PRODUCTION OF FERTILE XY ANIMALS FROM XY ES CELLS

• US 20110307968A1
• Filed: 06/10/2011
• Published: 12/15/2011
• Est. Priority Date: 06/11/2010
• Status: Active Grant

• Current Assignee: Regeneron Pharmaceuticals Incorporated
• Original Assignee: Regeneron Pharmaceuticals Incorporated
• Inventors: AUERBACH, Wojtek, Poueymirou, William, Frendewey, David, Valenzuela, David, DeChiara, Thomas
• Granted Patent: US 9,149,026 B2
• Time in Patent Office: Days
• US Class Current: 800/18
• CPC Class Codes: A01K 2207/12 Animals modified by administration of exogenous cells
• CPC Class Codes: A01K 2217/052 inducing gain of function
• CPC Class Codes: A01K 2217/054 inducing loss of function
• 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 2267/0331 Animal model for proliferative diseases
• 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 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2500/60 Buffer, e.g. pH regulation, osmotic pressure
• CPC Class Codes: C12N 2517/02 Cells from transgenic animals
• CPC Class Codes: C12N 2517/10 Conditioning of cells for in vitro fecondation or nuclear transfer
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• 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
• CPC Class Codes: C12N 9/0091 oxidizing metal ions (1.16)

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.

Claims

1. A method for making a fertile female XY mouse 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 maintaining mouse ES cells in culture,

wherein the base medium exhibits a characteristic selected from the group consisting of;

an osmolality of from 200 mOsm/kg to less than 329 mOsm/kg, a conductivity of about 11-13 mS/cm, a salt of an alkaline metal and a halide in a concentration of about 50-110 mM, a carbonic acid salt concentration of about 17-30 mM, a total alkaline metal halide salt and carbonic acid salt concentration of about 85-130 mM, and a combination of any two or more thereof;

(b) introducing the donor XY mouse ES cell into a host embryo;

(c) gestating the host embryo; and

,

(d) obtaining an XY female mouse progeny, wherein upon attaining sexual maturity the XY female mouse is fertile.

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

3. The method of claim 2, wherein the genetic modification comprises one or more 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.

4. The method of claim 3, wherein the genetic modification is a knock-out of a STEAP2 gene.

5. The method of claim 1, wherein the base medium exhibits 50±

5 mM NaCl, 26±

5 mM carbonate, and 218±

22 mOsm/kg.

6. The method of claim 5, wherein the base medium exhibits about 3 mg/mL NaCl, 2.2 mg/mL sodium bicarbonate, and 218 mOsm/kg.

7. The method of claim 1, wherein the base medium exhibits 87±

5 mM NaCl, 18±

5 mM carbonate, and 261±

26 mOsm/kg.

8. The method of claim 7, wherein the base medium exhibits about 5.1 mg/mL NaCl, 1.5 mg/mL sodium bicarbonate, and 261 mOsm/kg.

9. The method of claim 1, wherein the base medium exhibits 110±

5 mM NaCl, 18±

5 mM carbonate, and 294±

29 mOsm/kg.

10. The method of claim 9, wherein the base medium exhibits about 6.4 mg/mL NaCl, 1.5 mg/mL sodium bicarbonate, and 294 mOsm/kg.

11. The method of claim 1, wherein the base medium exhibits 87±

5 mM NaCl, 26±

5 mM carbonate, and 270±

27 mOsm/kg.

12. The method of claim 11, wherein the base medium exhibits about 5.1 mg/mL NaCl, 2.2 mg/mL sodium bicarbonate, and 270 mOsm/kg.

13. The method of claim 1, wherein the base medium exhibits 87±

5 mM NaCl, 26±

5 mM carbonate, 86±

5 mM glucose, and 322±

32 mOsm/kg.

14. The method of claim 13, wherein the base medium exhibits about 5.1 mg/mL NaCl, 2.2 mg/mL sodium bicarbonate, 15.5 mg/mL glucose, and 322 mOsm/kg.

15. A method of producing a transgenic mouse homozygous for a genetic modification in the F1 generation, comprising crossing an F0 XY fertile female mouse of claim 1 with a cohort F0 XY male mouse, wherein the F0 XY fertile female mouse and the F0 XY male mouse each is heterozygous for the genetic modification, and obtaining a F1 progeny mouse that is heterozygous for the genetic modification.

16. The method of claim 15, wherein the genetic modification comprises one or more 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.

17. The method of claim 16, wherein the F0 XY fertile female mouse is made according to claim 6.

18. A transgenic mouse homozygous for a genetic modification produced according to claim 15.

19. A fertile female XY mouse produced according to claim 1.

20. The fertile female XY mouse of claim 19, wherein the base medium exhibits 50±

5 mM NaCl, 26±

5 mM carbonate, and 218±

22 mOsm/kg.