Method for obtaining primordial germ cells through androgenesis haploid stem cell induction

Abstract

The invention relates to the technical field of cell culture, in particular to a method for obtaining primordial germ cells through androgenesis haploid stem cell induction. The method provided by the invention is an efficient and stable PGC in-vitro culture system, the androgenetic haploid stem cells are pre-induced in a specific pretreatment culture solution and then transferred to a GK15 culture solution for induction culture, the primordial germ cells can be obtained through induction, the proportion of the primordial germ cells reaches 73% after 4 days, and the culture time is 3 days. Compared with the prior art (3%), the improvement is extremely remarkable, and a new material is provided for beef cattle breeding.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a method for obtaining primordial germ cells by inducing androgenic haploid stem cells. Background Art

[0002] Primordial germ cells (PGCs) are the precursor cells of germ cells during the embryonic period. They develop into highly specialized germ cells to pass genetic material and epigenetic information to offspring, thereby ensuring the continuation of the species. The development of PGCs is dynamic and complex, including a series of processes such as specialization, migration, proliferation, sex differentiation, and extensive epigenetic reprogramming. In mammals, the formation of PGCs usually occurs during the gastrula stage of embryonic development. After the mammalian embryo is implanted in the uterus, under the joint action of the BMP signaling pathway and the Wnt signaling pathway, the proximal ectoderm cells differentiate to produce PGCs. PGCs are a group of precursor cells of gametes, induced from pluripotent stem cells, and can provide another source of gametes for reproduction. They help to deeply understand the basic mechanisms of reproductive biology, including sex determination, cell differentiation, and the development of germ cells, and can provide a theoretical basis for the optimization of breeding strategies.

[0003] In beef cattle breeding, this technology has broad application prospects. By gene editing or selective breeding of PGC, individuals with specific traits can be quickly obtained, greatly shortening the breeding cycle, and can also be stably inherited to offspring to achieve genetic improvement of beef cattle. By gene editing of PGC, excellent genes can be introduced into offspring to ensure that these genes are retained and expressed in beef cattle breeding during the breeding process. In addition, through the study of PGC, new methods of controlling the sex of offspring can be found, so as to select beef bulls or dairy cows in cattle breeding and improve economic benefits.

[0004] However, there are still huge challenges in the acquisition, cultivation and transplantation of PGCs, especially in large livestock such as beef cattle. The process of isolating and purifying PGCs from embryos is complicated, and the survival rate is low. It is also difficult to establish an efficient and stable PGC in vitro culture system, which limits the in-depth research and application of PGCs. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a method for obtaining primordial germ cells by inducing androecological haploid stem cells. The androecological haploid stem cells obtained by the present invention can be efficiently separated and cultured into bovine PGCs, providing new materials for beef cattle breeding.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a method for obtaining primordial germ cells by inducing androgenic haploid stem cells, comprising the following steps: Pre-inducing the haploid stem cells in a pre-treatment culture medium to obtain pre-induced haploid stem cells; the pre-treatment culture medium is based on N2B27 culture medium and also includes the following components: KSR with a volume concentration of 5%, BMP4 with a concentration of 80-120 ng / mL, CHIR99021 with a concentration of 8-10 μM, IWR1 with a concentration of 2-5 μM, and Y-27632 with a concentration of 8-12 μM; The pre-induced androgenic haploid stem cells are cultured in GK15 culture medium to obtain primordial germ cells; the GK15 culture medium is based on GMEM culture medium and also includes the following components: KSR with a volume concentration of 15%, 1× non-essential amino acids, 1× GlutaMAX, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 50 U / mL penicillin / streptomycin, 200 ng / ml BMP4, 100 ng / mL SCF, 50 ng / ml epidermal growth factor, 10 ng / mlL hLIF, 6 μM CHIR, 2.5 μM IWR1 and 10 μM Y27632.

[0007] Preferably, the pre-induction time is 24 hours, the pre-induction temperature is 37°C, the pre-induction environmental humidity is saturated humidity, and the volume concentration of CO2 in the pre-induction environment is 5%; the culture time is 4 days, the culture temperature is 37°C, the culture environmental humidity is saturated humidity, and the volume concentration of CO2 in the culture environment is 5%.

