A kind of sheep parthenogenetic haploid stem cell culture medium and its application

Abstract

The present invention relates to the field of biotechnology, and in particular to a sheep parthenogenetic haploid stem cell culture medium and its application. Sheep mature oocytes are parthenogenetically activated to obtain sheep parthenogenetic embryos, the sheep parthenogenetic embryos are cultured in vitro to blastocysts, and then the sheep parthenogenetic blastocysts are cultured in culture medium to obtain sheep parthenogenetic haploid stem cells. The culture medium includes mTeSR1 basal culture medium and FGF2, Activin A, CHIR99021 and XAV939, and each 500 mL mTeSR1 basal culture medium contains 5‑10 ng / mL FGF2, 0.5‑1 μM Activin A, 1‑2.5 μM CHIR99021, and 0.5‑1 μM XAV939. The present invention provides a culture medium suitable for sheep parthenogenetic haploid stem cell culture, obtains sheep parthenogenetic haploid stem cells, and establishes a sheep parthenogenetic haploid stem cell cell line. The entire genome of sheep parthenogenetic haploid stem cells is derived from oocytes, and they have the self-replication ability and pluripotency of stem cells. The resulting cell line is a haploid cell line and can be used for genetic screening at the cellular level.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a sheep parthenogenetic haploid stem cell culture fluid and application thereof. Background Art

[0002] Mammals are all diploid. During sexual reproduction, sperm and egg combine, and half of the genetic material of the offspring comes from the father and half from the mother. The acquisition of haploid embryos requires human intervention. Under chemical or electrical stimulation, the oocyte can initiate parthenogenetic activation and develop into a blastocyst. This embryo contains only one set of chromosomes. Parthenogenetic haploid embryonic stem cells (PG-haESCs) can be isolated from the parthenogenetic blastocyst. This stem cell has the advantages of pluripotency and penetrance, and can be used as an excellent vector for gene editing, which plays a huge role in livestock breeding. At present, PG-haESCs have been obtained in animals such as crab-eating macaques and mice for basic and applied biomedical research. However, in the sheep species, the establishment of PG-haESCs cell lines is still blank.

[0003] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a sheep PG-haESCs culture medium and its application, by providing a culture medium suitable for sheep PG-haESCs culture for culturing sheep PG-haESCs, establishing a sheep PG-haESCs cell line, and filling the gap in sheep PG-haESCs cell line.

[0005] The technical solution of the present invention is as follows:

[0006] The first aspect of the present invention provides a sheep parthenogenetic haploid stem cell culture medium, wherein the culture medium comprises mTeSR1 basal culture medium and exogenous additives, the exogenous additives comprise FGF2, Activin A, CHIR99021 and XAV939, and each 500 mL of mTeSR1 basal culture medium contains 5-10 ng / mL FGF2, 0.5-1 μM Activin A, 1-2.5 μM CHIR99021, and 0.5-1 μM XAV939.

[0007] The second aspect of the present invention provides a use of the sheep parthenogenetic haploid stem cell culture medium of the present invention in preparing sheep parthenogenetic haploid stem cells.

[0008] The third aspect of the present invention provides a method for preparing sheep parthenogenetic haploid stem cells, comprising:

[0009] Step 1, in vitro maturation of oocytes obtained from sheep ovaries;

[0010] Step 2, selecting mature oocytes that have expelled the first polar body for parthenogenetic activation to obtain sheep parthenogenetic embryos;

[0011] Step 3, culturing the sheep parthenogenetic embryo in vitro to obtain a sheep parthenogenetic blastocyst;

[0012] Step 4: Cultivate the sheep parthenogenetic blastocyst in the sheep parthenogenetic haploid stem cell culture medium of the present invention to obtain sheep parthenogenetic haploid stem cells.

[0013] Furthermore, the step 1 specifically includes: culturing the oocytes obtained from sheep ovaries in an in vitro maturation solution for 20-22 hours, removing the cumulus granulosa cells using hyaluronidase, and selecting the mature oocytes that have expelled the first polar body.

