Production of fertile XY animals from XY ES cells

• US 9,149,026 B2
• Filed: 06/10/2011
• Issued: 10/06/2015
• 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
• Primary Examiner(s): Noble, Marcia S
• Assistant Examiner(s): Hsu, Chi-Feng
• Application Number:
  US13/157,728
• Publication Number:
  US 20110307968A1
• Time in Patent Office: 1,579 Days
• US Class Current: 1/1
• 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 and breeding a fertile female XY mouse in an F0 generation, comprising:

(a) maintaining donor XY mouse ES cells 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 donor XY mouse ES cells do not comprise a Tdy-negative sex reversal genetic modification and wherein the base medium comprises an osmolality of 218 mOsm/kg to 322 mOsm/kg, a sodium chloride (NaCl) concentration of 50-110 mM and a sodium bicarbonate concentration of 13-25 mM;

(b) introducing 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;

(d) obtaining an F0 XY mouse progeny comprising a F0 XY phenotypically female mouse, wherein upon attaining sexual maturity the F0 XY female mouse is fertile; and

(e) breeding the F0 XY fertile female mouse to produce progeny.

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 a substitution of one or more nucleic acids.

4. The method of claim 2, wherein the genetic modification is a knock-out of a STEAP2 gene.

5. The method of claim 1, wherein the base medium comprises 87±

5 mM NaCl, 18±

5 mM sodium bicarbonate, and 261±

26 mOsm/kg.

6. The method of claim 1, wherein at least 34% of the F0 XY mouse progeny derived from the donor XY mouse ES cell are phenotypically female XY mice.

7. The method of claim 1, wherein at least 15% of the F0 XY mouse progeny derived from the donor XY mouse ES cell are phenotypically female XY mice.

8. The method of claim 1, wherein all of the females derived from the donor XY mouse ES cell in the F0 generation have a XY genotype.

9. The method of claim 1, wherein the breeding comprises crossing the F0 XY fertile female mouse with a cohort F0 XY male mouse, wherein the F0 XY fertile female mouse and the F0 XY male mouse each is heterozygous for a genetic modification, and obtaining an F1 progeny mouse that is homozygous for the genetic modification.