Methods and applications for directed differentiation of porcine pluripotent stem cells to sperm-like cells

Abstract

The application discloses a method for directionally inducing differentiation of porcine pluripotent stem cells into sperm-like cells and application thereof, and belongs to the technical field of biotechnology.The method comprises the following steps: inducing ex vivo porcine gastrulated epiblast stem cells into mesendodermal precursor cells, wherein the mesendodermal precursor cells express OCT4 protein, NANOG protein, SOX17 protein, PRDM1 protein, EOMES protein and TBXT protein.The primitive germ cell-like cells prepared by the method provided in the application can be further developed into sperm-like cells.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method and application for the directed induction of porcine pluripotent stem cells into sperm-like cells. Background Technology

[0002] Mammalian male germ cell (GC) development is a complex and ordered multi-step differentiation process, mainly comprising three distinct stages: primordial germ cell (PGC) specialization, sex determination and spermatogonial development, and subsequent spermatogenesis. Meiotic recombination events during haploid formation create genomic diversity, accelerating species evolution. Over the past two decades, pluripotent stem cells (PSCs) in mice and rats have been induced into primordial germ cell-like cells (PGCLCs), which subsequently differentiate into functional gametes. However, rodents have shorter lifespans, and their embryos are oval-cylindrical; their GC lineage specialization and developmental mechanisms differ significantly from those in humans and other species. Therefore, research findings in mice cannot be directly applied to clinical and livestock production and reproduction.

[0003] Recent research has shown that pluripotent stem cells from humans and non-human primates can be successfully induced into progenitor cell line cells (PGCLCs), which can then differentiate into anterior spermatogonia or early oogonia through epigenetic reprogramming in appropriate environments. However, due to ethical, moral, technical, and legal limitations, conducting in vivo functional studies on human PGCLCs is largely impractical. Compared to other laboratory animals, pigs are not only an important economic livestock but are also considered a reliable and representative biomedical model for studying human physiology, anatomy, and metabolism. Therefore, establishing a pig in vitro germ cell differentiation system not only has comparative biological significance but can also promote regenerative medicine, animal breeding, endangered species protection, and germplasm resource preservation. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to induce porcine gastrulated epiblast stem cells (pEpiSCs) to differentiate into porcine primordial germ cell-like cells (pPGCLCs) through mesoendothelial progenitor cells (pMEPCs), and then obtain sperm-like cells through reticulum testis transplantation. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: This invention provides a method for inducing porcine gastrulation epiblast stem cells into mesoendothelial precursor cells, comprising the following steps: inducing isolated porcine gastrulation epiblast stem cells into mesoendothelial precursor cells, wherein the mesoendothelial precursor cells express OCT4 protein, NANOG protein, SOX17 protein, PRDM1 protein, EOMES protein and TBXT protein.

[0006] SOX17 is SRY-box transcription factor 17, and its amino acid sequence is NCBI Reference Sequence:XP_001928411.1 (13-MAY-2017).

[0007] OCT4 (also called POU5F1) is a POU5 class homeobox 1, and its amino acid sequence is NCBI ReferenceSequence:NP_001106531.1 (MAM 02-APR-2025).

[0008] NANOG is a Nanog homeobox protein, and its amino acid sequence is NCBI Reference Sequence: NP_001123443.2 (03-NOV-2023).

[0009] PRDM1 is a zinc finger protein 1 with a PR domain. Its amino acid sequence is NCBI Reference Sequence: XP_005659397.1 (13-MAY-2017).

[0010] EOMES is the germin gene, and its amino acid sequence is NCBI Reference Sequence: XP_003132129.2 (13-MAY-2017).

[0011] TBXT is T-box transcription factor T, also known as brachyyury, and its amino acid sequence is NCBI Reference Sequence: XP_020930431.1 (13-MAY-2017).

[0012] GSC is the goosecoid homeobox gene, and its amino acid sequence is NCBI Reference Sequence: XP_003482361.2 (13-MAY-2017).

[0013] GATA4 is GATA-binding protein 4, and its amino acid sequence is NCBI Reference Sequence: NP_999458.1 (02-APR-2025).

[0014] The porcine gastrulation epiblast stem cells are derived from porcine embryos on day 10 of development. These porcine gastrulation epiblast stem cells express core pluripotent transcription factors, are positive for alkaline phosphatase staining, and have the ability to form embryoid bodies and teratomas, as well as to randomly differentiate into three germ layers. They exhibit multi-lineage differentiation characteristics and are in an intermediate pluripotent stage.

[0015] The mesoendothelial progenitor cells express SOX17, PRDM1, EOMES, and TBXT proteins, while the porcine gastrulation epiblast stem cells do not express SOX17, PRDM1, EOMES, and TBXT proteins.

[0016] The mesoendothelial precursor cells are cells with the potential to differentiate into primordial germ cells.

[0017] In the above method, the induction time for inducing isolated porcine gastrulation epiblast stem cells into mesoendothelial precursor cells is 30-42 hours.

[0018] SOX17 was significantly overexpressed and SOX2 (NCBI ReferenceSequence: NP_001116669.1, 02-APR-2025) was significantly underexpressed 30-42 h after differentiation of porcine gastrulation epiblast stem cells. OCT4 was also co-expressed.

[0019] In the above method, the induction is carried out by composition one, which includes an activator of the cell signaling pathway Activin / NODAL and an agonist of the cell signaling pathway Wnt / β-catenin.

[0020] The Activin / NODAL cell signaling pathway plays an important role in early embryonic development, cell fate determination, and germ layer differentiation.

[0021] The Wnt / β-catenin pathway is a series of biochemical reactions triggered by Wnt protein ligands, which ultimately lead to the accumulation of the transcriptional coactivator β-catenin in the cell nucleus and the initiation of transcription of specific genes.

[0022] Agonists are substances that mimic endogenous signaling molecules and trigger receptor-mediated signal transduction. Activators are substances that enhance the inherent biochemical activity of target proteins and / or enzymes.

[0023] The activators of the Activin / NODAL cell signaling pathway include, but are not limited to, activin A.

[0024] Agonists of the Wnt / β-catenin cell signaling pathway include, but are not limited to, CHIR99021.

[0025] In some specific embodiments of this invention, the content of activator A is 50-100 ng / mL, and the content of WNT agonist is 0.3-1 μM.

[0026] In one specific embodiment of the present invention, CHIR99021 is a product of Selleckchem, named GSK-3α / β inhibitor, with catalog number S1263.

[0027] In one specific embodiment of the present invention, the activator A is a PeproTech product with product number 120-14E.

[0028] In some specific embodiments of the present invention, the culture medium used in the induction process contains, in addition to composition one, a ROCK inhibitor (Y-27632) and aRB27 basal medium.

