PRODUCTION OF FERTILE XY FEMALE ANIMALS FROM XY ES CELLS

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

1-20. (canceled)

21. 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 an F0 XY mouse progeny.

22. The method of claim 21, wherein the base medium further comprises sodium chloride in a concentration of 3.0-6.4 mg/mL.

23. The method of claim 22, 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.

24. The method of claim 23, wherein the base medium further comprises 4.5 mg/mL glucose.

25. The method of claim 22, 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.

26. The method of claim 22, 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.

27. The method of claim 22, 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.

28. The method of claim 22, 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.

29. The method of claim 21, wherein the donor XY mouse ES cell comprises a genetic modification.

30. The method of claim 21, wherein the maintaining the donor XY mouse ES cell in step (a) further comprises genetically modifying the donor XY mouse ES cell.

31. The method of claim 29, 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.

32. The method of claim 29, wherein the genetic modification is a knockout of a STEAP2 gene.

33. The method of claim 29, wherein the genetic modification is a deletion in whole or in part of an endogenous nucleic acid sequence.

34. The method of claim 29, wherein the genetic modification is a substitution of one or more nucleic acids.

35. The method of claim 29, wherein the genetic modification is replacement of an endogenous nucleic acid sequence with a heterologous nucleic acid sequence.

36. The method of claim 29, wherein the genetic modification is a knockout.

37. The method of claim 29, wherein the genetic modification is a knock-in.

38. The method of claim 29, wherein the donor XY mouse ES cell is heterozygous for the genetic modification.

39. The method of claim 21, wherein the ratio of ES cell-derived pups to total pups generated in the F0 generation is greater than 23%.

40. The method of claim 39, wherein the ratio of ES cell-derived pups to total pups generated in the F0 generation is at least 40%.

41. The method of claim 40, wherein the ratio of ES cell-derived pups to total pups generated in the F0 generation is at least 51%.

42. The method of claim 41, wherein the ratio of ES cell-derived pups to total pups generated in the F0 generation is at least 61%.

43. The method of claim 42, wherein the ratio of ES cell-derived pups to total pups generated in the F0 generation is at least 72%.

44. The method of claim 43, wherein the ratio of ES cell-derived pups to total pups generated in the F0 generation is at least 87%.

45. The method of claim 44, wherein the ratio of ES cell-derived pups to total pups generated in the F0 generation is at least 91%.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0060] FIG. 1 shows the results of breeding using F0 generation female XY mice made from different XY ES cell clones.

[0061] FIG. 2 shows generation of XY female mice from ES cells incubated with low-salt DMEM, DMEM, or low-salt DMEM supplemented with FS (Wnt-3a-conditioned media, i.e., media conditioned by mouse L-cells transfected with a Wnt-3a-expression construct), NaCl, and NaHCO.sub.3.

DETAILED DESCRIPTION

[0062] All publications cited in this disclosure are hereby incorporated by reference.

[0063] The phrase base medium or base media includes a base medium known in the art (e.g., DMEM) that is suitable for use (with added supplements) in growing or maintaining ES cells in culture. Base media suitable for making a fertile XY female (i.e., low-salt DMEM) differs from base media typically used to maintain ES cells in culture. For purposes of discussing base media in general, base media that are not suitable for making fertile XY females are described in this section as DMEM and in the following table (e.g., typical DMEM media). For purposes of discussing base media suitable for making fertile XY females, the phrase low-salt DMEM is used. Differences between base media typically used to maintain ES cells in culture (e.g., DMEM) and base media suitable for making fertile XY females (e.g., low-salt DMEM) are articulated herein. The phrase low-salt DMEM is used for convenience; suitable DMEM for making fertile XY females exhibits characteristics not limited to low-salt, but includes those described herein. For example, the DMEM shown in Table 1 can be made suitable for making fertile XY females by altering the sodium chloride and/or sodium bicarbonate concentrations as provided for herein, which will also result in a different osmolality and a different conductivity as compared with the DMEM shown in Table 1. An example of base medium is Dulbeco's Modified Eagle's Medium (DMEM), in various forms (e.g., Invitrogen DMEM, Cat. No. 11971-025) (Table 1). A suitable low-salt DMEM is available commercially as KO-DMEM (Invitrogen Cat. No. 10829-018). Base medium is typically supplemented with a number of supplements known in the art when used to maintain cells in culture for use as donor cells. Such supplements are indicated as supplements or +supplements in this disclosure.