[0014] Furthermore, the step 2 specifically includes: placing the selected mature oocytes that have expelled the first polar body in 10-15 μmol / L ionomycin for 10 min, placing them in 2-5 mmol / L N6,N6-dimethylaminopurine (6-DMAP), and incubating at 38.5°C and 5% CO 2 The sheep parthenogenetic embryos were obtained by culturing for 4-6 hours under saturated humidity conditions.

[0015] Furthermore, the step 3 specifically includes: transferring the sheep parthenogenetic embryo to an in vitro SOF-BSA culture medium for culturing for 7.5 days, replacing the new SOF-BSA culture medium every 48 hours, and obtaining a sheep parthenogenetic blastocyst.

[0016] Furthermore, the step 4 specifically includes: culturing the sheep parthenogenetic blastocyst in the sheep parthenogenetic haploid stem cell culture medium of the present invention, and sorting out the sheep parthenogenetic haploid stem cells by flow cytometry every 5 generations of culture.

[0017] Furthermore, step 4 specifically includes: removing the zona pellucida from the sheep parthenogenetic blastocyst under the action of pronase, and then culturing the sheep parthenogenetic haploid stem cell culture medium described in the present invention; staining the cultured stem cells with Hoechst 33342; then sieving the cells to remove adhesion cells, and then sorting out the sheep parthenogenetic haploid stem cells using a flow cytometer.

[0018] A fourth aspect of the present invention provides a sheep parthenogenetic haploid stem cell, wherein the stem cell is prepared using the method for preparing sheep parthenogenetic haploid stem cells described in the present invention.

[0019] Beneficial effects: The present invention provides a culture medium suitable for culturing sheep PG-haESCs, and sheep parthenogenetic haploid stem cells are prepared using the provided sheep PG-haESCs culture medium, and a sheep parthenogenetic haploid stem cell cell line is established. The entire genome of the sheep parthenogenetic haploid stem cells described in the present invention is derived from oocytes, and has the self-replication ability and pluripotency of stem cells. The obtained cell line is a haploid cell line, which can be used for gene screening at the cellular level. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a morphological picture of the sheep parthenogenetic blastocyst obtained by in vitro culture in Example 1. The scale bar is 100 μm.

[0021] Figure 2 This is a morphological picture of the first generation (P1 generation) of sheep PG-haESCs established from sheep parthenogenetic blastocysts in Example 1. The scale bar is 100 μm.

[0022] Figure 3 This is a diagram showing the purification results of haploid stem cells separated by flow cytometry from sheep PG-haESCs in Example 1, after spontaneous diploidization of stem cells has been removed.

[0023] Figure 4 This is an immunofluorescence image of the sheep PG-haESCs expressing the pluripotency marker genes OCT4, SOX2, and OTX2 in Example 1. The scale bar is 50 μm.

[0024] Figure 5 This is a diagram showing the results of the karyotype experiment of sheep PG-haESCs in Example 1, with a scale bar of 25 μm. DETAILED DESCRIPTION

[0025] The present invention provides a sheep PG-haESCs culture medium and its application. To make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] Compared with existing parthenogenetic haploid stem cells, there is no suitable culture medium for sheep PG-haESCs, and the culture medium of parthenogenetic haploid stem cells of cynomolgus monkeys or mice cannot construct a stable cell line. Based on this, the present invention provides a culture medium suitable for the culture of sheep PG-haESCs, which plays a vital role in the culture and acquisition of sheep PG-haESCs.

[0027] Specifically, an embodiment of the present invention provides a sheep PG-haESCs culture medium, wherein the culture medium includes mTeSR1 basal culture medium and exogenous additives, the exogenous additives include FGF2, Activin A, CHIR99021 and XAV939, and each 500 mL of mTeSR1 basal culture medium contains 5-10 ng / mL FGF2, 0.5-1 μM Activin A, 1-2.5 μM CHIR99021, and 0.5-1 μM XAV939.

[0028] The embodiment of the present invention provides a culture medium suitable for sheep PG-haESCs culture, uses the provided sheep PG-haESCs culture medium to prepare sheep parthenogenetic haploid stem cells, and establishes a sheep parthenogenetic haploid stem cell cell line. The entire genome of the sheep parthenogenetic haploid stem cells described in the embodiment of the present invention is derived from oocytes, has the self-replication ability and pluripotency of stem cells, and the obtained cell line is a haploid cell line, which can be used for gene screening at the cellular level.