[0008] Preferably, before the androgenic haploid stem cells are pre-induced in the pre-treatment culture medium, the pre-induced container is treated with recombinant human fibronectin.

[0009] Preferably, before culturing the pre-induced androgenic haploid stem cells in the GK15 culture medium, the method further comprises: digesting the pre-induced androgenic haploid stem cells.

[0010] Preferably, the reagent used in the digestion treatment includes Accutase.

[0011] Preferably, the androecological haploid stem cells are bovine androecological haploid stem cells.

[0012] Preferably, the primordial germ cells express the SOX17 gene, the OCT4 gene and the NANOG gene, but do not express the SOX2 gene.

[0013] Preferably, the primordial germ cells have reduced expression levels of lysine dimethylation at position 9 of histone H3 and 5-methylcytosine, and increased expression levels of lysine 3 methylation at position 27 of histone H3.

[0014] Beneficial effects: The method provided by the present invention is an efficient and stable PGC in vitro culture system. Primordial germ cells can be induced by pre-inducing haploid stem cells in a specific pre-treated culture medium and then transferring them to GK15 culture medium for induction culture. The induction ratio in 4 days reaches 73%, which is a very significant improvement compared with the prior art (3%). No haploid cells were found in the inspection process of PGC, providing new materials for beef cattle breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0016] Figure 1 Schematic diagram of the construction of PGC-specific dual reporter gene vector and electrophoresis results; Figure 2 Schematic diagram of the process of inducing haploid stem cells into primordial germ cells and the fluorescence results; Figure 3 The results of flow cytometry analysis of haploid stem cells cultured for different days; Figure 4 These are the results of laser confocal microscopy observation of bovine haploid stem cells. DETAILED DESCRIPTION

[0017] The present invention provides a method for obtaining primordial germ cells by inducing androgenic haploid stem cells, comprising the following steps: Pre-inducing the haploid stem cells in a pre-treatment culture medium to obtain pre-induced haploid stem cells; the pre-treatment culture medium is based on N2B27 culture medium and also includes the following components: KSR with a volume concentration of 5%, BMP4 with a concentration of 80-120 ng / mL, CHIR99021 with a concentration of 8-10 μM, IWR1 with a concentration of 2-5 μM, and Y-27632 with a concentration of 8-12 μM; The pre-induced androgenic haploid stem cells are cultured in GK15 culture medium to obtain primordial germ cells; the GK15 culture medium is based on GMEM culture medium and also includes the following components: KSR with a volume concentration of 15%, 1× non-essential amino acids, 1× GlutaMAX, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 50 U / mL penicillin / streptomycin, 200 ng / mL BMP4, 100 ng / mL SCF, 50 ng / mL epidermal growth factor, 10 ng / mL hLIF, 6 μM CHIR, 2.5 μM IWR1 and 10 μM Y27632.

[0018] The present invention pre-induces the solitary haploid stem cells in a pre-treated culture medium to obtain pre-induced solitary haploid stem cells. As an embodiment, the solitary haploid stem cells are bovine solitary haploid stem cells. As an embodiment, the present invention adopts the method described in steps 2 and 3 of Example 1 in Chinese patent CN119120578A to culture the bovine solitary haploid stem cells. The method provided by the present invention can efficiently separate and obtain bovine solitary haploid stem cells.

[0019] As an embodiment, before the androgenic haploid stem cells are pre-induced in the pre-treatment culture medium, the pre-induced container is treated with recombinant human fibronectin. As an embodiment, the pre-induced container can be a culture dish.

[0020] As an embodiment, the pretreatment culture medium of the present invention uses N2B27 culture medium as the base culture medium, and also includes the following components: 5% KSR by volume, 100 ng / mL BMP4, 6 μM CHIR99021, 2.5 μM IWR1 and 10 μM Y-27632.