[0064] The term supplements or the phrase +supplements, includes elements added to base medium for growing or maintaining donor cells in culture, e.g., for maintaining pluripotency or totipotency of donor cells in culture. For example, media supplements suitable for growing or maintaining non-human ES cells in culture include fetal bovine serum (FBS), glutamine, penicillin and streptomycin (e.g., penstrep), pyruvate salts (e.g., sodium pyruvate), nonessential amino acids (e.g., MEM NEAA), 2-mercaptoethanol, and LIF.

[0065] In various embodiments of media for maintaining non-human donor cells in culture, to about 500 mL of base medium the following supplements are added: about 90 mL FBS (e.g., Hylcone FBS Cat. No. SH30070.03), about 2.4 millimoles of glutamine (e.g., about 12 mL of a 200 mM glutamine solution, e.g., Invitrogen Cat. No. 25030-081), penicillin:streptomycin (e.g., 60,000 units of Penicillin G sodium and 60 mg of streptomycin sulfate, with about 51 mg of NaCl; e.g., about 6 mL of Invitrogen pennstrep, Cat. No. 15140-122), about 0.6 millimoles of sodium pyruvate (e.g., 6 mL of 100 mM sodium pyruvate, Invitrogen Cat. No. 11360-070), about 0.06 millimoles of nonessential amino acids (e.g., about 6 mL of MEM NEAA, e.g., MEM NEAA from Invitrogen Cat. No. 11140-050), about 1.2 mL 2-mercaptoethanol, and about 1.2 micrograms of LIF (e.g., about 120 microliters of a 10.sup.6 units/mL LIF preparation; e.g., about 120 microliters of Millipore ESGRO-LIF, Cat. No. ESG1107). When composing base media for maintaining XY ES cells for making fertile XY females, typically the same supplements in about the same amounts are employed, but the composition of the base medium will differ (from DMEM, e.g., from the medium described in the table above) and the difference(s) correspond to the difference(s) taught herein.

[0066] In some embodiments, supplements include Wnt-conditioned media, e.g., Wnt-3a conditioned media.

[0067] The term animal, in reference to donor cells and/or host embryos, includes mammals, fishes, and birds. Mammals include, e.g., humans, non-human primates, rodents (e.g., mice, rats, hamsters, guinea pigs), livestock (e.g., bovine species, e.g., cows, steer, etc.; ovine species, e.g., sheep, goats, etc.; and porcine species, e.g., pigs and boars). Birds include, e.g., chickens, turkeys, ostrich, geese, ducks, etc. The phrase non-human animal, in reference to donor cells and/or host embryos, excludes humans.

[0068] In various embodiments, the donor cell and/or the host embryo are not from one or more of the following: Akodon spp., Myopus spp., Microtus spp., Talpa spp. In various embodiments, the donor cell and/or the host embryo are not from any species of which a normal wild-type characteristic is XY female fertility. In various embodiments, where a genetic modification is present in the donor cell or the host embryo, the genetic modification is not an XYY or XXY, a Tdy-negative sex reversal, Tdy-positive sex reversal, an X0 modification, an aneuploidy, an SRY translocation or modification, an fgf9.sup./ genotype, or a SOX9 modification.

Overview

[0069] Methods for making nonhuman animals, e.g., mice, from donor ES cells and host embryos are known in the art. Donor ES cells are selected for certain characteristics that enhance the ability of the cells to populate a host embryo and thus contribute in part or in substantial part to an animal formed by the donor ES cells and the host embryo. The animal formed may be male or female, based in large part on the genotype of the ES cell (e.g., XY or XX).

[0070] The majority of ES cell liens for making mice have a male XY genotype. Because of the dominance of the Y chromosome in mammalian sex determination, XY ES cells, when introduced into a host embryo and gestated, nearly always result in the first generation (F0) in phenotypically male animals that are chimeras, i.e., that contain cells derived from the male donor ES cell (XY) and cells derived from the host embryo, which can be either male (XY) or female (XX). To the extent that phenotypic females are observed in the F0 generation, these typically arise from the introduction of XY ES cells into a female XX embryo that results in a chimera whose ES cell contribution is insufficient to masculinize the embryonic genital ridge. In most cases such female chimeras do not produce oocytes derived from the XY ES cells and, therefore, are not capable of transmitting the ES cell genome to the next generation. In rare cases, female chimeras do not produce oocytes derived from the XY ES cells; these females can transmit the ES cell genome to the next generation (see, e.g., Bronson et al. (1995) High incidence of XXY and XYY males among the offspring of female chimeras from embryonic stem cells, Proc. Natl. Acad. Sci USA 92:3120-3123).

