female into which
the embryo is introduced under conditions that result in
development of live offspring, thereby producing a mutant
mouse.
28. The method of claim 27, wherein in (a) introducing is carried
out by injection.
29. The method of claim 28, wherein microinjection is piezo
microinjection.
30. The method of claim 29, wherein the at least one mutation in
genomic DNA is a gene knockout or exogenous DNA incorporated into
the genomic DNA.
31. A mouse embryo produced from a mouse tetraploid blastocyst
having incorporated therein mutant mouse non-inbred ES cells.
32. The mouse embryo of claim 31, wherein the non-inbred ES cells
are selected from the group consisting of: V6.5 cells; 129B6 cells;
F1.2-3 cells; V8.1 cells; V17.2 cells and V30.11 cells.
33. The mouse embryo of claim 31, wherein the mutant mouse
non-inbred ES cells comprise at least one mutation selected from
the group consisting of: transgenes which are cDNA, genes or
portions thereof; targeted mutations, random mutations, conditional
mutations, targeted insertions of foreign genes, YAC sized
transgenes, BAC sized transgenes, and all or part of a
chromosome.
34. The mouse embryo of claim 33, wherein the at least one
alteration in genomic DNA is a gene knockout or exogenous DNA
incorporated into the genomic DNA.
35. A method of producing a mouse, comprising: (a) introducing
mouse non-inbred ES cells into mouse tetraploid blastocysts,
thereby producing mouse blastocysts containing mouse non-inbred ES
cells; (b) maintaining the product of (a) under conditions that
result in production of embryos; (c) introducing an embryo into a
pseudopregnant female; and (d) maintaining the female into which
the embryo is introduced under conditions that result in
development of live offspring, thereby producing a mouse.
36. The method of claim 35, wherein in (a) introducing is carried
out by injection.
37. The method of claim 36, wherein microinjection is piezo
microinjection.
38. A mouse embryo produced from a mouse tetraploid blastocyst
having incorporated therein mouse non-inbred ES cells.
39. The mouse embryo of claim 38, wherein the non-inbred ES cells
are selected from the group consisting of: V6.5 cells; 129B6 cells;
F1.2-3 cells; V8.1 cells; V17.2 cells and V30.11 cells.
40. A method of identifying a drug to be administered to treat a
condition in a mammal in which the condition occurs, comprising:
(a) producing, using the method of claim 14, a mutant mouse that is
a model of the condition; (b) administering to the mutant mouse a
drug to be assessed for its effectiveness in treating or preventing
the condition; and (c) assessing the ability of the drug to treat
or prevent the condition, wherein if the drug reduces the extent to
which the condition is present or progresses, the drug is a drug to
be administered to treat the condition.
41. A method of producing a mutant non-human mammal, wherein
pluripotent cells comprising at least one mutation in genomic DNA
are introduced into tetraploid blastocysts of the same mammalian
species under conditions that result in production of an embryo and
the resulting embryo is transferred into a foster mother which is
maintained under conditions that result in development of live
offspring, wherein the pluripotent cells are non-inbred pluripotent
cells.
42. A method of producing a mutant mouse that is derived from a
single non-inbred ES cell clone, comprising breeding a mutant male
mouse and a mutant female mouse, wherein (a) the male mouse or an
ancestor thereof and (b) the female mouse or an ancestor thereof
were produced from the same non-inbred male ES cell and the female
mouse is an XO female.
43. The method of claim 42, wherein the non-inbred cell clone is a
non-inbred F1 cell clone.
44. A method of producing XO F1 ES cells, comprising: (a)
introducing into male F1 ES cells a negative selection marker,
under conditions appropriate for insertion of the negative
selection marker in the Y chromosome of male F1 ES cells, thereby
producing a mixture of male F1 ES cells comprising male F1 ES cells
in which the negative selection marker is inserted in the Y
chromosome and other male F1 ES cells, some of which do not contain
a Y chromosome; and (b) subjecting the resulting mixture to
conditions that result in the death of male F1 ES cells in which
the Y chromosome has the negative selection marker inserted therein
and do not result in the death of male F1 ES cells that lack a Y
chromosome and are XO F1 ES cells, thereby producing XO F1 ES
cells.
45. An XO female mouse produced by introducing XO F1 ES cells into
tetraploid mouse blastocysts under conditions that result in
production of an embryo and transferring the resulting embryo into
a foster mother which is maintained under conditions that result in
development of live offspring, wherein the live offspring are XO
female mice.
46. A method of producing a mutant mouse strain, comprising
breeding a mutant male mouse and a mutant female mouse, wherein (a)
the mutant male mouse or an ancestor thereof and (b) the mutant
female mouse or an ancestor thereof were derived from the same
non-inbred male mouse ES cell clone and the mutant female mouse is
an XO female.
47. The method of claim 46, wherein the non-inbred male ES cell is
an F1 male mouse ES cell.
48. The method of claim 47, wherein the mutant XO female mouse or
an ancestor thereof was derived from an male mouse F1 ES cell by
knocking out the Y chromosome of the F1 ES cell, thereby producing
an XO F1 ES cell; introducing the XO F1 ES cell into a tetraploid
mouse blastocyst under conditions that result in production of an
embryo and transferring the resulting embryo into a foster mother
which is maintained under conditions that result in development of
live offspring, thereby producing an XO female offspring.
49. A method of identifying XO F1 ES cells, comprising screening a
population of F1 ES cells for F1 ES cells that are XO F1 ES cells,
wherein said population is a mixture of XO F1 ES cells and XY F1 ES
cells.
50. The method of claim 49, wherein screening is carried out to
identify F1 ES cells in which spontaneous loss of the Y chromosome
has occurred, thereby producing XO F1 ES cells.
51. The method of claim 50, wherein said population is a population
of wildtype F1 ES cells or a population of mutant F1 ES cells.
52. The method of claim 51 wherein the F1 ES cells are mouse
cells.
53. The method of claim 51, wherein screening is carried out using
a Y chromosome probe against repetitive elements or PCR
amplification.
