Methods for producing a mouse XY embryonic (ES) cell line capable of producing a fertile XY female mouse in an F0 generation

• US 9,902,971 B2
• Filed: 06/26/2015
• Issued: 02/27/2018
• Est. Priority Date: 06/26/2014
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: Frendewey, David, Droguett, Gustavo, Gagliardi, Anthony, Kuno, Junko, Auerbach, Wojtek, Valenzuela, David M.
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US14/751,807
• Publication Number:
  US 20150376651A1
• Time in Patent Office: 977 Days
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/02 Animal zootechnically ameliorated
• CPC Class Codes: A01K 2267/0393 Animal model comprising a reporter system for screening tests
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2500/60 Buffer, e.g. pH regulation, osmotic pressure
• CPC Class Codes: C12N 2517/00 Cells related to new breeds of animals
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

Methods and compositions are provided for generating targeted genetic modifications on the Y chromosome or a challenging target locus. Compositions include an in vitro culture comprising an XY pluripotent and/or totipotent animal cell (i.e., XY ES cells or XY iPS cells) having a modification that decreases the level and/or activity of an Sry protein; and, culturing these cells in a medium that promotes development of XY F0 fertile females. Such compositions find use in various methods for making a fertile female XY non-human mammal in an F0 generation.

Claims

1. A method for making a mouse XY embryonic stem (ES) cell line capable of producing a fertile XY female mouse in an F0 generation, comprising:

(a) modifying a mouse XY embryonic stem (ES) cell to comprise a genetic modification comprising a deletion that inactivates the endogenous mouse Sry gene, wherein the mouse XY ES cell comprises a Y chromosome from a 129S6 strain; and

(b) culturing the modified mouse XY ES cell under conditions to produce a mouse XY ES cell line capable of producing a fertile XY female mouse in an F0 generation, wherein upon introduction of the modified mouse XY ES cell into a host embryo and following gestation of the host embryo to produce F0 mice, at least 60% of the F0 mice are XY females which upon attaining sexual maturity are fertile.

3. The method of claim 1, wherein the mouse XY ES cell is isolated from a mouse that is a cross between a 129S6 strain and a C57BL/6 strain.

4. The method of claim 3, wherein the mouse XY ES cell is isolated from a hybrid embryo produced by crossing a female C57BL/6NTac mouse to a male 129S6/SvEvTac mouse.

5. The method of claim 4, wherein the mouse XY ES cell is a VGF1 mouse ES cell.

6. The method of claim 1, wherein the mouse XY ES cell is not cultured in a feminizing medium.

7. The method of claim 1, wherein the culturing step comprises culturing the modified mouse XY ES cell in a medium comprising:

(1) a base medium comprising about 3 mg/mL sodium chloride and about 2.2 mg/mL sodium bicarbonate and having an osmolality of about 218 mOsm/kg; and

(2) supplements that maintain the modified mouse ES cell in culture.

8. The method of claim 1, wherein the genetic modification further comprises an insertion of one or more nucleotides, a substitution of one or more nucleotides, or a combination thereof.

9. The method of claim 8, wherein the genetic modification further comprises a knockout;

a knockin;

a replacement of an endogenous nucleic acid sequence with a homologous, orthologous, or heterologous sequence;

or a combination thereof.

10. The method of claim 8, wherein the genetic modification further comprises an insertion of a nucleic acid encoding a selectable marker and/or a nucleic acid encoding a reporter gene operably linked to a promoter active in the mouse XY ES cell.

11. The method of claim 8, wherein the genetic modification further comprises an insertion of a nucleic acid encoding a reporter gene operably linked to the endogenous Sry promoter.

12. The method of claim 1, further comprising modifying the XY ES cell to comprise at least one additional targeted genetic modification of a polynucleotide of interest.

13. The method of claim 1, wherein the genetic modification is generated by introducing into the mouse XY ES cell a nuclease agent that induces a nick or a double-strand break at a recognition site on the Y chromosome.

14. The method of claim 1, wherein the genetic modification is generated by introducing into the XY ES cell a targeting vector comprising an insert polynucleotide flanked by first and second homology arms corresponding to first and second target sites at a target genomic locus on the Y chromosome, wherein the mouse XY ES cell is modified to comprise the insert polynucleotide at the target genomic locus.

15. The method of claim 1, wherein the genetic modification is generated by introducing into the XY ES cell:

(i) a nuclease agent that induces a nick or a double-strand break at a recognition site on the Y chromosome; and

(ii) a targeting vector comprising an insert polynucleotide flanked by first and second homology arms corresponding to first and second target sites at a target genomic locus on the Y chromosome,

wherein the mouse XY ES cell is modified to comprise the insert polynucleotide at the target genomic locus.

16. The method of claim 13, wherein the nuclease agent is a zinc finger nuclease (ZFN), a Transcription Activator-Like Effector Nuclease (TALEN), a meganuclease, or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein and a guide RNA (gRNA).