[0071] Phenotypically female mice with an XY genotype can arise as the result specific mutations. See, e.g., Lovell-Badge et al. (1990) XY female mice resulting from a heritable mutation in the primary testis determining gene, Tdy, Development 109:635-646; see also, Colvin et al. (2001) Male-to-Female Sex Reversal in Mice Lacking Fibroblast Growth Factor 9, Cell 104(6):875-889 (Fgf/ XY females that die at birth from lung hypoplasia). The South American Akadon spp. of rodents comprise XY females (see, e.g., Hoekstra et al. (2000) Multiple origins of XY female mice (genus Akodon): phylogenetic and chromosomal evidence, Proc. R. Soc. Lond. B 267:1825-1831), but ES cell lines from such mice are generally not available and not widely used, if at all.

[0072] In some instances, e.g., using the VELOCIMOUSE method (see, e.g., U.S. Pat. Nos. 7,659,442, 7,576,259, 7,294,754, and Poueymirou et al. (2007) F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses, Nat. Biotech. 25(1):91-99; each hereby incorporated by reference), it is possible to obtain F0 generation mice that are fully derived from the donor ES cell. Under normal circumstances and standard experimental conditions, XY donor ES cells produce only phenotypically male fully ES cell-derived mice, while ES cells that are XX or X) (XY ES cells that have lost the Y chromosome) produce only phenotypically female fully ES cell-derived mice. To produce mice with homozygous targeted mutations from the male and female fully ES cell-derived mice requires two subsequent generations of breeding to first produce the F1 generation heterozygous male and females that when intercrossed have the potential to produce homozygous progeny in the F2 generation.

[0073] The inventors have devised a method for making a phenotypically female fertile XY mouse from an XY donor cell (e.g., an XY donor cell derived from a phenotypically male mouse) and a suitable host embryo. The method comprises making such a mouse in the F0 generation, which allows for forming a breeding pair (a male F0 and a female F0) in the F0 generation. This is particularly useful where the donor cell comprises a heterozygous genetic modification, and a homozygous mouse is desired. Although this disclosure illustrates the invention in the context of making phenotypically female fertile XY mice from donor mouse XY ES cells, the methods and compositions described herein may be applied to make phenotypically female XY fertile nonhuman animals from any suitable nonhuman cell (e.g., an iPS cell, an ES cell, or a pluripotent cell) and any suitable nonhuman embryo.

[0074] Methods and compositions are described that include conditions for maintaining a donor cell such that when the donor cell is used to generate an animal by introducing the donor cell into a host embryo, the animal so generated includes a phenotypically female fertile XY animal. A phenotypically female fertile XY animal includes an animal that exhibits sufficient phenotypically female characteristics to ovulate and to gestate an embryo upon fertilization of an ovum produced by ovulation in the animal, including to gestate an embryo to term and give birth to a live-born animal.

[0075] The inventors have devised a method that results, in various embodiments at least about 10%, 15%, 20%, or 25% or more of the time, in birth of a fertile female XY mouse from an XY mouse ES cell.

Animal Husbandry

[0076] In one aspect, a method is provided for generating a female animal from a sperm cell and an egg cell, comprising maintaining the sperm cell and/or the egg cell in a medium comprising low-salt base medium for one, two, three, or four or more days prior to fertilization, contacting the sperm cell and the egg cell under conditions that permit fertilization to form a fertilized egg, implanting the fertilized egg in a suitable host for gestation, gestating in the host, and obtaining a litter comprising a female animal.

[0077] In one embodiment, the fertilized egg is further maintained in the medium comprising low-salt base medium for one, two, three, or four or more days prior to implantation in the suitable host.

[0078] In one aspect, a method is provided for favoring the generation of a female animal from a fertilized egg or an embryo, comprising maintaining the fertilized egg or embryo in a medium comprising low-salt base medium for one, two, three, or four or more days prior to implantation in a suitable host, implanting the fertilized egg or embryo into a suitable host for gestation, gestating the fertilized egg or embryo in the host, and obtaining a litter comprising a female animal.