54. A method of isolating XO F1 ES cells from XY F1 ES cells,
comprising (a) screening a population of F1 ES cells for loss of
the Y chromosome, wherein said population is a mixture of XO F1 ES
cells and XY F1 ES cells; and (b) isolating F1 ES cells lacking the
Y chromosome, thereby isolating XO F1 ES cells.
55. The method of claim 54, wherein said population is a population
of wildtype F1 ES cells or a population of mutant F1 ES cells.
56. The method of claim 55, wherein the F1 ES cells are mouse
cells.
57. The method of claim 55, wherein screening is carried out using
a Y chromosome probe against repetitive elements or PCR
amplification.
58. The method of claim 55, wherein the F1 ES cells is generated by
introducing into male F1 ES cells a negative selection marker under
conditions appropriate for insertion of the negative selection
marker in the Y chromosome of male F 1 ES cells, thereby producing
a mixture of F1 ES cells comprising male F1 ES cells in which the
negative selection marker is inserted in the Y chromosome and other
male F1 ES cells, some of which do not contain a Y chromosome; and
subjecting the resulting F1 ES cells to conditions that result in
the death of male F1 ES cells in which the Y chromosome has the
negative selection marker inserted therein and do not result in the
death of male F1 ES cells that lack a Y chromosome.
59. A method of producing an XO female non-human mammal, comprising
introducing XO F1 ES cells identified using the method of claim 49
into tetraploid blastocysts of the same mammalian species under
conditions that result in production of an embryo and transferring
the resulting embryo into a foster mother which is maintained under
conditions that result in development of live offspring, thereby
producing an XO female non-human mammal.
60. The method of claim 59, wherein said XO F1 ES cells are
wildtype XO F1 ES cells or mutant XO F1 ES cells.
61. The method of claim 44, wherein said XO F1 ES cells are mouse
cells and the non-human mammal is a mouse.
62. A non-human mammal produced by the method of claim 59.
63. A non-human mammal produced by the method of claim 60.
64. A mouse produced by the method of claim 61.
65. A method of producing an XO female mouse, comprising
introducing mouse XO F1 cells identified using the method of claim
49 into tetraploid mouse blastocysts under conditions that result
in production of an embryo and transferring the resulting embryo
into a foster mother which is maintained under conditions that
result in development of live offspring, thereby producing an XO
female mouse.
66. The method of claim 65, wherein the mouse XO F1 ES cells are
wildtype mouse XO F1 ES cells or mouse mutant XO F1 ES cells.
67. A mouse produced by the method of claim 65.
68. A mouse produced by the method of claim 66.
69. An XO female mouse produced by (a) introducing mouse XO F1 ES
cells into tetraploid mouse blastocysts under conditions that
result in production of an embryo, said mouse XO F1 ES cells
identified by screening a population of mouse F1 ES cells for F1 ES
cells that are XO F1 ES cells, wherein said population is a mixture
of mouse XO F1 ES cells and mouse XY F1 ES cells; and (b)
transfecting the resulting embryo into a foster mother which is
maintained under conditions that result in development of live
offspring, wherein the live offspring are XO female mice.
70. The XO female mouse of claim 69, wherein screening is carried
out to identify F1 ES cells in which spontaneous loss of the Y
chromosome has occurred, thereby producing XO F1 ES cells.
71. The XO female mouse of claim 70, wherein said population of
mouse F1 ES cells is a population of mouse wildtype F1 ES cells or
a population of mouse mutant F1 ES cells.
72. The method of claim 70, wherein screening is carried out using
a Y chromosome probe against repetitive elements or PCR
amplification.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/755,003, filed Jan. 5, 2001, which claims
the benefit of the filing date of U.S. Provisional Application No.
60/234,378, filed Sep. 20, 2000 and the filing date of U.S.
Provisional Application No. 60/255,970, filed Dec. 15, 2000. The
entire teachings of the referenced applications are incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0003] In the past two decades, considerable effort has been
invested in producing non-human mammals, such as mutant or
transgenic mammals, such as mice, and during that time, a variety
of methods have been developed. In order to produce a desired
mutant animal, such as a mouse, using any of the presently
available methods, one must first produce chimeras and breed the
chimeras to produce homozygous offspring; production of offspring
which are not chimeric requires two breeding cycles for each gene.
This is the case for each mutation to be introduced and, if
offspring exhibiting more than one mutation are desired, additional
breeding cycles are required. For example, if mutant mice bearing
six different alterations (e.g., six different genes) are to be
produced, approximate breeding time will be two years. Producing
desired genetically manipulated mammals, even those for which the
breeding cycle is relatively brief, requires considerable time, as
well as resources, using current methods. It would be very valuable
if a more efficient method of producing mutant mammals, such as
mice, were available.
SUMMARY OF THE INVENTION
[0004] The present invention relates to methods of producing
non-human mammals, which can be mutant non-human mammals or
non-mutant non-human mammals, such as mice, that do not require
production of chimera or chimeric offspring (offspring that consist
of cells that are derived from more than one zygote). The invention
is a method of producing non-human mammals, such as mutant or
non-mutant mice, by tetraploid blastocyst complementation using
non-inbred pluripotent cells or cell lines, such as non-inbred ES
cells or cell lines.
[0005] The present method makes it possible to include multiple
mutations or alterations in the same pluripotent cells (e.g.,
embryonic stem (ES) cells) or cell lines (e.g., ES cell lines)
before producing an animal from the ES cells or ES cell lines. The
present invention relates to methods of producing non-human mammals
that, thus, avoid the time-consuming step of breeding chimera to
produce the desired offspring. As is evident from the work
described herein, mutant or targeted offspring, particularly mice,
that are entirely derived from ES cells or ES cell lines and
survive postnatally have been produced without the need to produce
chimeric intermediates. Mutations introduced into the non-inbred
pluripotent cells or cell lines can be non-random or targeted
alterations or can be random or non-targeted alterations. The
products of either approach are referred to herein as mutant. In
those embodiments in which mutations are non-random or targeted,
the resulting products can also be referred to as targeted (e.g.,
targeted ES cells, targeted ES cell lines, targeted non-human
mutant mammals, such as targeted mutant mice). Alterations can be
of a variety of types, including deletion, addition, substitution,
or modification of all or a portion of DNA (e.g., a gene,
regulatory element) in the ES cells. These alterations include
addition of a gene or gene portion not normally present in the ES
cells or ES cell lines. Non-mutant mice that are derived entirely
from ES cells or ES cell lines and survive postnatally can also be
produced using the method described. The present methods of
producing mice, particularly mutant mice, make it possible to
produce offspring, particularly mutant offspring, very efficiently,
particularly in comparison with other methods.