[0029] In some specific embodiments of the present invention, the ROCK inhibitor (Y-27632) is a product of Selleckchem with catalog number S1049.

[0030] In some specific embodiments of the present invention, the ROCK inhibitor (Y-27632) is present in the aforementioned culture medium at a concentration of 5-10 μM.

[0031] The aRB27 basal medium consisted of: 1×B27 supplement, 1% penicillin-streptomycin, 1% GlutaMax, and 0.1 mM β-mercaptoethanol, with the balance being Advanced RPMI 1640 medium (Thermo Fisher, 12633012).

[0032] The B27 supplement is a product of Thermo Fisher Scientific, catalog number 12587010, with a concentration of 50×.

[0033] The present invention also provides a method for inducing mesoendodermal precursor cells into primordial germ cell-like cells, comprising the following steps: preparing mesoendodermal precursor cells according to the aforementioned method; inducing the mesoendodermal precursor cells into primordial germ cell-like cells; wherein the primordial germ cell-like cells express marker proteins, the marker proteins including NANOS3 protein, SOX17 protein, OCT4 protein, NANOG protein, PRDM1 protein, and TFAP2C protein.

[0034] NANOS3 for nanos C2HC Type zinc finger 3, its amino acid sequence is: MAPLCWCPLHPVSWTMHSFGRCIFGGAAASPPVTIRNLPQPAPPSSHPLGGIRRELTAQTPGLQREKGRGRGKGIEGRSLGWLGFFSLSALSPGTLCPAMGTFNLWTDYLGLARLVGALRGEEEPETRLDPQ PAPVPGPEGQRSPESSPAPERLCSFCKHNGESRAIYQSHVLKDEAGRVLCPILRDYVCPQCGATRERAHTRRFCPLTSQGYTSVYSYTTRNSAGKKLARPDKARTQDSGHRRGGGGGGASTGSKGAGKSSGTSPSPCCPSTSA.

[0035] TFAP2C is the transcription factor AP-2γ, and its amino acid sequence is NCBI Reference Sequence: NP_001116673.1(24-SEP-2024).

[0036] Primordial germ cell-like cells are cells that resemble primordial germ cells in the body in terms of gene expression, molecular characteristics, epigenetic characteristics, and developmental potential.

[0037] In the above method, the induction time for inducing the mesoderm precursor cells into primordial germ cell-like cells is 12-24 hours.

[0038] In the above method, the induction is carried out by composition two, which includes bone morphogenetic protein 4 (BMP4), human leukemia inhibitory factor (hLIF), stem cell factor (SCF), and epidermal growth factor (EGF).

[0039] In some specific embodiments of the present invention, the content of bone morphogenetic protein 4 is 200-500 ng / mL, the content of human leukemia inhibitory factor is 10 ng / mL, the content of stem cell factor is 100 ng / mL, and the content of epidermal growth factor is 50 ng / mL.

[0040] In some specific embodiments of the present invention, the bone morphogenetic protein 4 (BMP4) is a product of PeproTech with catalog number 120-05.

[0041] In some specific embodiments of the present invention, the human leukemia inhibitory factor (hLIF) is a product of PeproTech with catalog number 300-05.

[0042] In some specific embodiments of the present invention, the stem cell factor (SCF) is a PeproTech product, catalog number 300-07.

[0043] In some specific embodiments of the present invention, the epidermal growth factor (EGF) is a PeproTech product with catalog number AF-100-15.

[0044] In some specific embodiments of the present invention, the culture medium used to induce the mesoderm precursor cells into primordial germ cell-like cells includes, in addition to Composition 2, a ROCK inhibitor (Y-27632) and aRB27 basal medium. The concentration of Y-27632 is 10 μM. The composition of the aRB27 basal medium is as described above.

[0045] This invention provides a method for inducing porcine gastrulation epiblast stem cells into primordial germ cell-like cells, comprising the following steps: first, preparing mesoderm precursor cells according to the aforementioned method, and then preparing primordial germ cell-like cells according to the aforementioned method.

[0046] The present invention provides a product for generating sperm-like cells in the testicular reticulum of a recipient animal, the product comprising primordial germ cell-like cells prepared according to the aforementioned method.

[0047] Sperm-like cells are cells that resemble natural sperm cells in morphology, gene expression, or function.

[0048] Mesoendothelial precursor cells or primitive germ cell-like cells prepared by the aforementioned methods are also within the scope of protection of this invention.

[0049] The present invention also provides compositions, said compositions being any of the following: M1) A composition for inducing porcine gastrulated epiblast stem cells into mesoendothelial precursor cells, said composition comprising the aforementioned composition one and isolated porcine gastrulated epiblast stem cells; M2) A composition for inducing mesoendodermal precursor cells into primordial germ cell-like cells, said composition comprising the aforementioned composition two and mesoendodermal precursor cells prepared according to the aforementioned method; M3) A composition for inducing porcine gastrulation epiblast stem cells into primordial germ cell-like cells, said composition comprising the aforementioned composition one, the aforementioned composition two and isolated porcine gastrulation epiblast stem cells; M4) is a composition consisting of composition one and composition two.

[0050] Composition 1 and Composition 2 are packaged separately, as are porcine gastrulated epiblast stem cells and / or mesoderm precursor cells.

[0051] The present invention also provides any of the following applications of the aforementioned mesoderm precursor cells or primordial germ cell-like cells: A1) Construct mesoendothelial precursor cells with SOX17 or NANOS3 gene knock-in; A2) Prepare products for constructing mesoendodermal precursor cells with SOX17 or NANOS3 gene knock-in; A3) Produces mesoendothelial precursor cells; A4) Products prepared from mesoderm precursor cells; A5) Produces primordial germ cell-like cells; A6) Prepare products that produce primordial germ cell-like cells; A7) Produces sperm-like cells; A8) Prepare products that produce sperm-like cells.

[0052] A9) Prepare products for treating diseases caused by a lack of sperm cells or insufficient sperm activity.