[0079] In one aspect, the methods and compositions of the invention are employed to make a female pet, a female domesticated farm animal, a female animal as a scientific research subject, or an animal of an endangered species. In one embodiment, the animal is a mouse, rat, hamster, monkey, ape, cat, dog, cow, horse, bull, sheep, goat, pig, deer, and bison.

EXAMPLES

Example 1

Donor XY ES Cells and Host Embryos

[0080] Donor Cells and Host Embryos. Donor ES cells were 129S6C57B16/F1 hybrid ES cells. The donor ES cells were frozen in freezing medium containing 10% DMSO until use. Once thawed, donor ES cells were maintained in base medium and supplements as described below. Host embryos were from Swiss Webster (SW) mice, and were maintained in KSOM medium (Millipore) until use. Eight-cell embryos were obtained as previously described (Poueymirou et al. (2007) Nature Biotech. 25(1):91-99; U.S. Pat. Nos. 7,659,442, 7.576,259, and 7,294,754).

[0081] DMEM ES cells: ES cells prepared and frozen in DMEM were thawed in DMEM, grown for three days, and microinjected into host embryos in DMEM.

[0082] Low-salt DMEM ES cells: ES cells prepared and frozen in low-salt DMEM were thawed in low-salt DMEM (KO-DMEM), grown for three days, and microinjected into host embryos in DMEM.

[0083] FS low-salt DMEM: ES cells prepared and frozen in low-salt DMEM were thawed and maintained in low-salt DMEM (440 mL)+10% Wnt-3a-conditioned media (FS) (60 mL), and microinjected into host embryos in DMEM.

[0084] Low-salt DMEM+NaCl+NaHCO.sub.3: ES cells prepared and frozen in low-salt DMEM with added NaCl (1,300 mg/L) and NaHCO.sub.3 (1,500 mg/L) and microinjected into host embryos in DMEM.

[0085] 10% Wnt-3a-conditioned media (FS): Wnt-3a-conditioned media was made from cultures of mouse L cells transformed with a Wnt-3a expression vector (ATCC CRL-2647). The L cells are grown according to ATCC instructions (except that KO-DMEM is used in place of DMEM), in a FibraStage (New Brunswick) system.

Example 2

Making F0 Generation Mice Derived from Donor ES Cells

[0086] Generating F0 Generation Mice. Donor ES cells were introduced into 8-cell stage pre-morula host embryos using the VELOCIMOUSE method, as described previously (Poueymirou et al. (2007) Nature Biotech. 25(1):91-99; U.S. Pat. Nos. 7,659,442, 7,576,259, and 7,294,754), except that the mouse ES cells were maintained in the base medium plus supplements as described herein. For microinjection, ES cells were grown and microinjected into the embryos, and the embryos were cultured overnight in either KSOM or DMEM medium prior to implantation into surrogate mothers.

Example 3

F0 Generation Fertile Female Mice from Donor XY ES Cells

[0087] In a typical protocol, ES cells are thawed in the presence of KO-DMEM and grown for one passage (about 5 five days). Passaged cells are then electroporated with a gene targeting vector and then placed under selection for 10 days in a medium comprising KO-DMEM (Invitrogen Cat. No. 10829-018). Drug-resistant cells are harvested and expanded in a medium comprising KO-DMEM, then frozen. For microinjection, cells are thawed in KO-DMEM and grown for 3 days in KO-DMEM, then microinjected into embryos in DMEM. The embryos are then introduced into surrogate mothers for gestation.

[0088] Mouse pups were initially characterized as male or female based on the appearance of external genitalia in order to select breeding pairs.

[0089] FIG. 1 shows that F0 XY females exhibit a high rate of fertility. Twenty-one out of 33 F0 XY females produced litters.

Example 4

Comparing DMEM with Low-Salt DMEM

[0090] Osmolality was measured on a Advanced Model 3250 Single-Sample Osmometer. Conductivity was measured on a Mettler Toledo GmbH SevenMulti ECN # 15055 conductivity meter.