[0006] The present invention also relates to a method for deriving
fertile XO female mice from non-inbred (F1) mouse male ES cells or
non-inbred (F1) mouse male cell lines and a method of deriving
males and females carrying all genetic alterations introduced into
a single non-inbred ES clone, such as a targeted non-inbred mouse
ES cell clone. Breeding of the mutant males and females allows the
production of a mutant mouse strain derived from a single
non-inbred ES cell clone, such as a targeted (e.g., multiply
targeted ES cell clone), without outcrossing the mutant animal with
a wildtype partner, as is required in presently available
methods.
[0007] The present invention also relates to non-human mammals,
particularly mutant non-human mammals, such as mutant mice,
produced by the methods; cells obtained from the mutant or
non-mutant non-human mammals and cell lines produced from these
cells. A particular embodiment is cells obtained from mutant or
non-mutant mice produced by a method of the present invention;
cells obtained from the mice and cell lines produced from such
cells. The invention further relates to a method of producing
blastocysts useful in the method of producing mutant or non-mutant
mammals, such as mouse blastocysts (non-mutant or mutant) useful
for producing non-mutant or mutant mice by the method described
herein and blastocysts produced by the method.
[0008] The invention also relates to methods of identifying XO F1
ES cells (e.g., mouse XO F1 ES cells) comprising screening a
population of F1 ES cells, such as a population of wildtype or
mutant F1 ES cells, for F1 ES cells that are XO F1 ES cells. In a
particular embodiment, screening is carried out to identify F1 ES
cells in which spontaneous loss of the Y chromosome has occurred,
resulting in XO F1 ES cells. By population of F1 ES cells is meant
a mixture of XO F1 ES cells and XY F1 ES cells.
[0009] In particular embodiments, mutant non-human mammals (e.g.,
mutant mice) are produced to mimic or serve as a model for a
condition (e.g., a neurological, muscular or respiratory condition,
cancer, viral infection, arthritis,) that occurs in another
species, such as in humans. They are used to identify new drugs
that have a therapeutic or preventive effect on the condition or
assess the ability of known drugs to act as therapeutics or
preventatives. Thus, the present invention encompasses methods in
which the mutant non-human mammals (particularly mutant mice) are
used, such as in a method of screening to identify a new drug that
inhibits the occurrence of (prevents the onset, reduces the extent
or severity of) or reverses a condition caused by or associated
with the genetic alteration(s) and a method of screening known
drugs for those that inhibit onset of or reverse such conditions.
Drugs identified by methods in which the mutant mammals of the
present invention are used are also the subject of this invention.
These include drugs that inhibit onset of a condition (prevent the
onset or reduce the extent to which the condition is established or
severity of the condition), referred to as preventatives or
prophylactic drugs and drugs that reverse (partially or completely)
or reduce the extent or duration of the condition once it has
occurred.
DETAILED DESCRIPTION OF THE INVENTION
[0010] As described herein, Applicants have demonstrated that
genetic background is a crucial parameter controlling postnatal
survival of offspring that are entirely derived from ES cells or ES
cell lines. That is, heterozygosity of the genome of the
pluripotent donor cell (e.g., heterozygosity of the donor ES cell
genome) is critical for postnatal survival of offspring whose
development is achieved without the contribution of normal cells
derived from the host embryo. Further, Applicants have demonstrated
that non-human mammals, particularly mice, can be generated without
the need to first create a chimeric intermediate. The ability to
derive offspring (e.g., mice) directly from ES cells or ES cell
lines without the need to produce chimeric intermediates is a
distinct advantage, not only because it avoids the time consuming
and expensive step of producing chimera, but also because it
facilitates the generation of offspring with multiple genetic
alterations. The generation of Fl ES cell-tetraploid mice provides
a simple procedure for directly deriving animals with complex
genetic alterations without the need to create a chimeric
intermediate. The tetraploid technology in combination with the use
of F1 cells or F1 cell lines allows assembly or production of
multiple genetic alterations in the same ES cell clone by
consecutive gene targeting cycles in vitro. The resulting multiply
targeted F1 ES cell clone is introduced into tetraploid blastocysts
to produce an embryo that is then transferred to an appropriate
foster mother and permitted to develop to term. Thus, a transgenic
animal with one or multiple desired or selected genetic alterations
can be generated without the need for production of chimeric
founders and outbreeding with wild type mice.
[0011] Also described herein is a strategy for deriving fertile XO
females from F1 male ES cells or F1 male ES cell lines and a method
of breeding a mutant mouse strain derived from a given multiply
targeted ES cell clone without outcrossing the mutant animal with a
wild type partner. This avoids time consuming and costly
outcrossing, which would otherwise be necessary. Because each F1 ES
cell line is of a given sex--usually male--it would not be possible
to breed a mutant mouse strain derived from a given multiply
targeted ES cell clone without outcrossing, using presently
available methods. As described herein, however, outcrossing is no
longer required, in view of the fact that it is possible to
generate mutant males and females from a single targeted male ES
cell clone by selection for loss of one Y chromosome, resulting in
generation of XO ES cells. In one embodiment, a negative selection
marker (e.g., a negative selection gene, such as a Herpes Tk gene)
is introduced into the Y chromosome of F1 male ES cells, as
described further below, and the resulting cells are subject to
selection with an agent (e.g., gancyclovir) which kills all cells
carrying the Y chromosome. Cells that are not killed have lost the
Y chromosome and, thus, are XO. This enables subsequent generation
of males and females carrying identical genetic alterations, as
described further below.