[0053] The advantages of this invention lie in the construction of a germ cell gene-targeted integration fluorescent reporter system using pig epiblast stem cell lines (pEpiSCs). A two-dimensional (2D) adherent monolayer culture method is employed, and based on the transient mesendoderm precursor cell (MEPC) stage, a large number of pPGCLCs are rapidly induced. The obtained pPGCLCs are equivalent to early E14 PGCs in vivo. When male pPGCLCs are transplanted into pre-pubescent testes of pigs lacking endogenous germ cells (GCs) via ultrasound-mediated transplantation, they participate in spermatogenesis. Collected sperm cells can be fertilized via intracytoplasmic sperm injection (ICSI). The embryos produced by ICSI can not only establish new embryonic stem cell lines (ESCs), but are then transplanted into estrous sows, where they can develop into fetuses in vivo. Attached Figure Description

[0054] Figure 1This study aims to determine the pedigree fate of porcine E10-E14 embryos. a) is a UMAP visualization of porcine embryo single-cell transcriptome sequencing (scRNA) data and published porcine embryo datasets obtained earlier in the inventors' laboratory. Colors and shapes represent different embryonic days and cell lineages, respectively. n=542 single cells. EPI: epiblast, PGC: primordial germ cells. b) is a UMAP plot showing the distribution and expression of pluripotency and lineage-specific genes. The gradient from white to red indicates expression from low to high. c) is a violin plot showing the average expression level of pluripotency and lineage-specific genes associated with each cell fate identified in extended data plot a. d) is a bubble chart showing the expression patterns of selected genes in different cell types. The size of the dots represents the percentage of significant expression (…). logP), and the color of the dots represents the average expression level of the corresponding genes. e is the gene ontology (GO) enrichment analysis of upregulated genes in E12 and E13_SOX17 positive cells compared with E10 and E11_EPI.

[0055] Figure 2 This is the result of stem cell sex determination. Lane 1 is for D2000 molecular markers; lanes 2 and 4 are for pZFX gene detection (band size 506 bp); lanes 3 and 5 are for pSRY gene detection (band size 400 bp). Lanes 2 and 3 are 1-EPI cell lines, which express both pZFX and pSRY genes and are therefore male stem cell lines; lanes 4 and 5 are 2-EPI cell lines, which express only pZFX gene and are therefore female stem cell lines.

[0056] Figure 3 This image shows the results of direct induction of porcine gastrulated epiblast stem cells (pEpiSCs) into primordial germ cells. a) Schematic diagram of three-dimensional (3D) induction of pEpiSCs into primordial germ cells (PGCs). b) Bright-field images of male pEpiSCs differentiating into PGCs via embryoid bodies within 0-4 days. Scale bar: 500 μm. c) Gene expression changes during PGC induction; error bars represent ±SD (n=3 biological replicates). P The value was calculated using Dunnett's multiple comparison test; ns, not significant. * P <0.05, *** P <0.001, where NANOS3 The differences between D1, D2, D3, and D4 and pEpiSCs were not significant, among which... SOX17 The differences between D1, D2, D3, and D4 and pEpiSCs were not significant, among which... TFAP2C The differences between D3 and D4 and pEpiSCs were not significant.

[0057] Figure 4 For pigs SOX17 and NANOS3 Verification of the cleavage efficiency of three target sgRNAs of the gene. 'a' represents the sgRNA target cleavage site on chromosome 4. SOX17 Sanger sequencing results. b shows the PCR amplification of the region spanning the sgRNA target site, which is close to the SOX17 stop codon and was used for T7 endonuclease I (T7E1) detection. c shows the Sanger sequencing results of the sgRNA target cleavage site NANOS3 on chromosome 2.

[0058] Figure 5 For reporter gene insertion identification. a) Screening of SOX17-targeting clones using genomic PCR of edited pEpiSCs. The integration of the reporter gene in the clones was screened using the internal primer pSOX17-3×Flag-P2A-NLS-tdTomato. #1-#7 are edited pEpiSCs, #8 is a wild-type pEpiSC. b) Screening of NANOS3-targeting clones using genomic PCR of edited pEpiSCs. The integration of the reporter gene in the clones was screened using the internal primer pNANOS3-3×Flag-P2A-NLS-tdTomato. #1-#17 are edited pEpiSCs, #18 is a wild-type pEpiSC.

[0059] Figure 6 for SOX17 - P2A - tdTomato Verification of homozygous knock-in pEpiSCs. a is... SOX17 - P2A - tdTomato ( ST Representative images of clonal morphology (left) and wild-type (WT) pEpiSCs, and alkaline phosphatase (ALP) staining (right). Scale bar, 400 μm. b represents males. ST The karyotype of pEpiSCs is 37+XY. c represents the difference between the wild-type and the qRT-PCR detection result. ST Expression of core pluripotency markers in pEpiSCs. Data are presented as mean ± standard deviation (n = 3 biological replicates). P The values ​​were calculated using Dunnett's multiple comparison test. ns, not significant. d is... ST Representative immunostaining (IF) images of core pluripotency markers (NANOG, OCT4, and SOX2) in pEpiSCs. Scale bar, 50 μm. e represents... STRepresentative images of tdTomato fluorescence and co-immunostained GATA4 and OCT4 in pEpiSCs-derived endoderm. Scale bar, 50 μm. f represents the contrast between the two images. ST Co-immunoimmunization analysis of pEpiSCs-derived endoderm showed co-localization of ST, SOX17, and 3×Flag fluorescence, indicating that the reporter gene faithfully mimics the pig's... SOX17 Endogenous gene expression patterns. Cell nuclei stained with DAPI. Scale bar, 50 μm.

