Production of fertile XY female mice from XY mouse ES cells

Patent metadata

Field	Value
Publication number	US-9885058-B2
Application number	US-201715486210-A
Country	US
Kind code	B2
Filing date	Apr 12, 2017
Priority date	Jun 11, 2010
Publication date	Feb 6, 2018
Grant date	Feb 6, 2018

Abstract

Methods and compositions are described for making phenotypically female fertile animals from XY donor cells and suitable host embryos. Culture media and methods are provided for maintaining XY donor cells in culture that after introduction into a host embryo and gestation in a suitable host will result in fertile XY female animals. Methods and compositions are described for making fertile female animals in an F0 generation from a donor XY cell and a host embryo, as are methods for making F1 progeny that are homozygous for a modification from a heterozygous F0 fertile male and a heterozygous F0 fertile female sibling.

First claim

We claim:

1. A method for increasing the efficiency of generating embryonic stem (ES) cell-derived mice in an F0 generation, comprising:
(a) maintaining a donor XY mouse ES cell in a medium comprising:
(i) a base medium; and
(ii) supplements suitable for growing the mouse ES cells in culture and maintaining pluripotency,
wherein the base medium comprises sodium bicarbonate in a concentration of 1.5-2.2 mg/mL, comprises fetal bovine serum, and has an osmolality of 218-322 mOsm/kg;

(b) injecting a donor XY mouse ES cell from step (a) into a pre-morula stage host mouse embryo;
(c) introducing the host mouse embryo of step (b) into a recipient female mouse and gestating the host mouse embryo; and
(d) obtaining F0 XY mouse progeny, wherein the ratio of ES-cell-derived pups to total pups generated in the F0 generation is greater than the ratio of ES-cell-derived pups to total pups generated in the F0 generation produced from donor XY ES cells maintained in a medium having an osmolality of greater than 322 mOsm/kg.

2. The method of claim 1 , wherein the base medium further comprises sodium chloride in a concentration of 3.0-6.4 mg/mL.

3. The method of claim 2 , wherein the base medium comprises 3 mg/mL sodium chloride and 2.2 mg/mL sodium bicarbonate and has an osmolality of 218 mOsm/kg.

4. The method of claim 3 , wherein the base medium further comprises 4.5 mg/mL glucose.

5. The method of claim 2 , wherein the base medium comprises 5.1 mg/mL sodium chloride and 1.5 mg/mL sodium bicarbonate and has an osmolality of 261 mOsm/kg.

6. The method of claim 2 , wherein the base medium comprises 6.4 mg/mL sodium chloride and 1.5 mg/mL sodium bicarbonate and has an osmolality of 294 mOsm/kg.

7. The method of claim 2 , wherein the base medium comprises 5.1 mg/mL sodium chloride and 2.2 mg/mL sodium bicarbonate and has an osmolality of 270 mOsm/kg.

8. The method of claim 2 , wherein the base medium comprises 5.1 mg/mL sodium chloride, 2.2 mg/mL sodium bicarbonate, and 15.5 mg/mL glucose and has an osmolality of 322 mOsm/kg.

9. The method of claim 1 , wherein the donor XY mouse ES cell comprises a genetic modification.

10. The method of claim 1 , wherein the maintaining the donor XY mouse ES cell in step (a) further comprises genetically modifying the donor XY mouse ES cell.

11. The method of claim 9 , wherein the genetic modification comprises one or more of a deletion in whole or in part of an endogenous nucleic acid sequence, a substitution of one or more nucleic acids, a replacement of an endogenous nucleic acid sequence with a heterologous nucleic acid sequence, a knockout, and a knock-in.

12. The method of claim 9 , wherein the genetic modification is a knockout of a STEAP2 gene.

13. The method of claim 9 , wherein the genetic modification is a deletion in whole or in part of an endogenous nucleic acid sequence.

14. The method of claim 9 , wherein the genetic modification is a substitution of one or more nucleic acids.

15. The method of claim 9 , wherein the genetic modification is replacement of an endogenous nucleic acid sequence with a heterologous nucleic acid sequence.

16. The method of claim 9 , wherein the genetic modification is a knockout.

17. The method of claim 9 , wherein the genetic modification is a knock-in.

18. The method of claim 9 , wherein the donor XY mouse ES cell is heterozygous for the genetic modification.

19. The method of claim 1 , wherein the ratio of ES cell-derived pups to total pups generated in the F0 generation is greater than 23%.

20. The method of claim 19 , wherein the ratio of ES cell-derived pups to total pups generated in the F0 generation is at least 40%.

21. The method of claim 20 , wherein the ratio of ES cell-derived pups to total pups generated in the F0 generation is at least 51%.

22. The method of claim 21 , wherein the ratio of ES cell-derived pups to total pups generated in the F0 generation is at least 61%.

23. The method of claim 22 , wherein the ratio of ES cell-derived pups to total pups generated in the F0 generation is at least 72%.

24. The method of claim 23 , wherein the ratio of ES cell-derived pups to total pups generated in the F0 generation is at least 87%.

25. The method of claim 24 , wherein the ratio of ES cell-derived pups to total pups generated in the F0 generation is at least 91%.

26. The method of claim 1 , wherein the base medium further comprises Wnt-3a conditioned medium.

Assignees

• Regeneron Pharma

Inventors

• Auerbach Wojtek
• Dechiara Thomas
• Poueymirou William
• Frendewey David
• Valenzuela David

Classifications

• C12N2517/10

  Conditioning of cells for in vitro fecondation or nuclear transfer · CPC title

• A01K2217/075

  inducing loss of function, i.e. knock out · CPC title

• A01K2217/052

  inducing gain of function · CPC title

• C12N15/873 Primary

  Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos · CPC title

• A01K2267/0331

  Animal model for proliferative diseases · CPC title