[0091] The effect of low-salt DMEM and of DMEM (each with supplements) on the formation of F0 generation XY females from XY ES cells was studied. Table 2 shows the osmolality and conductivity values of base media with and without additional salts and/or supplements. The indicator +supplements=addition (to 0.5 L of base medium) of the following: 90 mL Hyclone FBS (Cat. No. SH30070.03), 12 mL of Invitrogen glutamine solution (Cat. No. 25030-081), 6 mL of Invitrogen Pen Strep (Cat. No. 15140-122), 6 mL of Invitrogen sodium pyruvate (Cat. No. 11360-070), 6 mL of MEM NEAA (Invitrogen Cat. No. 11140-050), 1.2 mL 2-mercaptoethanol, and 120 microliters of Millipore ESGRO-LIF (Cat. No. ESG1107).

[0092] FIG. 2 shows a comparison of XY ES cells grown in different media prior to microinjections into host embryos. XY ES cells grown and maintained in low-salt DMEM and then injected into embryos produced XY females. XY ES cells grown and maintained in low-salt DMEM supplemented with NaCl and NaHCO.sub.3 and then injected into embryos produced no XY females. This demonstrates that XY female production is promoted when XY ES cells are maintained in low-salt DMEM, and that the sex ratio of XY ES cells can be controlled by altering the salt concentration of the base medium. Adding a Wnt-3a-conditioned medium (10% FS) to a low-salt DMEM increased the frequency of production of F0 XY females.

[0093] Furthermore, the efficiency of generating ES cell-derived mice in the F0 increased when the ES cells were maintained in Low-salt DMEM. The ratio of ES cell-derived pups to total pups generated in the F0 generation increased from about 23% for ES cells maintained in DMEM, to 61% for ES cells maintained in low-salt DMEM, to 72% for ES cells maintained in low-salt DMEM supplemented with 10% Wnt-3a-conditioned media. See FIG. 2.

TABLE-US-00001 TABLE 2 Comparison of DMEM and Low-salt DMEM Physical Characteristics Conductivity Osmolality Medium for ES Cells (mS/cm) (mOsm/kg) Low-salt DMEM, alone 12.84 270 DMEM, alone 15.40 337 Low-salt DMEM + NaHCO.sub.3 + NaCl, alone 15.82 342 Low-salt DMEM, + supplements 12.75 279 DMEM, + supplements 14.91 330 Low-salt DMEM + NaHCO.sub.3 + NaCl, + 15.29 335 supplements

Example 5

Analysis of F0 Generation Mice

[0094] Coat Color. Mice were analyzed for coat color contribution from donor XY ES cells (agouti) and host embryo (white). None of the F0 generation mice exhibited any coat color contribution from host embryos.

[0095] Gender. F0 generation pups were identified as female or male by visual inspection of the external genitalia. F0 pups were assigned gender and paired for breeding based on visual inspection.

[0096] Genotyping. The presence of an X chromosome was detected using a TAQMAN QPCR assay specific for a sequence on the X chromosome. The presence of Y chromosome was detected using a TAQMAN QPCR assay specific for a sequence on the Y chromosome. The genotyping of phenotypically female F0 generation mice indicated a single copy of the X chromosome and a single copy of the Y chromosome in those phenotypically female mice tested.

[0097] Karyotyping. Six F0 generation XY females were karyotyped. Karyotyping results indicated that all six had a normal X and a normal Y chromosome.

[0098] XY Female Reproductive Anatomy. Several F0 generation XY females were examined for internal reproductive organs. All of the F0 XY females examined appeared to have normal female internal reproductive organs. Tissue samples from each reproductive organ (ovary, oviduct, uterus) were genotyped, and the results indicated that the tissues had a uniform XY genotype.

Example 6

Analysis of the Effect of Osmolality on Efficiency of Generating ES Cell-Derived Pups and XY Females

[0099] To determine the effect of osmolality on the generation of XY females from XY ES cells maintained in low-salt, low-carbonate DMEM, glucose was added to low-salt, low-carbonate DMEM to bring the osmolality to within that of DMEM. Osmolality was measured on a Advanced Model 3250 Single-Sample Osmometer.