[0060] Figure 7 pEpiSCs were induced to differentiate into porcine middle endoderm precursor cells (pMEPCs). SOX17 - P2A - tdTomato ( ST ) Schematic diagram of pEpiSCs inducing differentiation into porcine middle endoderm cells (ME). b is... ST Bright-field images (top) and real-time ST fluorescence images (bottom) of pEpiSCs during differentiation into MEs via monolayer culture. Scale bar: 200 μm. c represents the relative intensity of SOX17 protein during ME differentiation. d represents the ST fluorescence intensity during pEpiSCs differentiation as analyzed by flow cytometry. + Cell proportion. e represents the mRNA expression levels of SOX17 and core pluripotency markers during ME differentiation, analyzed temporally. SOX17 There was no significant difference between 12 h and pEpiSCs. f represents the expression levels of markers in the protostome, mesoderm, and early PGCs during ME differentiation, as detected by qRT-PCR. GSC There was no significant difference between 12 h and pEpiSCs. GATA4 There was no significant difference between 12 h and pEpiSCs. FOXA2 There was no significant difference between 12 h and pEpiSCs. TET2 There was no significant difference between 12 h and pEpiSCs. g represents time-series IF analysis showing OCT4 co-expression during differentiation of ST reporter cell lines into ME. White arrows indicate individual ST cells. + Cells (red); clusters of yellow arrows point to individual OCT4 cells. + Cells (green). White dashed lines represent ST. + Strong expression but OCT4 + Cells with weak expression, and the purple dashed line represents ST. + Weak expression but OCT4 + Cells with strong expression. Scale bar, 50 μm. h is... Figure 7 The results of f were quantified, showing the co-expression of ST during the 0-48 hour process. + OCT4 +Cell percentage. Total cell number was calculated using nuclear counting (DAPI). Image i shows a representative co-immunostained image of ST-positive cells and SOX2 during ME differentiation. White arrows indicate individual ST cells. + Cells (red). Clusters of yellow arrows indicate individual SOX2 cells. + Cells (green). White dashed lines represent SOX17. + Strong expression and SOX2 + Cells with weak expression. Scale bar, 50 μm. j represents the relative intensity of OCT4 and SOX2 proteins during ME differentiation, where... OCT4 There were no significant differences between pEpiSCs and 12h, 24h, 36h, and 48h. k is a schematic diagram illustrating that during ME differentiation, the OCT4 heterodimer chaperone exchanges from SOX2 to SOX17 and enters the mesoendodermal lineage. l shows representative immunostaining images of NANOG and OCT4, ST and PRDM1, and TBXT during ME differentiation. White arrows indicate individual STs. + Cells. White dashed lines highlight cells co-expressing ST. + PRDM1 + Cells. Cell nuclei counterstained with DAPI. Scale bar, 50 μm. Scale bar, 50 μm. m represents the relative intensities of NANOG, PRDM1, and TBXT proteins during ME differentiation. n represents representative immunostaining images of mesoderm markers in 36-hour ST+ pMEPCs, where... NANOG There were no significant differences between pEpiSCs and pEpiSCs at 12h, 24h, and 36h. Scale bar, 50μm. The scatter plot compares the mean gene expression levels between pEpiSCs (left) and pMEPCs (right). Adjusted for... p Differentially expressed genes (DEGs) were identified using threshold values ​​padj < 0.05 and absolute log2-fold change ≥ 1. Upregulated genes (log2-fold change ≥ 1) are shown in red, downregulated genes (log2-fold change ≤ -1) in blue, and insignificant genes in gray. p represents significant functional entries associated with differentially upregulated genes in pMEPCs compared to pEpiSCs, as shown by gene ontology (GO) enrichment analysis. Error bars for c, e, f, j, and m are shown as ± standard deviation (n = 3 biological replicates). P The values ​​were calculated using Dunnett's multiple comparison test.

[0061] Figure 8Specialization of porcine primordial germ cell-like cells (pPGCLCs). a) Schematic diagram of the differentiation protocol for porcine primordial germ cell-like cells (pPGCLCs). b) Bright field (top) and real-time ST fluorescence (bottom) images of cells during ST pMEPCs monolayer culture 2. Scale bar, 200 μm. c) Time-series analysis of mRNA expression levels of pluripotency markers and early PGCs markers during pPGCLC differentiation, where... STELLA There was no significant difference between pMEPCs and pEpiSCs. NANOS3 There was no significant difference between pMEPCs and pEpiSCs. TFAP2C The differences between pMEPCs and pEpiSCs at 6h, 12h, and 18h ​​were not significant. PRDM14 There was no significant difference between pMEPCs and pEpiSCs. TFCP2L1 There were no significant differences between pMEPCs and pEpiSCs at 6h, 12h, and 18h. d shows bright-field (left) and real-time ST fluorescence (right) images of pPGCLCs cultured in ST pMEPCs monolayers for 24h. Scale bar, 200μm. e shows flow cytometry analysis of ST fluorescence in pPGCLCs at 24h of differentiation. + Cell proportions. f shows the expression of OCT4 during pPGCLC differentiation using ST fusion reporter cell lines and cytokine induction over 0-48 h, analyzed by time-series immunofluorescence staining. Representative images are shown at 0 h, 12 h, 24 h, and 36 h. Scale bar, 50 μm. g shows the relative intensity of OCT4 protein expression during pPGCLC differentiation. h shows the expression of PRDM1 and TFAP2C during pPGCLC differentiation using ST fusion reporter cell lines and cytokine induction over 0-48 h, analyzed by time-series immunofluorescence staining. Representative images are shown at 0 h, 12 h, 24 h, 36 h, and 48 h. Scale bar, 50 μm. i shows the relative intensity of SOX17, PRDM1, and TFAP2C protein expression during pPGCLC differentiation. j shows representative immunostaining images of TNAP, MKI67, and NANOS3 in ST+ pPGCLCs. Scale bar, 50 μm. k represents high levels of positive alkaline phosphatase (ALP) staining in the cytoplasm of NANOS3-tdTomato+ pPGCLCs. Scale bar, 50 μm. l represents representative co-immunostained images of SOX17, OCT4, NANOG, SOX2, and PRDM1 in NANOS3-tdTomato+(N3T) pPGCLCs. Scale bar, 50 μm. m represents the staining in pEpiSCs and pPGCLCs. H19 / I GF2 and SNRPN Methylation status of differentially methylated regions (DMRs). Each row represents a single allele. Black circles represent methylated CpG islands, and white circles represent unmethylated CpG islands. n is a representative image of OCT4 and TET2 co-immunostained in pPGCLCs compared to pEpiSCs. Scale bar, 50 μm. o is the relative intensity of TET2 protein in differentiated pPGCLCs compared to pEpiSCs. p is a representative image of OCT4 and 5-methylcytosine (5-mC) co-immunostained in differentiated pPGCLCs. Scale bar, 50 μm. q is the relative intensity of 5-mC protein expression in pPGCLCs compared to pEpiSCs. r is a representative image of OCT4 and H3K27me3 co-immunostained in differentiated pPGCLCs. Scale bar, 50 μm. s represents the relative intensity of H3K27me3 protein expression in pPGCLCs compared to pEpiSCs. t represents a representative image of OCT4 and H3K9me2 co-immunostained pPGCLCs compared to pEpiSCs. Scale bar, 50 μm. u represents the relative intensity of H3K9me2 protein in differentiated pPGCLCs compared to pEpiSCs. For c, i, and g, error bars represent ±SD (n=3 biological replicates), calculated using Dunnett's multiple comparison test. P Values. For o, q, s, and u, the error bars represent ±SD (n=3 biological replicates). P The values ​​were calculated using a two-tailed unpaired Student t-test.

[0062] Figure 9 Transcriptome expression profiling of pPGCLCs. a) Heatmap showing specific markers for a given cell cluster. b) Heatmap of correlation coefficients among pEpiSCs, pMEPCs, and pPGCLCs. c) Scatter plot comparing mean gene expression levels between pEpiSCs (left) and pPGCLCs (right). Differentially expressed genes (DEGs) were identified by an adjusted p-value threshold of padj < 0.05 and an absolute log2-fold change ≥ 1. Upregulated genes (log2-fold change ≥ 1) are shown in red, downregulated genes (log2-fold change ≤ -1) in blue, and insignificant genes in gray. d) Gene Ontology (GO) enrichment analysis showing significant functional entries associated with differentially upregulated genes in pPGCLCs compared to pMEPCs. e is a UMAP plot showing the integration of individual epiblast cells (EPI, E10-E14), primordial germ cells (PGCs, E14 and E31), pEpiSCs, pMEPCs, NANOS3-tdTomato+ PGCLCs, and D2.5 somatic cells. n=702 cells. f is a heatmap of selected marker genes in a specified cell cluster.