[0021] As an embodiment, the pre-induction time is 24 hours, the pre-induction temperature is 37° C., the pre-induction environmental humidity is saturated humidity, and the volume concentration of CO 2 in the pre-induction environment is 5%.

[0022] After obtaining the pre-induced androgenic haploid stem cells, the present invention cultured the pre-induced androgenic haploid stem cells in a GK15 culture medium to obtain primordial germ cells. As an embodiment, before culturing the pre-induced androgenic haploid stem cells in the GK15 culture medium, the method further includes: digesting the pre-induced androgenic haploid stem cells. As an embodiment, the reagent used in the digestion treatment includes Accutase.

[0023] The GK15 culture medium of the present invention is based on GMEM culture medium, and also includes the following components: KSR with a volume concentration of 15%, 1× non-essential amino acids, 1×GlutaMAX, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 50 U / mL penicillin / streptomycin, 200 ng / mL BMP4, 100 ng / mL SCF, 50 ng / mL epidermal growth factor, 10 ng / mL hLIF, 6 μM CHIR, 2.5 μM IWR1 and 10 μM Y27632.

[0024] As an embodiment, the culture time is 4 days, the culture temperature is 37° C., the culture environment humidity is saturated humidity, and the volume concentration of CO 2 in the culture environment is 5%.

[0025] As an embodiment, the primordial germ cells express the SOX17 gene, the OCT4 gene and the NANOG gene, but do not express the SOX2 gene.

[0026] As an embodiment, the expression levels of dimethylation of lysine 9 and 5-methylcytosine of histone H3 in the primordial germ cells are reduced, and the expression level of methylation of lysine 3 at position 27 of histone H3 is increased.

[0027] The method provided by the present invention is an efficient and stable PGC in vitro culture system. Primordial germ cells can be induced by pre-inducing haploid stem cells in a specific pre-treated culture medium and then transferring them to GK15 culture medium for induction culture. The induction ratio in 4 days reaches 73%, which is a very significant improvement compared with the prior art (3%). In addition, no haploid cells were found in the inspection process of PGC, providing new materials for beef cattle breeding.

[0028] To further illustrate the present invention, a method for obtaining primordial germ cells by inducing androgenic haploid stem cells provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1 1. The method described in steps 2 and 3 of Example 1 in Chinese patent CN119120578A is used to culture bovine haploid stem cells.

[0030] 2. In order to determine the ability of bovine haploid stem cells to differentiate into primordial germ cells in vitro, the primordial germ cell-specific genes PRDM1 (also known as BLIMP1) and TFAP2C were selected, and the reporter fluorescent gene was connected to the target gene to construct a PRDM1-tdTomato (PT) and TFAP2C-mNeonGreen (TG) dual reporter gene recombinant vector in vitro ( Figure 1 A and C); the sgRNA in the CRISPR-Cas9 gene editing system is designed for the stop codon region of PRDM1 and TFAP2C, and the sgRNA is used to act with Cas9 to bind to the target gene and specifically cut the stop codon region of the target gene, causing the double-strand break of the target DNA in the region near the stop codon, causing the cell to automatically start homologous recombination repair. The present invention transfects the synthesized dual-reporter gene linear fragment into the target cell, which can be used as a template for DNA repair, thereby guiding the target gene to synthesize DNA according to the sequence of the dual-reporter vector when accompanying DNA repair, and finally realizes that PRDM1 and TFAP2C carry fluorescent reporter genes in the bovine haploid stem cells respectively, and accompany the differentiation process of the bovine haploid stem cells to primordial germ cells. PRDM1 and TFAP2C are expressed with fluorescence at the same time, and the differentiation ability of the bovine haploid stem cells to primordial germ cells is verified by fluorescence. The specific steps are as follows: 1) The target fragments tdTomato and mNeonGreen were synthesized by Shanghai Bioengineering, and the specific nucleotide sequences are as follows: tdTomato(SEQ ID NO.1): mNeonGreen (SEQ ID NO.2): 5'--3'.