[0100] Donor XY ES cells were maintained in low-salt, low-carbonate, high glucose DMEM containing inter alia 5.1 mg/ml NaCl, 2.2 mg/ml NaHCO.sub.3, and 15.5 mg/ml glucose, having an osmolality of 322 mOsm/kg (DMEM-LS/LC/HG). Upon transfer of said ES cells into embryos per the VELOCIMOUSE method (supra), 15% of all resultant ES cell-derived F0 progeny were phenotypically female XY mice. As a negative control, in the F0 generation, no phenotypically female XY mice were derived from ES cells maintained in DMEM (DMEM: 6.4 mg/ml NaCl, 3.7 mg/ml NaHCO.sub.3, and 4.5 mg/ml glucose; 329 mOsm/kg). This 15% F0 XY female result lies between the 0% F0 XY females from DMEM-derived ES cells (329 mOsm/L) and the 27.8% F0 XY female mice derived from ES cells maintained in low-salt, low carbonate DMEM (DMEM-LS/LC: 5.1 mg/ml NaCl, 2.2 mg/ml NaHCO.sub.3, and 4.5 mg/ml glucose; 270 mOsm/kg). Thus, one interpretation is that osmolality provides some of the feminization effect, but not all. An alternative explanation is that the low salt and/or low carbonate provides the feminization effect, and high glucose impedes to some extent the feminization of XY ES cells. See Table 3.

[0101] Furthermore, the efficiency of generating ES cell-derived mice (Table 3) in F0 when the ES cells were maintained in DMEM-LS/LC/HG (i.e., about 40%) was greater than that for ES cells maintained in DMEM (i.e., about 22%), but not quite as high as that for ES cells maintained in DMEM-LS/LC (i.e., about 51%). See Table 3.

TABLE-US-00002 TABLE 3 Effect of Osmolarity, Salt, and Carbonate on ES-cell Derived Pups and F0 XY Females Osmol- ES- ality NaCl NaHCO.sub.3 Glucose Derived ES-derived pups (mOsm/ (mg/ (mg/ (mg/ pups/Total XY XY Media kg) mL) mL) mL) pups male female DMEM 329 6.4 3.7 4.5 13/58 13/13 0/13 (22.4%) (0%) DMEM- 270 5.1 2.2 4.5 36/71 26/36 10/36 LS/LC (50.7%) (27.8%) DMEM- 322 5.1 2.2 15.5 20/50 17/20 3/20 LS/LC/ (40%) (15%) HG DMEM- 218 3.0 2.2 4.5 53/58 35/53 18/53 VLS/LC (91.4%) (34.0%) DMEM- 261 5.1 1.5 4.5 50/57 33/50 17/50 LS/VLC (87.7%) (34%) DMEM- 294 6.4 1.5 4.5 49/68 35/49 14/49 VLC (72.1%) (28.6%)

Example 7

Analysis of the Effect of Salt Concentration on Efficiency of Generating ES Cell-Derived Pups and XY Females

[0102] To determine the effect of salt concentration or ionic strength on the generation of XY females from XY ES cells, ES cells were maintained in very low salt (DMEM-VLS/LC: 3.0 mg/mL NaCl, 2.2 mg/mL NaHCO.sub.3, 4.5 mg/mL glucose, at 218 mOsm/kg). Upon transfer of said ES cells into embryos per the VELOCIMOUSE method (supra), 34% of all resultant ES cell-derived F0 progeny were phenotypically female XY mice; a slight increase over the DMEM-LS/LC control level of 27.8%. Interestingly, 91.4% of the F0 pups resulting from the transfer of ES cells maintained in DMEM-VLS/LC media were ES cell-derived; whereas only 50.7% and 22.4% were ES cell-derived in the DMEM-LS/LC and DMEM controls, respectively.

[0103] In another experiment, ES cells were maintained in high salt and low carbonate media (DMEM-HS/VLC: 6.4 mg/mL NaCl, 1.5 mg/mL NaHCO.sub.3, 4.5 mg/mL glucose, at 294 mOsm/kg). Upon transfer of said ES cells into embryos per the VELOCIMOUSE method (supra), 28.6% of all resultant ES cell-derived F0 progeny were phenotypically female XY mice; a slight increase over the DMEM-LS/LC control level of 27.8%. Interestingly, 72.1% of the F0 pups resulting from the transfer of ES cells maintained in DMEM-HS/VLC media were ES cell-derived; whereas only 50.7% and 22.4% were ES cell-derived in the DMEM-LS/LC and DMEM controls, respectively.

[0104] These results confirm that low salt and/or low carbonate contribute both to the increase in proportion of ES cell-derived F0 progeny as well as F0 XY females. (See Table 3.)