[0063] Figure 10 For pPGCLCs in porting to NANOS3 After gene knockout of the porcine testicular reticulum, sperm-like cells were generated. a) Schematic diagram of sperm-like cell generation from N3T / AG pPGCLCs via ultrasound-mediated testicular reticulum transplantation. b) Statistical analysis of spermatogenesis efficiency after pPGCLC transplantation. c) Slow, positive-pressure delivery of pPGCLCs by introducing an ultrasound-guided injection needle into the testicular reticulum cavity. White arrows indicate the mediastinum in the testicular reticulum, while clusters of white arrows indicate the needle inserted into the reticulum. d) Longitudinal section of the recipient porcine testis 4 months after pPGCLC transplantation. Scale bar, 5 mm. e) Transplantation of pPGCLCs to... NANOS3 Histological analysis of the testes of recipient pigs 4 months after KO. White arrow clusters indicate germ cells, while black arrows indicate supporting cells. Scale bar: left 100μm, right 25μm. f: Histological analysis of epididymal tissue sections from recipient pigs injected with pPGCLCs 4 months after transplantation. White arrow clusters indicate putative sperm cells. Scale bar: left 400μm, right 100μm. g: Representative genotyping results of NANOS3 KO recipient pig testes 4 months after pPGCLC transplantation. PC: positive control (N3T+ pPGCLCs), NC: negative control (WT pEpiSCs). h: Representative IF images of recipient pig testicular sections 4 months after transplantation, showing the expression of designated key GC markers (red: DDX4, ACROSIN) and DAPI (blue). Scale bar: left 50μm, right 50μm. Figure 10 μm. i represents 4 months of age. NANOS3 Representative IF images of KO pig testicular sections, showing no expression of key GC markers (DDX4, ACROSIN). Scale bar, 50 μm. j: Representative IF images of recipient pig epididymal sections 4 months post-transplantation, showing expression of key sperm markers (red: PNA) and DAPI (blue). Scale bar, 50 μm.

[0064] Figure 11 Haploid characteristics and origin of sperm-like cells from recipient pigs were detected. a) Representative IF images of sperm cells from semen collected from NANOS3 knockout recipient pigs transplanted with pPGCLCs, showing the expression of key sperm markers (green: ACROSIN) and DAPI (blue). Scale bar, 10 μm. b) Detection of sperm-related genes in semen from NANOS3 KO recipient pigs using RT-PCR. PRM1 , PRM2 , ACROSIN and TNP1Expression analysis was performed. c shows the proportion of pPGCLCs-derived haploid cells in semen collected from 13-month-old NANOS3KO recipient pigs after transplantation, analyzed by flow cytometry. Wild-type testicular tissue isolated from pig Sertoli cells (SCs) served as a control group. d shows that the DNA from recipient pig semen collected from the recipient pigs contained a mixture of signals from the recipient pig and the pPGCLCs donor at the examined S0090 microsatellite locus. e shows... NANOS3 Representative agarose gel images of genotypes. Genomic DNA extracted from sperm cells isolated from semen collected from NANOS3 knockout pigs at different time points after transplantation was used for genotyping. Controls included N3T pEpiSCs (positive control) and blood from NANOS3 knockout recipient pigs (negative control). Molecular weight (MW) is the DNA gradient of D2000bp. f is a schematic diagram of PCR-based genotyping of NANOS3 alleles, using DNA samples including: N3T + Blood samples from pPGCLCs and NANOS3 KO boars, and semen samples from a recipient boar (#2205902) that received pPGCLCs. g shows sex determination of a single pPGCLCs-derived sperm cell using nested PCR. h shows sex determination of a single wild-type sperm cell using nested PCR. i shows analysis of selected single N3T pEpiSCs and single N3T... + Genomic insertion fragments at the NANOS3 site in pPGCLC and single probable sperm cells 3×Flag - P2A - NLS - tdTomato j represents sperm cells derived from pPGCLCs and wild-type (WT) sperm. H19 / IGF2 and SNRPN Methylation status of differentially methylated regions (DMRs). Each row represents a single allele. Black circles indicate methylated CpG islands, and white circles indicate unmethylated CpG islands. All representative data were obtained from at least three independent experiments. J. Proposed sperm cells from semen collected from NANOS3 KO recipient pigs were stained with Hochest 33342. Scale bar: 100 μm on the left, 25 μm in the middle and right.

[0065] Figure 12 Functional validation of sperm-like cells originating from pPGCLCs. a) Schematic diagram: Pig embryos produced in vitro via intracytoplasmic sperm injection (ICSI) and surgically transferred to obtain pig fetuses. b) Collection. NANOS3Fertilization micrographs of sperm cells knocked out from recipient pigs 5 months after transplantation and injected into porcine oocytes using intracytoplasmic sperm injection (ICSI) were observed, showing meiotic recovery, polar body expulsion, pronuclear formation, zygote fusion, anaphase, and two-cell stage. Cell nuclei were stained with DAPI. Scale bar, 50 μm. c shows the in vitro development rate statistics of ICSI-injected embryos produced from pPGCLCs-derived sperm cells, including two-cell stage embryos and E7 blastocysts. n=5. d shows a representative IF image of E7 blastocysts produced by ICSI. The inner cell mass (SOX2 positive) is shown in green, the trophoblast (CDX2 positive) in red, and the nucleus (DAPI) in blue. Scale bar, 50 μm. e shows the total number of cells in the inner cell mass and trophoblast of E7 blastocysts produced by porcine ICSI. n=15. f shows an image of E30 porcine fetuses derived from pPGCLCs-derived sperm cells. Scale bar, 5 mm. g shows the isolation and establishment of E30 fetal fibroblast cell lines produced by ICSI. Scale bar, 400 μm. h represents karyotype analysis of porcine fetal fibroblast cell lines derived from ICSI-produced E30 embryos. i represents genotyping of N3T and AG cells in E30 porcine fetal tissues. j represents the karyotypes of N3T and AG cells in ICSI-derived and wild-type E30 fetuses. SNRPN Bisulfite sequencing of differentially methylated regions. Each line represents a single allele. Black circles indicate methylated CpG islands, and white circles indicate unmethylated CpG islands. k represents the overall methylation status of ICSI-derived fetuses (NE1-NE6) and normal porcine fetuses (WE1-WE4) at E30-E40. l represents the methylation level at specific sites (PLAGL1 and ZNF791) in ICSI-derived porcine fetuses.