[0029] Among them, FGF2 plays an important role in regulating cell survival, division, differentiation and migration, and can regulate the self-renewal of various stem cells; Activin A plays an essential role in maintaining the self-renewal and pluripotency of stem cells; CHIR99021, as an inhibitor of glycogen synthase-3 (GSK3), can promote the differentiation of stem cells into specific cell types and promote the proliferation of stem cells; IWR1-1-endo inhibits the Wnt / β-catenin pathway by maintaining the stability of Axin, a destructor of the Wnt pathway, and can inhibit the differentiation of stem cells during stem cell culture. XAV939 is a specific blocker of the Wnt / β-catenin signaling pathway, which directly binds to the Wnt receptor and prevents the interaction between the Wnt protein and the receptor, thereby inhibiting the conduction of the Wnt signal.

[0030] An embodiment of the present invention provides a use of the above-mentioned sheep PG-haESCs culture medium in preparing sheep PG-haESCs.

[0031] The embodiment of the present invention provides a method for preparing sheep PG-haESCs, which comprises:

[0032] Step 1, maturing oocytes obtained from sheep ovaries in vitro;

[0033] Step 2, selecting mature oocytes that have expelled the first polar body for parthenogenetic activation to obtain sheep parthenogenetic embryos;

[0034] Step 3, culturing the sheep parthenogenetic embryo in vitro to obtain a sheep parthenogenetic blastocyst;

[0035] Step 4: Cultivate the sheep parthenogenetic blastocyst in the sheep parthenogenetic haploid stem cell culture medium described in the embodiment of the present invention to obtain sheep PG-haESCs.

[0036] The sheep PG-haESCs of the embodiment of the present invention are prepared by in vitro culture. First, oocytes obtained from sheep ovaries are matured in vitro, and then the mature oocytes are parthenogenetically activated to obtain sheep parthenogenetic embryos, and then the sheep parthenogenetic embryos are cultured in an in vitro culture medium to blastocysts to obtain sheep parthenogenetic blastocysts. Sheep parthenogenetic blastocysts are cultured in the culture medium described in the embodiment of the present invention to obtain sheep PG-haESCs.

[0037] The entire genome of the sheep PG-haESCs described in the embodiment of the present invention is derived from oocytes, and has the self-replication ability and pluripotency of stem cells. The obtained cell line is a haploid cell line and can be used for gene screening at the cellular level.

[0038] The sheep PG-haESCs obtained in the embodiment of the present invention can be applied to spindle transfer (ST), which can solve the problem of unclear genetic information caused by the sheep oocytes coming from slaughterhouses. Through the ST technology, the genetic material of the donor sheep oocytes is replaced with sheep PG-haESCs with clear genetic information. The reconstructed oocytes can be used for in vitro fertilization to obtain offspring with clear genetic information of the parents.

[0039] Furthermore, the step 1 specifically includes: culturing the oocytes obtained from sheep ovaries in in vitro maturation fluid for 20-22 hours, removing the cumulus granulosa cells using hyaluronidase (0.001 g / mL), and selecting mature oocytes that have expelled the first polar body (PB1) and are in the Metaphase II (M II) stage for parthenogenetic activation.

[0040] Each 10 mL of maturation solution includes 9 mL of a solution consisting of M199 culture medium and 10 mmol / L 4-hydroxyethylpiperazineethanesulfonic acid, and 1 mL of a solution consisting of fetal bovine serum, 0.01 μg / mL luteinizing hormone, and 0.01 μg / mL estradiol.

[0041] Furthermore, the step 2 specifically includes: activating the mature oocyte that has expelled the first polar body by ionomycin to obtain a sheep parthenogenetic embryo.

[0042] Mature oocytes were activated by ionomycin, and embryos that expelled the first polar body were selected to make it easier to obtain sheep PG-haESCs later.

[0043] Furthermore, the step 2 specifically comprises: placing the selected mature oocytes that have expelled the first polar body in 10-15 μmol / L I for 10 min, placing them in 2-5 mmol / L 6-DMAP, and incubating at 38.5°C and 5% CO 2 The sheep parthenogenetic embryos were obtained by culturing for 4-6 hours under saturated humidity conditions.