17. The method of claim 16, wherein the nuclease agent is the Cas protein and the gRNA, wherein the Cas protein is a Cas9 protein, and wherein the gRNA comprises:

(a) a CRISPR RNA (crRNA) that targets the recognition site, wherein the recognition site is immediately flanked by a Protospacer Adjacent Motif (PAM) sequence; and

(b) a trans-activating CRISPR RNA (tracrRNA).

18. The method of claim 17, wherein the gRNA targets a sequence comprising SEQ ID NO:

10, SEQ ID NO;

11, or SEQ ID NO;

12.

19. The method of claim 16, wherein the nuclease agent is the TALEN.

20. The method of claim 19, wherein the TALEN targets a sequence comprising SEQ ID NO:

72 and a sequence comprising SEQ ID NO;

73.

33. The method of claim 1, wherein upon introduction of the modified mouse XY ES cell into the host embryo and following gestation of the host embryo, at least 80% of the F0 mice are XY females which upon attaining sexual maturity are fertile.

34. The method of claim 1, wherein upon introduction of the modified mouse XY ES cell into the host embryo and following gestation of the host embryo, at least 90% of the F0 mice are XY females which upon attaining sexual maturity are fertile.

35. The method of claim 1, wherein upon introduction of the modified mouse XY ES cell into the host embryo and following gestation of the host embryo, at least 95% of the F0 mice are XY females which upon attaining sexual maturity are fertile.

40. The method of claim 15, wherein the first homology arm is from about 400-1000 base pairs, and the second homology arm is from about 400-1000 base pairs.

41. The method of claim 15, wherein the sum total of the first homology arm and the second homology arm is at least 10 kb.

2. A method for making a fertile XY female mouse in an F0 generation, comprising:

(a) introducing a mouse XY embryonic stem (ES) cell into a host embryo, wherein the XY ES cell comprises a Y chromosome from a 129S6 strain and a genetic modification comprising a deletion that inactivates the endogenous mouse Sry gene;

(b) gestating the host embryo; and

(c) obtaining F0 mice following gestation of the host embryo, wherein at least 60% of the F0 mice are XY females which upon attaining sexual maturity are fertile.

21. The method of claim 2, wherein the F0 XY female mouse is fertile when crossed to a wild-type mouse.

22. The method of claim 21, wherein the wild-type mouse is a C57BL/6 mouse.

23. The method of claim 2, wherein upon introduction of the modified mouse XY ES cell into the host embryo and following gestation of the host embryo, at least 80% of the F0 mice are XY females which upon attaining sexual maturity are fertile.

24. The method of claim 2, wherein upon introduction of the modified mouse XY ES cell into the host embryo and following gestation of the host embryo, at least 90% of the F0 mice are XY females which upon attaining sexual maturity are fertile.

25. The method of claim 2, wherein upon introduction of the modified mouse XY ES cell into the host embryo and following gestation of the host embryo, at least 95% of the F0 mice are XY females which upon attaining sexual maturity are fertile.

26. The method of claim 2, wherein the mouse XY ES cell is isolated from a mouse that is a cross between a 129S6 strain and a C57BL/6 strain.

27. The method of claim 26, wherein the mouse XY ES cell is isolated from a hybrid embryo produced by crossing a female C57BL/6NTac mouse to a male 129S6/SvEvTac mouse.

28. The method of claim 27, wherein the mouse XY ES cell is a VGF1 mouse ES cell.

29. The method of claim 2, wherein the genetic modification further comprises an insertion of one or more nucleotides, a substitution of one or more nucleotides, or a combination thereof.

30. The method of claim 2, wherein the XY ES cell is isolated from a hybrid embryo produced by crossing a female C57BL/6NTac mouse to a male 129S6/SvEvTac mouse, and upon introduction of the modified mouse XY ES cell into the host embryo and following gestation of the host embryo, at least 80% of the F0 mice are XY females which upon attaining sexual maturity are fertile.

31. The method of claim 30, wherein the XY ES cell is a VGF1 ES cell.

32. The method of claim 2, wherein the host embryo is a pre-morula stage embryo.

36. The method of claim 29, wherein the genetic modification further comprises a knockout;

a knockin;

a replacement of an endogenous nucleic acid sequence with a homologous, orthologous, or heterologous sequence;

or a combination thereof.

37. The method of claim 29, wherein the genetic modification further comprises an insertion of a nucleic acid encoding a selectable marker and/or a nucleic acid encoding a reporter gene operably linked to a promoter active in the mouse XY ES cell.

38. The method of claim 29, wherein the genetic modification further comprises an insertion of a nucleic acid encoding a reporter gene operably linked to the endogenous Sry promoter.

39. The method of claim 2, wherein the XY ES cell further comprises at least one additional targeted genetic modification of a polynucleotide of interest.