[0066] Figure 13Establishment and characterization of next-generation porcine embryonic stem cells (pESCs). a) Statistical analysis of pESC data derived from E7 blastocysts produced by ICSI. b) Outgrowth observation observed 10 days after seeding in 4-well plates, with P10 pESCs obtained through continuous passage. Scale bar: top 100 μm, bottom 400 μm. c) Representative image of alkaline phosphatase (ALP) staining in pESCs from ICSI blastocysts. Scale bar: 400 μm. d) Karyotype analysis of pESCs derived from ICSI blastocysts. e) Representative IF images of core pluripotency markers (NANOG, OCT4, SOX2, OTX2, and PRDM14) and the pluripotency surface marker CDH1 in ICSI blastocyst-derived pESCs under 3i / LAF culture conditions. Scale bar: 50 μm. f shows representative IF images of pluripotency surface markers (SSEA1, SSEA4, TRA1-60, and TRA1-81) in ICSI blastocyst-derived pESCs under 3i / LAF culture conditions. Scale bar, 50 μm. g shows a heatmap analysis of the correlation coefficient between ICSI-pESCs and pEpiSCs. h shows a schematic diagram of in vitro embryomorphic body (EBs) differentiation experiments. i shows the morphology of in vitro pESC-derived EBs (left image) and adherent growths of EBs (EBD, right image). Scale bar, 400 μm. j shows representative IF images of three germ layer markers (βIII-TUBULIN, α-SMA, and GATA4) 10 days after spontaneous differentiation of pESCs into EBs. Scale bar, 50 μm. k shows the differentiation of ICSI blastocyst-derived pESCs into N3T+ pPGCLCs. Scale bar, 100 μm. l is a representative IF image of N3T+ pPGCLCs derived from pESCs, showing the expression of key PGC markers (PRDM1, TFAP2C, NANOS3, and SOX17). Scale bar, 50 μm. Representative data from at least three independent experiments.

[0067] In the above figures, all relative expression levels (unspecified values) showed significant or highly significant differences unless otherwise specified. Detailed Implementation

[0068] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0070] The terminology used in this invention: Differentiation of the mesendoderm (ME): During mammalian embryonic development, the mesendoderm, also known as the primitive streak, is the transitional period between the differentiation of the mesoderm and the endoderm. Here, it specifically refers to the in vitro differentiation of epiblast stem cells into the mesendodermal lineage.

[0071] E12-E13 EPIs refer to the epiblast tissues and cells of a pig fetus on days 12-13 of gestation.

[0072] In the gene expression analyses of the following examples, total RNA was extracted using the RNAprep Cell / Bacteria Kit (Tiangen, DP430) or TRIzol reagent (Thermo Fisher, 15596026CN) according to the manufacturer's instructions. cDNA was then reverse transcribed using the 5×All-In-One RTMaster Mix Kit (abm, G486).

[0073] In the quantitative experiments described below, all experiments were performed in triplicate, and GAPDH and EEF1A1 were used as housekeeping genes for standardization. The average value was taken, and the results were analyzed using the following methods: .

[0074] The following examples used GraphPad Prism v6.01 statistical software to process the data. Experimental results are expressed as mean ± standard deviation. One-way ANOVA and two-tailed unpaired t-tests were used. ns indicates no significance. P <0.05 (*) indicates a significant difference.

[0075] All experiments involving mice and pigs were conducted in accordance with the ethics guidelines of China Agricultural University and approved by the Animal Ethics Committee of China Agricultural University (License No.: AW02113202-3-1). CD1 and BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) for use in feeder cell preparation and teratoma formation experiments.

[0076] NANOS3 - / -Bama pigs: Constructed by the Institute of Zoology, Chinese Academy of Sciences, and described in the non-patent literature "Wang, J. et al. Nanos3 knockout pigs to model transplantation and reconstruction of the germline. Cell Prolif. 56, e13463 (2023)", their name in the aforementioned literature is Nanos3knockout pigs. They are publicly available from China Agricultural University. This biological material is only for repeating the relevant experiments of this invention and cannot be used for other purposes. The preparation method is as follows: Using CRISPR / Cas9 technology, pig fetal fibroblasts in Bama pigs were targeted... NANOS3 A large segment of the exon 1 region of the gene was knocked out. The resulting knockout cell lines were then subjected to somatic cell nuclear transfer injection, followed by embryo transfer to obtain gene knockout pigs. The mutation details are as follows: cell lines #1 and #7 were both male, with knockout fragments of 708 bp and 679 bp, respectively; cell line #51 was female, with a knockout fragment of 713 bp. All recipient pigs used in this study were male and derived from cell line #7. NANOS3 - / - Bama pigs.

[0077] Large White sows used in the embryo transfer experiment were in estrus and were sourced from Hebei Mutai Livestock Co., Ltd. All animals were raised under conditions free from specific pathogens.

[0078] The histological analysis methods in the following examples were as follows: Tissues were dehydrated stepwise with a gradient of ethanol (70%, 80%, 90%, 95%, and 100%), then cleared in xylene and embedded in paraffin. All samples were serially sectioned to a thickness of 5 μm, attached to glass slides, dewaxed in xylene, and rehydrated with a gradient of ethanol (from high to low). They were then counterstained with hematoxylin (Sigma-Aldrich, MHS16) and eosin (Sigma-Aldrich, HT110116) and observed under a microscope (Leica, DM5500B).

[0079] The following examples illustrate the immunofluorescence assay method: Cells were fixed with 4% PFA at room temperature for 20 min or overnight at 4°C, then washed twice with DPBS for 3 min each time. Next, cells were permeabilized in 0.3% Triton X-100 / DPBS for 20 min, washed twice with DPBS for 3 min each time, and blocked with 1% BSA / 10% donkey serum for 40 min. Subsequently, cells were incubated overnight with a primary antibody at 4°C, washed three times with DPBS for 5 min each time. Immediately afterwards, cells were incubated with the appropriate secondary antibody at room temperature for 30 min, washed three times with DPBS for 5 min each time, counterstained with DAPI for 5 min, and finally washed with DPBS. Images were acquired using an Olympus IX71 fluorescence inverted microscope.