Example 8

Analysis of the Effect of Carbonate Concentration on Efficiency of Generating ES Cell-Derived Pups and XY Females

[0105] To determine the effect of carbonate concentration on the generation of XY females from XY ES cells, ES cells were maintained in low salt and very low carbonate media (DMEM-LS/VLC: 5.1 mg/mL NaCl, 1.5 mg/mL NaHCO.sub.3, 4.5 mg/mL glucose, at 261 mOsm/kg). Upon transfer of said ES cells into embryos per the VELOCIMOUSE method (supra), 34% of all resultant ES cell-derived F0 progeny were phenotypically female XY mice; a slight increase over the DMEM-LS/LC control level of 27.8%. Interestingly, 87.7% of the F0 pups resulting from the transfer of ES cells maintained in DMEM-LS/VLC media were ES cell-derived; whereas only 50.7% and 22.4% were ES cell-derived in the DMEM-LS/LC and DMEM controls, respectively.

[0106] These results confirm that low carbonate contributes both to the increase in proportion of ES cell-derived F0 progeny as well as F0 XY females. (See Table 3.)

Example 9

Phenotype of F0 XY Female Mice

[0107] F0 XY phenotypic female mice exhibited relatively normal phenotype attributes compared to F1 XX phenotypic female mice of the same strain. The XY female mice however did exhibit a larger range of values for each physical parameter. The body weight of the adult XY females ranged from about 15 grams to about 30 grams with an average of about 21.5 grams. The body weight of the adult XX females ranged from about 16 grams to about 17 grams with an average of about 16.8 grams.

[0108] The ratio of the distance between the anus and the genitals was determined and calculated as a ratio of body mass (anogenital distance (cm)/body mass (g)). The ratio for F0 XY females ranged from about 0.11 cm/g to about 0.24 cm/g with an average of about 0.16 cm/g. The ratio for F1 XX females ranged from about 0.17 cm/g to about 0.19 cm/g with an average of about 0.18 cm/g.

[0109] There was no significant difference between the relative masses of various organs (e.g., liver, kidneys, heart and lung, and spleen) for the XY female mice and the XX female mice. Relative masses are expressed as organ mass (mg)/body mass (g). The relative mass of the liver of the F0 XO females ranged from about 35 mg/g to about 50 mg/g with an average of about 42 mg/g. The relative mass of the liver of the F1 XX females ranged from about 37.5 mg/g to about 46.9 mg/g with an average of about 42.5 mg/g. The relative mass of the kidneys of the F0 XO females ranged from about 11.5 mg/g to about 15 mg/g with an average of about 13.4 mg/g. The relative mass of the kidneys of the F1 XX females ranged from about 12.6 mg/g to about 13.8 mg/g with an average of about 13.7 mg/g. The relative combined mass of the heart and lungs of the F0 XO females ranged from about 14.3 mg/g to about 18.9 mg/g with an average of about 16.1 mg/g. The relative combined mass of the heart and lungs of the F1 XX females ranged from about 14.7 mg/g to about 16.1 mg/g with an average of about 15.9 mg/g. The relative mass of the spleen of the F0 XO females ranged from about 2.7 mg/g to about 6.6 mg/g with an average of about 3.3 mg/g. The relative mass of the spleen of the F1 XX females ranged from about 2.7 mg/g to about 4.0 mg/g with an average of about 3.8 mg/g.

[0110] The F0 XY female mice were shown to have relatively normal serum levels of electrolytes, enzymes, glucose, proteins, lipids and other indicia compared to syngeneic F1 XX females. The XY female mice however did exhibit a larger range of values for each mearsured serum parameter. The serum sodium levels of the adult XY females ranged from about 150 mEq/L to about 159 mEq/L; and the levels for the XX females ranged from about 148 mEq/L to about 155 mEq/L.

[0111] The serum potassium levels of the adult XY females ranged from about 0.7 mEq/L to about 7 mEq/L; and the levels for the XX females were about 0.7 mEq/L.

[0112] The serum chloride levels of the adult XY females ranged from about 111 mEq/L to about 121 mEq/L; and the levels for the XX females ranged from about 113 mEq/L to about 120 mEq/L.

[0113] The serum calcium levels of the adult XY females ranged from about 7 mEq/L to about 9 mEq/L; and the levels for the XX females were about 7 mEq/L.

[0114] The serum alkaline phosphatase levels of the adult XY females ranged from about 124 U/L to about 285 U/L; and the levels for the XX females ranged from about 191 U/L to about 236 U/L.

[0115] The serum alanine aminotransferase levels of the adult XY females ranged from about 21 U/L to about 285 U/L; and the levels for the XX females ranged from about 13 U/L to about 34 U/L.