[0012] The invention described herein relates to a method of
producing non-human mammals, which can be mutant or non-mutant
animals, such as mutant or non-mutant mice. As described herein, it
has now been shown that mutant non-human mammals can be produced
without the intermediate step of producing chimeric animals which,
in presently available methods, must be bred to produce the desired
mutants. In particular, targeted or mutant mice have been produced
and the present invention is described in detail by describing
their production. However, the present invention is useful to
produce mutants or non-mutants of any non-human mammal for which
embryonic stem (ES) cells can be obtained.
[0013] The invention is, in one embodiment, a method of producing a
non-human mammal. The method comprises introducing non-inbred
pluripotent cells, such as non-inbred ES cells, into tetraploid
blastocysts of the same mammalian species, under conditions that
result in production of an embryo (at least one/one or more embryo)
and transferring the resulting embryo(s) into an appropriate foster
mother, such as a pseudopregnant female of the same mammalian
species. The resulting female is maintained under conditions that
result in development of live offspring, thereby producing a mutant
non-human mammal. The resulting non-human mammal is derived from a
single zygote (that which originally gave rise to the ES cells).
Such mammals are referred to herein as ES non-human mammals.
[0014] In another embodiment, the invention is a method of
producing a mutant non-human mammal. The method comprises
introducing non-inbred pluripotent cells, such as non-inbred ES
cells, comprising at least one mutation or alteration into
tetraploid blastocysts of the same mammalian species, under
conditions that result in production of an embryo (at least one/one
or more embryo) and transferring the resulting embryo(s) into an
appropriate foster mother, such as a pseudopregnant female of the
same mammalian species. The resulting female is maintained under
conditions that result in development of live offspring, thereby
producing a mutant non-human mammal. The resulting mutant non-human
mammal is derived from a single zygote (that which originally gave
rise to the ES cells). Such mammals are referred to herein as
mutant ES non-human mammals. The mutations or alterations can be
non-random or targeted or, alternatively, can be introduced
randomly or in a non-targeted manner.
[0015] A specific embodiment of the present invention is a method
of producing a targeted or mutant mouse, comprising: (a)
introducing mouse non-inbred pluripotent cells comprising at least
one alteration in genomic DNA into mouse blastocysts, preferably
tetraploid blastocysts, thereby producing mouse blastocysts
containing mouse non-inbred pluripotent cells; (b) maintaining the
product of (a) under conditions that result in production of
embryos; (c) introducing an embryo or embryos (at least one/one or
more embryos) into a foster mother, such as a pseudopregnant female
mouse; and (d) maintaining the female into which the embryo(s) were
introduced under conditions that result in development of live
offspring, thereby producing a mutant mouse. The mutant mouse is
also referred to herein as a mutant ES mouse. In one embodiment,
the non-inbred mouse pluripotent cells are non-inbred mouse ES
cells, such as F1 cells derived from two different strains of mice
or F2, F3 F4, etc. cells that can be derived from parents after
consecutive brother-sister matings. Alternatively, such cells can
be derived from parents after backcrossing an F1 strain to one of
the parent strain to obtain the first backcross generation (N1) and
by further backcrossing to obtain N2, N3, N4, etc. backcross
generations. As used herein, the term non-inbred ES cells
encompasses all of the hereinabove described ES cells and cell
lines. Derivation of non-inbred ES cells, with specific reference
to production of mouse ES cells, is described in detail in the
Examples.
[0016] In a further embodiment, the invention is a method of
producing a non-mutant mouse, referred to as a non-mutant ES mouse.
The method is carried out as described above for the production of
mutant ES mice, except that DNA in the non-inbred pluripotent
cells, such as non-inbred ES cells (e.g., non-inbred mouse cells)
has not been altered prior to their use. That is, the non-inbred ES
cells as obtained may contain alterations or mutations, but are not
further modified to produce non-random or random mutations. The
method comprises: (a) introducing mouse non-inbred pluripotent
cells into mouse blastocysts, preferably tetraploid blastocysts,
thereby producing mouse blastocysts containing mouse non-inbred
pluripotent cells; (b) maintaining the product of (a) under
conditions that result in production of embryos; (c) introducing an
embryo or embryos (at least one/one or more embryos) into a foster
mother, such as a pseudopregnant female mouse; and (d) maintaining
the female into which the embryo(s) were introduced under
conditions that result in development of live offspring, thereby
producing a non-mutant mouse.
[0017] A variety of methods can be used to introduce mouse
pluripotent cells, such as non-inbred mouse ES cells, into mouse
tetraploid blastocysts. In one embodiment, this is carried out by
injecting the non-inbred cells into tetraploid blastocysts, such as
by microinjection, particularly piezo microinjection. Other methods
can be used to introduce non-inbred ES cells into the blastocysts.
For example, the method described by Amano et al., or a
modification thereof, can be used (Amano, T. et al., Theriogenology
53, 1449-1458 (2000)). Alternatively, any other method, such as a
chemical method, which results in introduction of non-inbred ES
cells into tetraploid blastocysts can be used.
[0018] Non-inbred pluripotent cells, such as non-inbred ES cells,
used in the present method can contain at least one/one or more
genetic alterations or mutations. Alternatively, as described
above, non-inbred ES cells used can be non-mutant (have not been
altered, after they are obtained, to contain a genetic alteration
or mutation); such cells are used to produce non-mutant progeny by
the method of the present invention. The genetic alterations or
mutations that can be present in non-inbred ES cells used include,
but are not limited to, transgenes (cDNA, genes or portions
thereof), mutations (targeted or random), conditional mutations,
targeted insertions of foreign genes, YAC and BAC sized transgenes,
all or part of a chromosome, which may be from the same species as
the embryo or another species, such as from a human. They include
physical knockout of all or a part of a gene, functional knockout
of a gene, introduction of a functional gene and introduction of
DNA or a gene portion that changes the function/level of expression
of a gene present in the ES cell (e.g., a promoter, enhancer or
repressor). An important feature of the method of the present
invention is that multiple genetic alterations, which will
typically be consecutive genetic alterations but can also be
simultaneous, can be made in the non-inbred ES cells, thus
circumventing the need for breeding to combine multiple alterations
in one animal, as is required if presently-available methods are
used. Alterations can also be present in the non-inbred ES cells as
they are obtained from the zygote from which they are derived. As
used herein, the terms mutant non-inbred pluripotent cells, mutant
non-inbred ES cells and similar terms encompass cells which
comprise a mutation or mutations as obtained from the zygote which
gave rise to the cells and cells which are mutated or altered after
they are obtained from the zygote. Alterations can all be of the
same type (e.g., all introduction of exogenous DNA) or of more than
one type (e.g., introduction of exogenous DNA, gene knockout and
conditional gene knockout). They can also be a combination of
mutations present in the non-inbred ES cells as derived from a
zygote and mutations made after they are derived from a zygote. The
alterations made in genomic DNA of non-inbred ES cells can be
chosen to produce a phenotype that is similar to (mimics) a
condition that occurs in other species (e.g., humans) and the
resulting mutant mice can, thus, serve as a model for that
condition.