[0080] The karyotype analysis method in the following examples was as follows: When cells reached 70-80% confluence after passage, they were cultured in fresh medium containing 20 ng / mL KaryoMax Colcemid (Thermo Fisher, 15212012) for 2-3 h. The treated pEpiSCs were dissociated into single cells using Accutase and collected by centrifugation. After resuspending the cells, 0.075 M potassium chloride solution (pH 7.4) was added dropwise, and the cells were incubated at 37°C for 20 min. Then, the cells were fixed with freshly prepared methanol and glacial acetic acid (3:1) at 4°C for 3 min, followed by three fixation steps, each lasting 20 min. The fixed cells were dropped from a height onto pre-cooled, clean glass slides and then dried. The dried slides were stained with Giemsa stain, and chromosome diffusion images were taken under a microscope. The chromosome number was counted (at least 30 cells were analyzed).

[0081] The alkaline phosphatase staining method in the following examples was as follows: Cells in the logarithmic growth phase were washed with pre-warmed DPBS and fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. After three washes with DPBS, cells were stained with BCIP / NBT ALP buffer in the dark for 10 min. Cells were washed again with DPBS and observed using an inverted microscope.

[0082] The inventors revealed the dynamic process of germline fate emergence through experiments, integrating and analyzing single-cell resolution data of porcine embryos from day 10 (E10) to day 14 porcine epiblasts (pEPIs) (see...). Figure 1 (a) Around E10, core pluripotent transcription factors, such as..., were detected in pEPIs. OCT4 , NANOG , SOX2 and PRDM14 In E11's pEPIs, SOX2 and NODAL The expression of [something] was significantly downregulated. With further development to E12-E13, [something]... NANOG , OTX2 and PRDM14 Expression in EPIs is suppressed, while original markers, including SOX17 , PRDM1 , NODAL , Eomesodermin ( EOMES ), Goosecoid ( GSC ), GATA4 , GATA6 , PDGFRA , FOXA2 , GATA2 and GATA3 High expression of this gene indicates the onset of gastrulation (see...). Figure 1 (b and c). Given SOX17 It is the first factor regulating pPGC fate, and its expression can be detected in pEPIs of E12, increasing in E13. SOX17-positive cells in E12 and E13 EPIs co-express some lineage markers, such as... GATA6 , EOMES , NODAL , CHRD , FOXA2 , GATA4 , GSC and CER1 Similar to the original article in the report (see...) Figure 1 (d). These cells also showed significant expression of the Activin / NODAL and WNT signaling pathways. Gene ontology enrichment analysis revealed that, compared to E10 and E11 EPIs, the E12 and E13 SOX17-positive cell populations upregulated pathways including focal adhesion, tubular morphogenesis, gastrulation, endoderm development, and mesodermal formation. Therefore, the inventors defined these cells as pig mesendoderm precursor cells (pMEPCs) (see [reference needed]). Figure 1 (e) This cell refers to a cell state or type in pigs, which is different from pMEPCs mentioned later, which are cells induced to differentiate in vitro by pEpiSCs.