[0044] Furthermore, the step 3 specifically includes: transferring the sheep parthenogenetic embryo to an in vitro SOF-BSA culture medium for culturing for 7.5 days, replacing the new SOF-BSA culture medium every 48 hours, and obtaining a sheep parthenogenetic blastocyst.

[0045] The composition of the SOF-BSA culture medium is as follows: each 100 mL of SOF-BSA culture medium includes 0.6969 g of sodium chloride, 0.059 g of potassium chloride, 0.0176 g of dipotassium hydrogen phosphate, 0.0198 g of magnesium sulfate heptahydrate, 0.21 g of sodium bicarbonate, 0.008 g of sodium pyruvate, 0.0262 g of potassium chloride dihydrate, and 0.6 g of bovine serum albumin (BSA).

[0046] Furthermore, the step 4 specifically includes: culturing the sheep parthenogenetic blastocyst in the sheep PG-haESCs culture medium described in the embodiment of the present invention, and sorting out the sheep PG-haESCs by flow cytometry every 5 generations of culture.

[0047] Sheep PG-haESCs were obtained by preparing the first generation (P1) of sheep PG-haESCs in a culture medium suitable for sheep PG-haESCs culture, and then sorting the sheep PG-haESCs by flow cytometry every 5 generations to maintain the proportion of haploid stem cells.

[0048] Furthermore, step 4 specifically includes: after removing the zona pellucida from the sheep parthenogenetic blastocyst, culturing in the sheep parthenogenetic haploid stem cell culture medium described in the embodiment of the present invention; incubating the cultured stem cells with 10 ug / mL Hoechst33342 at 37°C for 30 min, shaking the cells every 10 min to ensure uniform staining; then sieving the cells to remove adherent cells, and then sorting out the sheep parthenogenetic haploid stem cells using a flow cytometer.

[0049] By sorting using flow cytometry, stem cells that spontaneously diploidized during cell culture were removed, the proportion of haploid stem cells was maintained, and the sheep PG-haESCs cell line was established.

[0050] The embodiment of the present invention provides sheep PG-haESCs, wherein the sheep PG-haESCs are prepared by the method for preparing sheep PG-haESCs described in the embodiment of the present invention.

[0051] The present invention will be further described below by means of specific examples.

[0052] Example 1

[0053] 1. In vitro culture of sheep parthenogenetic blastocysts

[0054] Oocytes were obtained from sheep ovaries by cutting and cultured in in vitro maturation medium for 20 h. The cumulus granulosa cells were removed by hyaluronidase (0.001 g / mL), and mature oocytes at the M II stage with the first polar body expelled were selected. The mature oocytes were treated with 12 μmol / L L for 10 min, placed in 5 mmol / L 6-DMAP, and incubated at 38.5°C and 5% CO 2 The sheep parthenogenetic embryos were cultured for 4 h under saturated humidity conditions. The sheep parthenogenetic embryos were then transferred to SOF-BSA culture medium for culture, and fresh SOF-BSA culture medium was replaced every 48 h. During this period, cleavage cells were picked out and cultured at 38.5°C and 5% CO 2 The cells were cultured under saturated humidity for 7.5 days until blastocysts were formed, and sheep parthenogenetic blastocysts were obtained (see Figure 1 as shown).

[0055] 2. Preparation of sheep PG-haESCs

[0056] A sheep PG-haESCs culture medium is provided, wherein the culture medium is prepared by the following method: 8 ng / mL FGF2, 0.8 μM Activin A, 2 μM CHIR99021, and 0.8 μM XAV939 are added to each 500 mL mTeSR1 basic culture medium.

[0057] The obtained sheep parthenogenetic blastocysts were removed from the zona pellucida under the action of pronase, transferred to feeder cells, and cultured in sheep PG-haESCs culture medium. The inner cell mass can grow on the feeder cells, thereby obtaining P1 sheep PG-haESCs (see Figure 2 As shown). The feeder layer cells refer to primary cells of mouse fetuses that are cultured, treated with mitomycin after they have grown all over the culture dish, the culture medium in the cell culture dish (high-glucose complete culture medium containing 10% fetal bovine serum) is discarded, and a culture medium containing 10 ug / mL mitomycin is added, and the cells are incubated in an incubator for 2 hours. After the incubation, the culture medium containing mitomycin is discarded, and the cells are washed 5 times with phosphate buffered saline (PBS), and the washed cells are digested, centrifuged, and frozen. The treatment process converts the primary cells of the mouse from fibroblasts into feeder layer cells that cannot proliferate, providing support and nourishment for embryonic stem cells.