[0019] A variety of methods, known to those of skill in the art,
can be used to alter or mutate inbred pluripotent (e.g., ES) cells
or cell lines to be used in the method of producing ES mice of the
present invention. For example, an appropriate vector or plasmid
can be used to introduce DNA into ES cells in order, for example,
to integrate DNA into genomic DNA, express foreign DNA in recipient
cells, cause recombination (homologous or nonhomologous) between
introduced DNA and endogenous DNA or knock out endogenous gene(s),
such as by means of the Cre-lox method. Alternatively, alterations
or mutations can be produced by chemical methods or radiation. Gene
targeting can also be used to produce mutant non-inbred pluripotent
cells or cell lines, such as mutant non-inbred ES cells or cell
lines. For example, the methodology described by Rideout and
co-workers can be used. See, Rideout, W. M. et al., Nature
Genetics, 24:109 (2000) and the references cited therein.
[0020] Tetraploid blastocysts can be produced by known methods,
such as that described by James and co-workers. James, R. M. et al,
Genet. Res. Camb., 60:185 (1992). See also Wang, Z-Q et al, Mech.
Dev., 62: 137 (1997) and the references cited therein.
[0021] The invention also relates to methods of identifying XO F1
ES cells, such as mouse XO F1 ES cells, by screening a population
of F1 ES cells, such as a population of wildtype or mutant F1 ES
cells, for F1 ES cells that are XO F1 ES cells. By population of F1
ES cells is meant a mixture of XO F1 ES cells and XY F1 ES cells.
In a particular embodiment, screening is carried out to identify F1
ES cells in which spontaneous loss of the Y chromosome has
occurred, resulting in XO F1 ES cells. The invention further
provides methods for isolating XO F1 ES cells from XY F1 ES cells,
comprising (a) screening a population of F1 ES cells (e.g.,
wildtype or mutant) for loss of the Y chromosome; and (b) isolating
F1 ES cells lacking the Y chromosome, thereby isolating XO F1 ES
cells. Screening can be carried out using a variety of methods
known and readily available in the art, such as using a Y
chromosome probe (e.g., against repetitive elements) in Southern
blot analysis or PCR amplification. F1 ES cells can be generated
using a variety of methods known and readily available in the art,
such as, for example, by limiting dilution subcloning or
transfection with exogenous DNA followed by appropriate selection.
By exogenous DNA is meant any DNA that is not endogenous to the
cells to be transfected or that does not already occur in the cells
to be transfected. An example of an exogenous DNA is a drug
selection marker.
[0022] Also the subject of this invention are mutant non-human
mammals (e.g., mutant ES mice) produced by the method described
herein; methods of producing non-human mammalian embryos; non-human
embryos produced by the method; and a method of identifying a drug
to be administered to treat a condition that occurs in a mammal,
such as a human. The method of producing mutant non-human mammalian
embryos comprises injecting non-human F1 ES cells into non-human
tetraploid blastocysts and maintaining the resulting tetraploid
blastocysts under conditions that result in formation of embryos,
thereby producing a mutant non-human mammalian embryo(s). In one
embodiment, the non-human mammalian embryo is a mutant mouse
embryo.
[0023] Another embodiment of the present invention is a method of
producing a non-human mammalian strain, such as a mouse strain,
particularly a mutant mouse strain, that is derived from a given
(single) ES cell clone, such as a mutant non-inbred ES cell clone,
without outcrossing with a wildtype partner. Until now, it has not
been possible to do so because each F1 ES cell line is of a given
sex (generally male). However, fertile XO females have been
produced from a male ES cell clone by in vitro selection for loss
of the Y chromosome. As a result, a mutant mouse strain carrying
all genetic alterations can be derived by breeding XY males and XO
females, both derived from the same targeted ES cell clone and of
identical genetic makeup without outbreeding of the mutant male to
a normal female. Production of XY and XO subclones from the same
clone, such as a clone carrying one or more genetic mutation or a
multiply targeted clone, makes it possible to generate males and
females carrying the same genetic alterations; such males and
females can then be used to produce genetically identical offspring
without the need for outbreeding.
[0024] For example, insertion of a negative selection marker (e.g.,
the Herpes Tk gene) on the Y chromosome of F1 ES cells makes it
possible to derive XY and XO subclones from an initial male ES cell
clone. The XO ES cells can be produced, for example, by inserting a
Herpes Tk gene onto the Y chromosome by homologous recombination
using known methods. For example, a vector that contains sequences
homologous to a Y-linked gene (such as the Sry gene, the Mov15 gene
or any other Y-linked gene) and expresses the Tk gene can be
produced and introduced into ES cells. The cells are maintained
under conditions that result in homologous recombination between
vector sequences and Y chromosome sequences. The Tk gene is, as a
result, introduced into the Y chromosome. The resulting cells can
then be targeted or otherwise genetically altered. To generate an
XO line from the clones, the cells are subjected to selection by
culturing in the presence of gancyclovir, which results in killing
of all cells carrying the Y chromosome into which the Tk gene has
been inserted. XO cells can be used to produce XO females, using
known methods. Similarly, XY cells can be used to produce males.
The resulting males and females can be bred to produce offspring
carrying the same genetic material (mutations) as the parents.