[0031] 2) Use PCR to splice tdTomato and mNeonGreen with the homology arm sequences selected from PRDM1 and TFAP2C to form the target linear fragment. Multiple rounds of PCR reactions are required during the construction process, as follows: The 1120 bp long base sequence upstream of the PRDM1 stop codon was selected as the left homology arm of the gene PRDM1, the 1440 bp long base sequence upstream of the TFAP2C stop codon was selected as the left homology arm of the gene TFAP2C, the 987 bp long base sequence downstream of the PRDM1 stop codon was selected as the right homology arm of the gene PRDM1, and the 1018 bp long base sequence downstream of the TFAP2C stop codon was selected as the right homology arm of the gene TFAP2C, and finally the fluorescent gene tdTomato was inserted between the left and right homology arms of the target gene PRDM1 to replace the stop codon of PRDM1, and the fluorescent gene mNeonGreen was inserted between the left and right homology arms of the target gene TFAP2C to replace the stop codon of TFAP2C, and the fluorescent gene tdTomato and mNeonGreen were co-expressed along with the expression of the PRDM1 and TFAP2C genes. The primers required for the PCR reaction of the homology arms are shown in Table 1.

[0032] Table 1 Primers required for PCR reaction of homology arms PCR reaction system for homology arms: 2× Phanta Flash Master Mix 25 μL, upstream primer F (10 μM) 2 μL, downstream primer R (10 μM) 2 μL, template DNA 1 μL, and ddH2O 20 μL; According to the above system, the left and right homologous arms of PRDM1 and TFAP2C were amplified to obtain the left and right homologous arm DNA of PRDM1 and TFAP2C. The upstream primer of the left homologous arm of PRDM1 is F1, and the downstream primer is R1, and the other homologous arms are similar. The template DNA comes from bovine haploid stem cells, and the bovine haploid stem cell DNA is extracted using FastPure Cell / Tissue DNA Isolation Mini Kit (Vazyme DC102).

[0033] The PCR reaction program of the homology arms was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 58°C for 5 s, extension at 72°C for 10 s, 35 cycles; and final extension at 72°C for 1 min.

[0034] PCR was used to splice the PRDM1 left homology arm DNA with the tdTomato sequence to obtain PRDM1 left homology arm + tdTomato DNA; PCR was then used to splice the left homology arm + tdTomato DNA with the PRDM1 right homology arm into a complete sequence (PRDM1 left homology arm + tdTomato + PRDM1 right homology arm). TFAP2C was spliced ​​with the mNeonGreen sequence in the same way to obtain TFAP2C left homology arm + mNeonGreen DNA; PCR was then used to splice the TFAP2C left homology arm + mNeonGreen DNA with the TFAP2C right homology arm into a complete sequence (TFAP2C left homology arm + mNeonGreen + TFAP2C right homology arm).

[0035] The PCR reaction system for connecting the left homology arm of PRDM1 to tdTomato was as follows: 2×Phanta Flash Master Mix 25 μL, upstream primer F1 (10 μM) 2 μL, downstream primer R5 (10 μM) 2 μL, PRDM1 left homology arm DNA 1 μL, tdTomato DNA 1 μL, and ddH2O 19 μL; The PCR reaction system for connecting the left homology arm of TFAP2C to mNeonGreen was as follows: 2×Phanta Flash MasterMix 25 μL, upstream primer F3 (10 μM) 2 μL, downstream primer R6 (10 μM) 2 μL, TFAP2C left homology arm DNA 1 μL, mNeonGreen DNA 1 μL, and ddH2O 19 μL; The PCR reaction program for the left homology arm connected to tdTomato or mNeonGreen was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 58°C for 5 s, extension at 72°C for 10 s, 35 cycles; final extension at 72°C for 1 min.