[0058] 3. Flow cytometry sorting and purification of sheep PG-haESCs

[0059] Haploid stem cells will spontaneously diploidize during the culture process. In order to maintain the proportion of haploid stem cells, haploid stem cells are sorted by flow cytometry every 5 generations of culture to maintain the proportion of haploid stem cells. Sheep PG-haESCs were incubated with 10 μg / mL Hoechst33342 at 37°C for 30 min. The cells were shaken every 10 min to ensure uniform staining. After staining, the cells were sieved to remove adherent cells, and then placed in the loading chamber of the BD FACS Ariall SORP flow cytometer for sorting. When sheep PG-haESCs were sorted using the BD FACS Ariall SORP flow cytometer, all sorting steps were performed aseptically. An 80 μm nozzle was selected, and the sheath fluid pressure was 45 psi. When the coefficient of variation (CV value) was about 2%, sorting began, and the sorting temperature was maintained at 4°C. The haploid stem cell population was collected in a centrifuge tube with culture medium, and the results are shown as follows. Figure 3 As shown in the figure, with the increase of sorting times, the proportion of haploids increased from 7.36% to 63.4%.

[0060] 4. Detection of pluripotency marker genes in sheep PG-haESCs

[0061] In order to verify whether sheep PG-haESCs have the pluripotency of stem cells, the expression of pluripotent stem cell marker genes Octamer-binding transcription factor (OCT4) (Santa Cruz, antibody ratio of 1:200), sex-determining region transcription factor 2 (SRY-box transcription factor 2, SOX2) (SantaCruz, antibody ratio of 1:200) and formative pluripotency marker Orthodenticle Homeobox2 (OTX2) (R&D Systems, antibody ratio of 1:250) were selected. Sheep PG-haESCs were cultured for 4 days, the culture medium was discarded, PBS was washed once, and 4% paraformaldehyde was added to fix the cells for 30 minutes. Then the cells were permeabilized, blocked, incubated with primary antibodies, secondary antibodies and 4',6-diamidino-2-phenylindole (DAPI), and finally anti-fluorescence quencher was added to seal the slides. Sheep PG-haESCs were observed using a NIKON A1R laser confocal microscope. Figure 4 As shown, sheep PG-haESCs express three pluripotent stem cell marker genes and have pluripotency, where Merged indicates integrated images and 4',6-diamidino-2-phenylindole (DAPI) indicates staining of cell nuclei.

[0062] 5. Karyotype analysis of sheep PG-haESCs

[0063] As a diploid animal, sheep have a total of 54 chromosomes. Karyotype analysis of sheep PG-haESCs was performed to verify the number of chromosomes. Sheep PG-haESCs with good growth status after sorting were selected, and fresh culture medium containing 0.2 μg / mL colchicine was added and cultured at 37°C and 5% CO. 2 Culture for 4 hours under saturated humidity conditions to arrest the cells in metaphase. Then discard the culture medium, digest the cells into a single cell suspension, add 0.56% (w / v) KCl solution preheated at 37°C, mix well, and treat hypotonic for 30 minutes. After centrifugation, discard the KCl solution, slowly add 8 mL of methanol-glacial acetic acid fixative precooled at -20°C, and fix twice, each time for 10 minutes. Resuspend the cells with an appropriate amount of fixative, and drip the cell suspension about 40 cm above the -20°C precooled slide. After the liquid dries, stain the cells and observe the number of chromosomes. The results are as follows Figure 5 As shown, there are 27 chromosomes in total, proving that this stem cell is a haploid stem cell.

[0064] It can be seen from this that the sheep haploid stem cells prepared by the culture medium provided by the present invention have pluripotency and haploidy, and are sheep parthenogenetic haploid stem cells.