Other negative selection markers, such as diphtheria toxin, can be
used and are introduced into the Y chromosome in a manner similar
to that described for introduction of the Tk gene. Alternatively,
XO ES cells can be produced by introducing DNA into XY ES cells,
such as by homologous recombination to functionally or physically
delete the Y chromosome. The desired XO cells can be identified,
for example, by use of a Y chromosome probe (e.g., for repetitive
elements) in Southern blot analysis. In addition, because the Y
chromosome is frequently lost spontaneously upon in vitro culture
of ES cells, the mutant male F1 ES cells can be passaged and
subclones screened for spontaneous loss of the Y chromosome. XO ES
cells can be identified, as described in Example 3, for example,
through Southern blot analysis in which a Y chromosome probe
against repetitive elements is used. XO ES cells can also be
identified using other methods known and readily available in the
art, such as, for example, by PCR. Fertile female mice have been
produced from male F1 ES cells that have a male karyotype and are
positive for Y-specific sequences by PCR in early passages. The
B6.times.Balb line, V30. 11 has produced 4/4 females who, as
judged, for example, by their coat color, should be totally derived
from male ES cells.
[0025] The method of the present invention is, thus, also a method
of producing a mutant mouse by breeding a mutant male mouse and a
mutant female mouse, wherein the male mouse (or an ancestor
thereof) and the female mouse (or an ancestor thereof) were
produced from the same F1 male ES cells or cell line, such as from
the same targeted male ES cell clone, and the female mouse is an XO
female. That is, the method is one of producing a mutant mouse
strain by breeding a mutant male mouse and a mutant female mouse
carrying identical genetic alterations as a result of having been
derived from a single targeted male F1 ES cell clone. The mutant
female mouse is XO and is produced (or is the progeny of an
ancestor which was produced) by selecting for loss of the Y
chromosome from a single (individual) male ES cell clone. The
present invention also encompasses female mice which are XO and
were produced from an F1 male ES cell or cell line, such as by
knocking out of the Y chromosome. It also encompasses progeny
produced by breeding a male and a female produced as described
herein and progeny thereof.
[0026] The mutant non-human mammals, such as mutant mice, can be
used as a model for a condition for which a preventive or
therapeutic drug is sought. A method of identifying a drug to be
administered to treat a condition in a mammal comprises producing,
using the method of the present invention, a mutant mouse that is a
model of the condition; administering to the mutant mouse a drug,
referred to as a candidate drug, to be assessed for its
effectiveness in treating or preventing the condition; and
assessing the ability of the drug to treat or prevent the
condition. If the candidate drug reduces the extent to which the
condition is present or progresses or causes the condition to
reverse (partially or totally), the candidate drug is a drug to be
administered to treat the condition.
[0027] The present invention is illustrated by the following
examples, which are not intended to be limiting in any way.
EXAMPLES
[0028] The following examples describe production of mice using
inbred ES cells from four different ES cell lines from three inbred
backgrounds (129/Sv, C57BL/6 and BALB/c) and six different F1 lines
(129/Sv.times.C57BL/6, C57BL.times.129/Sv, BALB/c.times.129/Sv,
129/Sv.times.M. castaneus, C57BL/6.times.BALB/c and
129/Sv.times.FVB); assessment of pups produced using the two types
of ES cells; and comparison of results obtained. The results show
that use of F1 ES cells consistently results in production of
viable mice, whether targeted or untargeted cells are used. They
also demonstrate that genetic background is a crucial parameter for
postnatal survival of pups derived from ES cells. Further, they
demonstrate that the method of the present invention has been
successfully used to produce mice that contain desired alterations
without the need to produce and breed chimera en route to producing
the desired non-chimeric pups.
[0029] Methods and Materials
[0030] The following methods and materials were used to produce
mouse pups.
[0031] Production of ES Cell Clones
[0032] Nuclear transfer of ES cell nuclei into enucleated metaphase
II oocytes was carried out as previously described (Wakayama, T. et
al., Nature, 394:369-374 (1998); Wakayama, T. & Yanagimachi,
R., Nature Genet., 22:127-128 (1999); Ogura, A. et al., Biol.
Reprod., 62:1579-1584 (2000); Rideout, W. M. et al., Nature Genet.,
24:109-110 (2000);Wakayama, T. et al., Proc. Natl. Acad. Sci. USA,
96: 14984-14989 (1999)). 1-3 hours after nuclear transfer oocytes
were activated for 5 hours with 10 mM Sr.sup.++ in Ca.sup.++ free
media in the presence of 5 mg/ml of Cytochalasin B. Embryos were
cultured in vitro to the blastocyst stage and transferred to
recipient mothers.
[0033] Embryo Culture
[0034] All embryo culture was carried out in microdrops on standard
bacterial petri-dishes (Falcon) under mineral oil (Squibb).
Modified CZB media (Chatot, C. L. et al., Biol. Reprod., 42:
432-440 (1990)) was used for embryo culture unless otherwise noted.
Hepes buffered CZB was used for room temperature operations while
long term culture was carried out in bicarbonate buffered CZB at
37.degree. C. with an atmosphere of 5% CO.sub.2 in air.
[0035] Recipient Females and Cesarean Section
[0036] Ten injected blastocysts were transferred to each uterine
horn of 2.5 days post coitum pseudopregnant Swiss females that had
mated with vesectomized males. Recipient mothers were sacrificed at
E 19.5 and pups were quickly removed from the uterus. After
cleaning fluid from their air passages, pups were placed under a
warming light and respiration was observed. Surviving pups were
fostered to lactating BALB/c albino mothers.
[0037] Culture of Embryonic Stem (ES) Cells
[0038] Derivation and culture of embryonic stem cells were carried
out as previously described (Nagy, A. et al., Development,
110:815-821 (1990)) with ES cell lines derived from both inbred and
F1 blastocysts. ES cells were cultured in DMEM with 15% FCS
containing 1000 U/ml Leukocyte Inhibiting Factor (LIF) on
gamma-irradiated primary feeder fibroblasts. For blastocyst
injection ES cells were trypsinized, resuspended in DMEM and
preplated on a standard 10 cm tissue culture dish for thirty
minutes to remove feeder cells and debris.