[0036] The PCR reaction system for PRDM1 left homology arm + tdTomato connected to right homology arm was: 2×Phanta FlashMaster Mix 25 μL, upstream primer F1 (10 μM) 2 μL, downstream primer R2 (10 μM) 2 μL, PRDM1 left homology arm + tdTomato DNA 1 μL, PRDM1 right homology arm DNA 1 μL and ddH2O 19 μL; The PCR reaction system of TFAP2C left homology arm + mNeonGreen connected to right homology arm was: 2×Phanta FlashMaster Mix 25 μL, upstream primer F3 (10 μM) 2 μL, downstream primer R4 (10 μM) 2 μL, TFAP2C left homology arm + mNeonGreen DNA 1 μL, TFAP2C right homology arm DNA 1 μL and ddH2O 19 μL; The PCR reaction program of the left homology arm + tdTomato or mNeonGreen connected to the right homology arm was: pre-denaturation at 95°C for 30s; denaturation at 95°C for 10s, annealing at 58°C for 5s, extension at 72°C for 10s, 35 cycles; final extension at 72°C for 1min.

[0037] 3) Construct the plasmid required for CRISPR-Cas9, the steps are as follows: select the BbsⅠ restriction site on the pSpCas9(BB)-2A-Puro(PX459) V2.0 (addgene, 62988) plasmid, use BbsⅠ to digest the plasmid linear fragment, and connect it with the synthesized sgRNA targeting the PRDM1 and TFAP2C stop codons to form a recombinant plasmid. The sgRNA sequence is as follows: PRDM1-sgRNA-F: 5'-caccctcgaggacttacgacttggcgagtca-3' (SEQ ID NO. 13); PRDM1-sgRNA-R: 5'-aaactgactcgccaagtcgtaagtcctcgag-3' (SEQ ID NO. 14); TFAP2C-sgRNA-F: 5'-caccctcgaggactcccagatcatctcgacc-3' (SEQ ID NO. 15); TFAP2C-sgRNA-R: 5'-aaacggtcgagatgatctgggagtcctcgag-3' (SEQ ID NO. 16); The ligation reaction system and conditions were as follows: Solution 1 (Solution Ⅰ) (Takara, 6022Q) 5 μL, plasmid linear fragment 1 μL, ddH2O 1 μL and sgRNA 3 μL; 16°C, overnight ligation.

[0038] 4) Through multiple rounds of PCR in step 2), the target fragment containing the homologous arms of the target gene can be obtained, and CRISPR-Cas9 technology can be used to perform homologous recombination to perform site-specific knock-in of the exogenous gene. The steps are as follows: The recombinant plasmid constructed in step 3) and the two double reporter gene recombinant linear vectors PRDM1 left homology arm + tdTomato + PRDM1 right homology arm (3592 bp), TFAP2C left homology arm + mNeonGreen + TFAP2C right homology arm (3223 bp) obtained in step 2) were co-transfected into the bovine haploid stem cells in step 1 through liposome transfection. After transfection, cell DNA was extracted and PCR reaction was used to determine whether the exogenous gene was integrated.

[0039] It was identified that tdTomato and mNeonGreen have been knocked into the PRDM1 and TFAP2C genes ( Figure 1 (B and D). When haploid stem cells are induced into PGC cells, PRDM1 and TFAP2C are specifically expressed, and the knocked-in fluorescent gene is also expressed, proving that haploid stem cells can be induced into PGCs.

[0040] Example 2 Induction of PGCs from haploid stem cells The bovine haploid stem cells knocked into the fluorescent gene in Example 1 were pre-induced, and the 12-well plates were treated with recombinant human fibronectin (FC010, Millipore). 5 Bovine haploid stem cells were placed in a pre-treated culture medium for 24 hours at 37°C, saturated humidity and 5% CO2. The pre-treated culture medium was prepared by adding BMP4 at a final concentration of 100 ng / mL, 6 μM CHIR99021, 2.5 μM IWR1 and 10 μM Y-27632 to N2B27 culture medium containing 5% (v / v) KSR (10828028, Thermo Fisher Scientific).