[0065] In summary, the present invention provides a kind of sheep PG-haESCs culture medium and its application, firstly, the oocytes obtained from sheep ovaries are matured in vitro, and then the mature oocytes are parthenogenetically activated by ionomycin to obtain sheep parthenogenetic embryos, and then the sheep parthenogenetic embryos are cultured in vitro culture medium to blastocysts to obtain sheep parthenogenetic blastocysts. Sheep parthenogenetic blastocysts are cultured in the culture medium described in the embodiment of the present invention to obtain sheep PG-haESCs. The culture medium includes mTeSR1 basal culture medium and exogenous additives, and the exogenous additives include FGF2, Activin A, CHIR99021 and XAV939, and each 500 mL basal culture medium contains 5-10 ng / mL FGF2, 0.5-1 μM Activin A, 1-2.5 μM CHIR99021, and 0.5-1 μM XAV939. In the process of sheep PG-haESCs culture, the proportion of sheep PG-haESCs can be maintained at about 63% by flow cytometry sorting.

[0066] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. Use of a sheep parthenogenetic haploid stem cell culture medium in preparing sheep parthenogenetic haploid stem cells, in, The culture medium consists of mTeSR1 basal culture medium and exogenous additives, wherein the exogenous additives are FGF2, Activin A, CHIR99021 and XAV939. 8 ng / mL FGF2, 0.8 μM Activin A, 2 μM CHIR99021 and 0.8 μM XAV939 are added to every 500 mL mTeSR1 basal culture medium.

2. A method for preparing sheep parthenogenetic haploid stem cells, characterized in that: include: Step 1, in vitro maturation of oocytes obtained from sheep ovaries; Step 2, selecting mature oocytes that have expelled the first polar body for parthenogenetic activation to obtain sheep parthenogenetic embryos; Step 3, culturing the sheep parthenogenetic embryo in vitro to obtain a sheep parthenogenetic blastocyst; Step 4, culturing the sheep parthenogenetic blastocyst in a sheep parthenogenetic haploid stem cell culture medium to obtain sheep parthenogenetic haploid stem cells; The culture medium consists of mTeSR1 basal culture medium and exogenous additives, wherein the exogenous additives are FGF2, Activin A, CHIR99021 and XAV939. 8 ng / mL FGF2, 0.8 μM Activin A, 2 μM CHIR99021 and 0.8 μM XAV939 are added to every 500 mL mTeSR1 basal culture medium.

3. The method for preparing sheep parthenogenetic haploid stem cells according to claim 2, characterized in that: The step 1 specifically comprises: culturing oocytes obtained from sheep ovaries in an in vitro maturation solution for 20-22 hours, removing cumulus granulosa cells using hyaluronidase, and selecting mature oocytes that have expelled the first polar body.

4. The method for preparing sheep parthenogenetic haploid stem cells according to claim 2, characterized in that: The step 2 specifically includes: placing the selected mature oocyte that has expelled the first polar body in 10-15 μmol / L ionomycin for 10 minutes, placing it in 2-5 mmol / L N6, N6-dimethylaminopurine, and culturing it at 38.5°C and 5% CO2 saturated humidity for 4-6 hours to obtain sheep parthenogenetic embryos.

5. The method for preparing sheep parthenogenetic haploid stem cells according to claim 2, characterized in that: The step 3 specifically includes: transferring the sheep parthenogenetic embryo to an in vitro SOF-BSA culture medium for culturing for 7.5 days, replacing the new SOF-BSA culture medium every 48 hours, and obtaining a sheep parthenogenetic blastocyst.

6. The method for preparing sheep parthenogenetic haploid stem cells according to claim 2, characterized in that: The step 4 specifically comprises: culturing the sheep parthenogenetic blastocyst in the sheep parthenogenetic haploid stem cell culture medium according to claim 1, and sorting out the sheep parthenogenetic haploid stem cells by flow cytometry after every 5 generations of culture.

7. The method for preparing sheep parthenogenetic haploid stem cells according to claim 6, characterized in that: The step 4 specifically comprises: removing the zona pellucida from the sheep parthenogenetic blastocyst under the action of pronase, and then culturing the sheep parthenogenetic haploid stem cell culture medium according to claim 1; staining the cultured stem cells with Hoechst 33342; then sieving the cells to remove the adherent cells, and then sorting out the sheep parthenogenetic haploid stem cells using a flow cytometer.

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