[0039] Preparation of Two Cell Embryos for Electrofusion
[0040] B6D2F1 females were superovulated by IP injection of 7.5 IU
PMS (Calbiochem) followed 46-50 hours later with 7.5 IU HCG
(Calbiochem). After administration of HCG, females were mated with
B6D2F1 males.). Fertilized zygotes were isolated 24 hours later.
Zygotes were left in Hepes buffered CZB with 0.1% bovine testicular
hyaluronidase for several minutes at room temperature to remove any
remaining cumulus cells. After washing, zygotes were transferred to
a new culture dish containing drops of bicarbonate buffered CZB and
placed at 37.degree. overnight to obtain two-cell embryos.
[0041] Preparation of Tetraploid Embryos by Electrofusion
[0042] 40 hours post HCG the blastomeres of two-cell embryos were
electrofused to produce one-cell tetraploid embryos. Electrofusion
was carried out on in inverted microscope using the lid of a petri
dish as a micro-manipulation chamber. Platinum wires were used as
both electrodes and micromanipulators to align two cell embryos for
fusion. A group of 15 two-cell embryos was placed on the stage in a
200 ml drop of M2 media (Sigma). Embryos were aligned with the
interface between their two blastomeres perpendicular to the
electrical field and a single electrical pulse of 100V with a
duration of 100 ms was applied to each individually. Manipulation
of a single group took less then five minutes. After electrofusion,
embryos were returned to CZB media at 37.degree. C. Embryos that
had not undergone membrane fusion within 1 hour were discarded.
[0043] Piezo Micromanipulator Injection of Tetraploid
Blastocyts
[0044] For microinjection, 5-6 blastocysts were placed in a drop of
DMEM with 15% FCS under mineral oil. A flat tip
microinjection-pipette with an internal diameter of 12-15 um was
used for ES cell injection. 15 ES cells were picked up in the end
of the injection pipette. The blastocyst to be injected was held in
the vicinity of the ICM with a standard holding pipette. The
injection pipette, containing the ES cells was pressed against the
zona opposite the inner cell mass. A brief pulse of the Piezo
(Primatech Pmm, Ibaraki, Japan) was applied and the injection
needle was simultaneously pushed through the zona and trophectoderm
layer into the blastocoel cavity. The ES cells were then expelled
from the injection pipette and pushed against the inner cell mass
of the blastocyst. After injection of the entire group, blastocysts
were returned to CZB media and placed at 37.degree. C. until
transfer to recipient females.
Example 1
Assessment of the Effects of Genetic Heterogeneity of Donor Cells
on Development of ES Cell-tetraploid Pups
[0045] The possible effect of genetic heterogeneity of the donor
cells on the development of ES cell-tetraploid pups was tested by
transferring inbred or F1 ES cells into tetraploid blastocysts and
assessing survival. Injection of ES cells into the blastocoel
cavity of tetraploid blastocysts was aided by the use of a
piezo-driven micromanipulator and the resulting composite embryos
were transferred to recipient females. 312 tetraploid blastocysts
were injected with four different inbred ES cell lines that gave
rise to 20 pups (6%) that were alive and active at cesarean
section. However, 17 of the 20 newborns died of respiratory failure
within 30 minutes. Of the three remaining pups, two were unable to
sustain respiration and died within the next few hours (Table 1).
Only one inbred ES cell-tetraploid pup was able to sustain
respiration and developed to adulthood. In contrast, of 344
tetraploid blastocysts injected with 6 different F1 ES cell lines,
60 (18%) developed to birth, 51 of which (85%) survived to
adulthood (Table 2). Thus, genetic heterogeneity of the donor ES
cells has a significant effect on long-term survival of both
nuclear clones and ES cell-tetraploid pups.
[0046] It has been previously shown that continued passage of ES
cells is detrimental to their developmental potency (Wang, Z. Q.,
et al., Mech. Dev., 62:137-145 (1997); Nagy, A. et al., Proc. Natl.
Acad. Sci. USA, 90:8424-8428 (1993)). In order to assess whether
continuous in vitro culture would impair the survival of F1 ES
cell-tetraploid pups, a 129Sv.times.C57BL/6 ES cell line (V6.5) was
kept continuously in culture and injected into tetraploid
blastocysts after prolonged passage. No impairment of postnatal
survival of the resulting ES cell-tetraploid pups was noted after
either 15 or 25 passages. In addition, F1 ES cell-tetraploid mice
were produced from cells that had been subjected to two consecutive
rounds of drug selection. First, selection with puromycin was used
for isolating cells that carried a targeted insertion of a
tet-transactivator gene in the Rosa26 locus. Second, hygromycin
selection was used to isolate cells with a tet-inducible promoter
driving expression of a hygromycin-thymidine kinase cassette in a
random locus. Injection of these double-selected cells into 20
tetraploid blastocysts resulted in one fall-term pup, which
survived to adulthood (Table 2). The results described herein
indicate that live, adult mice, entirely derived from ES cells can
be generated from F1 ES cells even after long-term passage of the
cells in culture or after consecutive rounds of drug selection.
Example 2
Histological Assessment of Lungs
[0047] ES cell-tetraploid pups derived from inbred ES cells
appeared to suffer from respiratory distress after delivery.
Histological analysis of both inbred and F1 completely ES cell
derived neonates was carried out. Examination of the lungs from
inbred ES cell-tetraploid pups revealed that the alveoli were not
inflated, while the lungs of newborns derived from F1 ES cells were
fully inflated. In addition, interstitial bleeding was often seen
in inbred ES cell derived mice. These observations suggest that the
failure to initiate breathing and/or sustain normal circulation
likely contributed to postnatal death of inbred ES cell-tetraploid
pups.
[0048] Results described herein demonstrate that genetic
heterozygosity is a crucial parameter influencing postnatal
survival of pups derived from ES cells by tetraploid embryo
complementation. Pups derived from inbred ES cells die perinatally
with a phenotype of respiratory failure. In contrast, the great
majority (80 to 85%) of pups derived from F1 ES cells survived to
adulthood. The observed respiratory phenotype appears to be due to
the inbred nature of the ES cell genome.