[0041] The pre-treated bovine haploid stem cells were digested with Accutase (A1110501, Thermo Fisher Scientific), and the pre-treated bovine haploid stem cells were placed in GK15 culture medium and cultured at 37°C, saturated humidity and 5% CO2 for 4 days to induce the haploid stem cells into PGC-like cells ( Figure 2In Figure A), the GK15 culture medium is based on GMEM culture medium, to which the following reagents are added at final concentrations: 15% (v / v) KSR, 1× non-essential amino acids, 1× GlutaMAX, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 50 U / ml penicillin / streptomycin, 200 ng / mL BMP4, 100 ng / mL SCF, 50 ng / mL epidermal growth factor, 10 ng / mL hLIF, 6 μM CHIR, 2.5 μM IWR1, and 10 μM Y27632.

[0042] The cells were observed under a fluorescence microscope on the 2nd, 3rd and 4th day of culture. Figure 2 Middle B. The results showed that red and green fluorescence could be observed after the second day of culture. Because the red and green fluorescence were connected to the primordial germ cell-specific expression genes PRDM1 and TFAP2C, respectively, the generation of fluorescence meant that the primordial germ cell-specific expression genes could begin to be expressed, proving that the induced culture system could quickly induce haploid stem cells into primordial germ cells.

[0043] Flow cytometry analysis was performed on the pre-treated bovine haploid stem cells cultured for different days. Figure 3 , where Hoechst represents DNA content, dG1 is the diploid G1 phase of the cell cycle, dS is the diploid S phase of the cell cycle, and dG2 is the diploid G2 phase of the cell cycle. The results showed that the number of PT+::TG+ positive cells continued to increase during PGC induction. On the 2nd, 3rd and 4th days of induction, 31%, 65% and 73% of the cells were PT+::TG+ positive, respectively. Compared with the previous literature (Shirasawa A, Hayashi M, Shono M, Ideta A, Yoshino T, Hayashi K. Efficientderivation of embryonic stem cells and primordial germ cell-like cells incattle. J Reprod Dev. 2024 Apr 4;70(2):82-95. doi: 10.1262 / jrd.2023-087. Epub2024 Feb 15. PMID: 38355134; PMCID: PMC11017101.), the proportion increased from 3% to 73%, which was a very significant improvement. After the start of differentiation induction, haploid cells automatically restored diploid characteristics, and no haploid cells were found during the PGC test. Therefore, the efficiency of inducing PGC by haploid stem cells is higher.

[0044] Example 3 Expression of marker genes Sox17 is a key transcriptional regulator for the development of definitive endoderm, Oct4, Sox2, and Nanog are specific genes for maintaining stem cell pluripotency, and PRDM1 and TFAP2C are primordial germ cell-specific genes.

[0045] The culture medium of the PGC cells induced and differentiated in Example 2 was discarded, paraformaldehyde was added to fix for 30 min, and then the cells were permeabilized and blocked with 3% (v / v) bovine serum albumin. Then, the antibodies of PGC endogenous PRDM1 and TFAP2C genes, PGC-specific expression genes SOX17, OCT4 and NANOG, and stem cell-specific expression gene SOX2 were incubated overnight at 4°C, washed 3 times with phosphate buffered saline (PBS), incubated with secondary antibodies at 37°C, and then stained with 4',6-diamidino-2-phenylindole (DAPI) for cell nuclei, and finally added with anti-fluorescence quencher for slicing. The sources of the primary antibodies used and the dilution ratios are as follows: SRY-box transcription factor 17, SOX17 (Santa Cruz), dilution ratio 1:200; Octamer-binding transcription factor, OCT4 (Santa Cruz), dilution ratio 1:200; Nanog Homeobox, NANOG (Santa Cruz), dilution ratio 1:100; SRY-box transcription factor 2, SOX2 (Santa Cruz), dilution ratio 1:250; The secondary antibody sources and dilution ratios used are as follows: AlexaFluor 594 goat anti-rabbit IgG, Thermo, dilution ratio 1:1000; AlexaFluor 488 goat anti-rabbit IgG, Thermo, dilution ratio 1:1000; AlexaFluor 594 goat anti-mouse IgG, Thermo, dilution ratio 1:1000.