[0116] The serum aspartate aminotransferase levels of the adult XY females ranged from about 42 U/L to about 190 U/L; and the levels for the XX females ranged from about 42 U/L to about 269 U/L.

[0117] The serum lipase levels of the adult XY females ranged from about 16 U/L to about 49 U/L; and the levels for the XX females ranged from about 21 U/L to about 26 U/L.

[0118] The serum glucose levels of the adult XY females ranged from about 227 mg/dL to about 319 mg/dL; and the levels for the XX females ranged from about 255 mg/dL to about 270 mg/dL.

[0119] The total serum protein levels of the adult XY females ranged from about 4.6 mg/dL to about 5.2 mg/dL; and the levels for the XX females ranged from about 4.6 mg/dL to about 4.8 mg/dL.

[0120] The serum albumin levels of the adult XY females ranged from about 3 mg/dL to about 3.5 mg/dL; and the levels for the XX females ranged from about 3.1 mg/dL to about 3.2 mg/dL.

[0121] The serum cholesterol (total) levels of the adult XY females ranged from about 58 mg/dL to about 108 mg/dL; and the levels for the XX females ranged from about 61 mg/dL to about 85 mg/dL.

[0122] The serum triglyceride levels of the adult XY females ranged from about 42 mg/dL to about 89 mg/dL; and the levels for the XX females ranged from about 39 mg/dL to about 48 mg/dL.

[0123] The serum HDL levels of the adult XY females ranged from about 29 mg/dL to about 57 mg/dL; and the levels for the XX females ranged from about 23 mg/dL to about 42 mg/dL.

[0124] The serum LDL levels of the adult XY females ranged from about 3.7 mg/dL to about 11 mg/dL; and the levels for the XX females ranged from about 3.7 mg/dL to about 13 mg/dL.

[0125] The blood urea nitrogen (BUN) levels of the adult XY females ranged from about 12 mg/dL to about 27 mg/dL; and the levels for the XX females ranged from about 18 mg/dL to about 21 mg/dL.

[0126] The serum magnesium levels of the adult XY females ranged from about 1.6 mg/dL to about 3.2 mg/dL; and the levels for the XX females were about 2.1 mg/dL.

[0127] The serum inorganic phosphate levels of the adult XY females ranged from about 5.1 mg/dL to about 10 mg/dL; and the levels for the XX females ranged from about 7.2 mg/dL to about 8.4 mg/dL.

[0128] The serum uric acid levels of the adult XY females ranged from about 0.9 mg/dL to about 3.5 mg/dL; and the levels for the XX females ranged from about 0.7 mg/dL to about 2.2 mg/dL.

Example 10

Production of Homozygous Genetically Modified Mouse in the F1 Generation

[0129] To determine whether F1 mice homozygous for a genetic modification could be made, F0 XY female mice containing at least one knocked-out allele of a STEAP2 gene was mated to XY male cohort containing the same STEAP2 gene knock-out. (The STEAP2 (Six transmembrane epithelial antigen of the prostate 2) gene encodes for a putative 6 membrane metalloreductase with ferrireductase and cupric reductase activity, and has been shown to stimulate the cellular uptake of both iron and copper in vitro. As a cell-surface antigen, STEAP2 is a potential diagnostic or therapeutic target in prostate cancer. STEAP2 was significantly elevated in both untreated primary and hormone-refractory prostate carcinomas than in benign prostate hyperplasias, suggesting that it may be involved in the development of prostate cancer. STEAP2 KO mouse has not been reported. See Ohgami et al., BLOOD, vol. 108(4):1388-1394, 2006.) The results are depicted in Table 4.

TABLE-US-00003 TABLE 4 Genotypes of STEAP2 F1 Cohorts from F0 XY Males F0 XY Females Sex Chromosomes STEAP2 Genotype (N/%) Sex Phenotype (N/%) Wt/wt Wt/KO KO/KO Female XX (7/15%) 2/4.3% 3/6.4% 2/4.3% XO (4/8.5%) 1/2.1% 1/2.1% 2/4.3% XY (0/0%) 0/0% 0/0% 0/0% Male XY (17/36%) 6/12.8% 6/12.8% 5/10.6% XXY (8/17%) 3/6.4% 3/6.4% 2/4.3% XYY (11/23.5%) 3/6.4% 3/6.4% 5/10.6%