[0049] The possibility of deriving mice directly from ES cells
without the production of a chimeric intermediate has great
potential for facilitating the generation of animals with multiple
genetic alterations. In conventional approaches, targeted ES cells
are injected into diploid blastocysts to generate chimeric
founders. The derivation of transgenic mice carrying the desired
mutant allele requires out-crossing these chimeras with wild type
mice. Thus, the generation of compound animals that combine
multiple desired alleles or transgenes in their genome entails
time-consuming and expensive cycles of crossing mice derived from
different chimeric founders. In contrast, the ES cell-tetraploid
technology in combination with F1 ES cells allows assembling
multiple genetic alterations in the same ES cell line by
consecutive gene targeting cycles in vitro prior to generating
mutant animals. The desired transgenic mice with numerous genetic
alterations can be derived in a single step by injecting the
multiply targeted F1 ES cells into tetraploid blastocysts. Finally,
unlike nuclear cloning technology, which has proven both difficult
to master and transfer from laboratory to laboratory, the ES
cell-tetraploid technology is easily adapted to any laboratory
currently creating chimeric mice by ES cell blastocyst
injection.
[0050] At present, the mechanisms that permit long-term survival of
clones and ES cell-tetraploid pups derived from F1 but not from
inbred ES cells are unclear. Though it is generally assumed that
"hybrid vigor" is an important parameter in animal survival under
various selective conditions, it is not apparent whether
wide-ranging chromosomal heterozygosity or heterozygosity at only a
few crucial modifier loci is required. Examining the potency of ES
cells that have been derived from backcrosses between F1 mice and
their parental inbred strains may clarify this question.
1TABLE 1 Survival of Inbred ES Cell-Tetraploid Pups Pups Pups Pups
respirating surviving alive at after to adult- ES cell 4N blasts
term C-section hood line Genotype injected (% Inj) (% Alive) (%
Alive) J1 129/Sv 120 9 (7.5) 0 0 V18.6 129/Sv 48 5 (10) 1 (20) 0
V26.2 C57BL/6 72 3 (4) 1 (33) 0 V39.7 BALB/c 72 3 (4) 1 (33) 1 (33)
Total Inbred 312 20 (6) 3 (15) 1 (5)
Example 3
Generation of Fertile Mice Carrying A Mutation of Interest.
[0051] A male targeted F1 ES cell line was made as described above.
The cell line was then screened by Southern blot for subclones that
have lost the Y chromosome. The probe used in the Southern blot to
identify targeted (mutant) ES subclones which have lost the Y
chromosome was a Y chromosome probe against repetitive elements.
This probe was previously described by Lamar, E. E. and Palmer, E.,
Cell, 37:171-177 (1984).
[0052] Subclones that have lost the Y chromosome were taken and
used to make mice by tetraploid embryo complementation as described
above. The female ES mice produced were shown to be fertile,
indicating that they can transmit the mutation of interest.
[0053] Male ES mice were produced from parent cell lines as
described above. The male ES mice produced were shown to be
fertile.
[0054] The loss of the Y chromosome, as shown by Southern blot
analysis, is a general phenomenon in several ES lines, as
demonstrated by the results shown in Table 3. Loss of the Y
chromosome occurs at a frequency that can easily and routinely be
screened for, as shown in Table 4.
[0055] Both male (generated from the Flpreporter targeted line) and
female (generated from subclone #315) mice carry the exact mutation
at the same locus in the genome and when intercrossed will directly
lead to homozygous mutant off-spring. Both male and female ES mice
were shown to be fertile.
[0056] While this invention has been particularly shown and
described with reference to particular embodiments thereof, it will
be understood by those skilled in the art that various changes
inform and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
2TABLE 2 Survival of F1 ES Cell-Tetraploid Pups Pups Pups Pups
respirating surviving alive at after to adult- ES cell 4N blasts
term C-section hood line Genotype injected (% Inj) (% Alive) (%
Alive) V6.5 C57BL/6 72 18 (25) 17 (94) 16 (89) X 129/Sv V6.5*
C57BL/6 60 11 (18) 9 (81) 9 (81) X 129/Sv V6.5** C57BL/6 20 1 (15)
1 (100) 1 (100) X 129/Sv 129B6 129/Sv 48 2 (4) 1 (50) 1 (50) x
C57BL/6 F1.2-3 129/Sv 48 4 (8) 3 (75) 3 (75) X M. Cast. V8.1 129/Sv
24 7 (30) 7 (100) 7 (100) x FVB V17.2 BALB/c 48 13 (27) 12 (92) 11
(85) X 129/Sv V30.11 C57BL/6 24 4 (30) 4 (100) 3 (75) X BALB/c
Total F1 344 60 (18) 54 (90) 51 (85) *ES cell subclone targeted at
the Rosa26 locus. **ES cell subclone serially targeted once at the
Rosa26 locus and once with a random insertion.
[0057]
3TABLE 3 Frequency of Y Chromosome Loss in WT and Targeted ES Cell
Lines # of # of subclones subclones lacking Y screened for repeats
ES cell line Genotype Passage # loss of Y (% of screened) V6.6
129svJae P8-9 448 8(1.8) x c57B6 B6129 C57B6 P6 146 4(2.7) x
129svJae J1 129svJae P7 289 5(1.7) V6.5LJGF1 129svJae 1X 210 3(1.4)
preport x trans c57B6 fection
[0058]
4TABLE 4 Production of Male and Female ES-Tetraploid Mice from
Targeted Cell Lines 4N Neonates alive Mice Sex of surviving Blasts
at C-Section Surviving to mice ES cell line* Inj (% blasts)
Adulthood Female Male FLP reporter 68 7(10.2) 3(4.4) 0 3 FLP
reporter Subclone #66 63 7(11.1) 6(9.5) 6 0 FLP reporter Subclone
#315 46 5(10.8) 5(10.8) 5 0 *The parent line (Flpreporter targeted
line) generated male mice while the two subclones tested # (#66 and
#315), which were identified as having lost the Y chromosome,
generated female mice.
* * * * *