[0046] The results were observed using a NIKON A1R laser confocal microscope. Figure 4Middle A. The results showed that on the fourth day of culture, the induced primordial germ cells could express PRDM1 and TFAP2C, but also the stemness-related genes Oct4 and Nanog, and the endoderm-specific gene Sox17. This is highly consistent with existing research results. Human primordial germ cells in the early stages of development express a series of genes related to stem cell pluripotency, such as OCT4 and NANOG, but do not express the key transcription factor gene SOX2. Instead, they express two other SOX family genes (SOX15 and SOX17).

[0047] At the same time, the expression of histone H3 lysine 9 dimethylation (H3K9me2, Abcam, dilution ratio 1:50), histone H3 lysine 27 methylation (H3K27me3, Abcam, dilution ratio 1:50) and 5-methylcytosine (5mC, Abcam, dilution ratio 1:50) was detected by immunofluorescence. The results are shown in Figure 4 Middle B. The results showed that compared with negative cells, the levels of DNA methylation (5mC) and H3K9me2 in induced primordial germ cells were reduced, while the levels of H3K27me3 were increased. Studies have confirmed that in primordial germ cells, H3K9me2 and 5mC undergo extensive demethylation to clear somatic epigenetic memory and establish germ cell-specific regulation; while H3K27me3 maintains the silencing state of key genes through dynamic regulation and maintains a high methylation state in the early stage of PGC. Figure 4 The results of B are also consistent with this conclusion, confirming that the primordial germ cells induced by the haploid stem cells meet the characteristics of primordial germ cells, proving that GK15 culture medium can accurately induce haploid stem cells to produce primordial germ cells In summary, in addition to the intra-species / inter-species chimera ability, haploid stem cells are also suitable for direct induction of PGCs in vitro.

[0048] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for obtaining primordial germ cells by inducing haploid stem cells, characterized in that: The following steps are involved: Pre-inducing the haploid stem cells in a pre-treatment culture medium to obtain pre-induced haploid stem cells; the pre-treatment culture medium is based on N2B27 culture medium and also includes the following components: KSR with a volume concentration of 5%, BMP4 with a concentration of 80-120 ng / mL, CHIR99021 with a concentration of 8-10 μM, IWR1 with a concentration of 2-5 μM, and Y-27632 with a concentration of 8-12 μM; The pre-induced androgenic haploid stem cells are cultured in GK15 culture medium to obtain primordial germ cells; the GK15 culture medium is based on GMEM culture medium and also includes the following components: KSR with a volume concentration of 15%, 1× non-essential amino acids, 1× GlutaMAX, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 50 U / ml penicillin / streptomycin, 200 ng / ml BMP4, 100 ng / ml SCF, 50 ng / mL epidermal growth factor, 10 ng / mL hLIF, 6 μM CHIR, 2.5 μM IWR1 and 10 μM Y27632.

2. The method according to claim 1, characterized in that The pre-induction time is 24 hours, the pre-induction temperature is 37°C, the pre-induction environmental humidity is saturated humidity, and the volume concentration of CO2 in the pre-induction environment is 5%; the culture time is 4 days, the culture temperature is 37°C, the culture environmental humidity is saturated humidity, and the volume concentration of CO2 in the culture environment is 5%.

3. The method according to claim 1 or 2, characterized in that: Before the androecium haploid stem cells are pre-induced in the pre-treatment culture medium, the pre-induced container is treated with recombinant human fibronectin.

4. The method according to claim 1 or 2, characterized in that: Before the pre-induced androgenic haploid stem cells are cultured in the GK15 culture medium, the method further comprises: digesting the pre-induced androgenic haploid stem cells.

5. The method according to claim 4, characterized in that The reagent used in the digestion treatment includes Accutase.

6. The method according to claim 1, characterized in that The androecological haploid stem cells are bovine androecological haploid stem cells.

7. The method according to claim 1, characterized in that The primordial germ cells express the SOX17 gene, the OCT4 gene and the NANOG gene, but do not express the SOX2 gene.

8. The method according to claim 1, characterized in that The primordial germ cells have decreased expression levels of lysine dimethylation at position 9 of histone H3 and 5-methylcytosine, and increased expression levels of lysine 3 methylation at position 27 of histone H3.

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