[0083] Primers used in this invention: pig EEF1A1-Fw:5'-AATGCGGTGGGATCGACAAA-3', Rv:5'-CACGCTCACGTTCAGCCTTT-3'; pig GAPDH-Fw:5'-TGGTGAAGGTCGGAGTGAAC-3', Rv:5'-CCTTGACTGTGCCGTGGAA-3'; pig NANOG-Fw:5′-CATCTGCTGAGACCCTCGAC-3′,Rv:5′-GGGCTTGTGGAAGAATCAGG-3′ pig OCT4-Fw:5′-CAAACTGAGGTGCCTGCCCTTC-3′,Rv:5′-ATTGAACTTCACCTTCCCTCCA-3′ pig SOX2-Fw:5′-CCAGAAGAACAGCCCAGACC-3′,Rv:5′-GCTTCTCCGTCTCCGACAAA-3′ pig STELLA-Fw:5′-CCCGCCTTTCAATCTGTCTCC-3′,Rv:5′-TCGCCGAACCGTGTATCGAA-3′ pig KLF4-Fw:5′-CATGAGTTGGGGGAGGGAAG-3′,Rv:5′-ACTCACCAAGCACCATCGTT-3′ pig TFCP2L1-Fw:5′-TGCACGAAGAGACCTTGACC-3′,Rv:5′-CGCGGATGGTACTCTTCACA-3′ pig PRDM14-Fw:5′-GAGGTGCCACACTTCCTGAACA-3′,Rv:5′-TCGGGTAGCAGAGAAGCATCCA-3′ pig TBXT-Fw:5′-GCTTCCCAGAGACCCAGTTC-3′,Rv:5′-GCGTAAGATTGGGAGTACCCG-3′ pig EOMES-Fw:5′-ACTCCCATGGACCTCCAGAA-3′,Rv:5′-TCGCTTACAAGCACTGGTGT-3′ pig GATA4-Fw:5′-CGACACCCTAATCTCGATATGTTT-3′,Rv:5′-TCATCTTGTGGTAGAGGCCG-3′ pig GSC-Fw:5′-TGCTGCCCTACATGAACGTG-3′,Rv:5′-TCATCGGTGAAGATGGTGCG-3′ pig FOXA2-Fw:5'-CCAACAAGATGCTGACCCTG-3',Rv:5'-AGGAAGCAGTCGTTGAAGGA-3'; pig PDGFRA-Fw:5'-GGTCACCTGTGCCGTCTTTA-3',Rv:5'-TTTGATGGACGGGACCTTGG-3'; pig NANOS3-Fw:5'-CTTCGAGACTACGTGTGCCC-3',Rv:5'-CGATGTCCAGAGTCCTGTGTC-3'; pig SOX17-Fw:5'-CGCACGGAGTTTGAACAATA-3',Rv:5'-CAGACGTCGGGGTAGTTACAG-3'; pig PRDM1-Fw:5'-CAGTGCCGTGAAGTTTCCA-3',Rv:5'-AAGGATGCCTCTGCCTGAAC-3'; pig TFAP2C-Fw:5'-AGGACTCCACATCCGTTTG-3',Rv:5'-TGGTCACCTTCAACCTCGT-3'; pig TET2-Fw:5'-GCCGAGTGATGAGAACAGACG-3',Rv:5'-GTCTGCTGCTGAATGTTGGC-3'; pig DNMT3B-Fw:5'-ATGACCGGCCTTTCTTCTGG-3',Rv:5'-AGCCGAAGATCCTGTTCATCC-3'; pig DAZL-Fw:5'-CTGCTGGGGAACAAAGGAGT-3',Rv:5'-CCACTGTCTGTATGCTTCGGT-3'; pig DDX4-Fw:5'-AGCTGATCGCATGCTGGATATGG-3',Rv:5'-TCCGCCCACTTGTCCAACAG-3'; pig STRA8-Fw:5'-CGTGTCTGCAGCCATCTCCC-3',Rv:5'-TCGGGGGTGGATACCAGTGA-3'; pig SYCP3-Fw:5'-GGAGAGTCAGTGGGTTCTGG-3',Rv:5'-GCCAGGCACCTTCCAGTTT-3'; pig PRM1-Fw:5'-AGATGTTGCCGCAGCCATAG-3',Rv:5'-AGTGCGGTGGTCTTGCTAC-3'; pig PRM2-Fw:5'-GACCAGGGCTGCAGACGGA-3',Rv:5'-TACTCAAGATCTCGTGGGCTCCT-3'; pig TNP1-Fw:5'-AAGCCCCTCATTTTGGCAGA-3',Rv:5'-GTCACAAGTGGGAGCGGTAA-3'; pig ACROSIN-Fw:5'-TCTTGCTGAACTCGCACTGG-3',Rv:5'-GTGGCTTCACCGGCTTATTG-3'; pig SRY-Fw:5'-GGGAAAGGCTCCTCACTATTT-3',Rv:5'-AGGGATACATCCTCTCCTCTAC-3'; pig ZFX-Fw:5'-GTGCTGCTTTGTCTTGGAATG-3',Rv:5'-GAGGGAGTTAGGTCTGGATACT-3'; pSOX17-sgRNA1-Fw:5'-caccGTAACTACCCCGACGTCTGA-3',Rv:5'-aaacTCAGACGTCGGGGTAGTTAC-3'; pSOX17-sgRNA2-Fw:5'-caccgTAACTACCCCGACGTCTGAC-3',Rv:5'-aaacGTCAGACGTCGGGGTAGTTAc-3'; pSOX17-sgRNA3-Fw:5'-caccGATGGGCACCCGTCAGACGT-3',Rv:5'-aaacACGTCTGACGGGTGCCCATC-3'; pSOX17-sgRNA test-Fw:5'-CGCACGGAGTTTGAACAATATC-3',pSOX17-sgRNA test-Rv:5'-TGACAGAGGCACTAGCAGAG-3'; pSOX17-5'arm-Fw:5'-cgaattgggcccggcgcgccACGCCTTAGCCTA-3',pSOX17-5'arm-Rv:5'-tggtccttatagtcggtaccGACGTCGGGGTAGTT-3'; pSOX17-3'arm-Fw:5'-ctatacgaagttatatcgatTGACGGGTGC-3',pSOX17-3'arm-Rv:caagctatgcatttaattaaTGATTGGCGAG-3'; pNANOS3-sgRNA1-Fw:5'-caccgAAAGGTGCCGGGAAGTCTTC-3',pNANOS3-sgRNA1-Rv:5'-aaacGAAGACTTCCCGGCACCTTTc-3'; pNANOS3-sgRNA2-Fw:5'-caccgCCTAAGAGGCTGGCGCGAGC-3',pNANOS3-sgRNA2-Rv:5'-aaacGCTCGCGCCAGCCTCTTAGGc-3'; pNANOS3-sgRNA3-Fw:5'-caccgTCCACTTCTGCCTAAGAGGC-3',pNANOS3-sgRNA3-Rv:5'-aaacGCCTCTTAGGCAGAAGTGGAc-3'; pNANOS3-sgRNA test-Fw:5'-AGAGTCCTATGGCCGCGAG-3',pNANOS3-sgRNA test-Rv:5'-GATCCGCGAAGGGTAGGTCG-3'; pNANOS3-5'arm-Fw:5'-gcgaattgggcccggcgcgccCATTTGGGTGCCCCGAGT-3',pNANOS3-5'arm-Rv:5'-tggtccttatagtcggtacc GGCAGAAGTGGAGGGACAGC-3'; pNANOS3-3'arm-Fw:5'-aatgtatgctatacgaagttatatcgatTGGCGCGAGCAGGAC-3',pNANOS3-3' arm-Rv:5'-gaatactcaagctat gcatttaattaaGGCACTCTAATTGCCTCTCCAC-3'; pig NANOS3-KO test-Fw:GGCAGGTGCATTTTTGGAGG-3′,pig NANOS3-KO test-Rv:5′-AGCAACAGATCCAGAAGACTGT-3′; EGFP-test-Fw:CGAGTAAACGGCCACAAGTTCAGC-3′,EGFP-test-Rv:5′-GATGGGGGTGTTCTGCTGGTAGTGG-3′ tdTomato-test-Fw:GACAACAACATGGCCGTCATCAAAG-3′,tdTomato-test-Rv:5′-GCTCGTCCATGCCGTACAGGAACAG-3′ S0090-Fw:CCAAGACTGCCTTGTAGGTGAATA-3′,Rv:5′-GCTATCAAGTATTGTACCATTAGG-3′ pig H19 / IGF2 DMR2 Inside-Fw:5'-AGGTGTTATTTTGTTTGTTGGT-3',pig H19 / IGF2DMR2 Inside-Rv:ATAAAATAACCTAAAAAAACTCAA-3' pig H19 / IGF2 DMR3 Inside-Fw:5'-GATTTTTAGGTTTGTTATTATTT-3',pig H19 / IGF2 DMR3 Inside-Rv:CAAATATTCAATAAAAAA ACCC-3'; pig SNRPN DMR Inside-Fw:5'-TTATTGTGAAGGATTTTGTGTTTTG-3',pig SNRPN DMRInside-Rv:5'-AAAAAATAACCTCAAACATCT CCAA-3' IS PCR primer:5′-AAGCAGTGGTATCAACGCAGAGT-3′; 3′P2 primer:5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3′. QP2 primer:5'-CAAGCAGAAGAGGCATACGA.

[0084] Antibodies used in immunofluorescence staining in this invention: Rabbit anti-NANOG (1:1000, Peprotech, 500-P236), Mouse anti-SOX2 (1:500, Santa Cruz Biotechnology, sc-365823), Goat anti-OCT3 / 4 (1:1000, Santa Cruz Biotechnology, sc-8628), Rabbit anti-PRDM14 (1:300, Abcam, ab187881), Goat anti-OTX2 (1:300, R&D Systems, AF1979), Mouse anti-SSEA1(MC480) (1:200, Cell Signaling Technology, 4744p), Mouse anti-SSEA4(MC813) (1:300, Abcam, ab16287), Mouse anti-TRA-1-60 (1:500, Cell Signaling Technology, 4746), Mouse anti-TRA-1-81 (1:500, Abcam, ab16289), Rabbit anti-H3K27me3 (1:300, Cell Signaling Technology, 9733s), Rabbit anti-H3K27me3 (1:300, Millipore, 07-449), Rabbit anti-H3K9me2 (1:300, Abcam, ab1220), Rabbit anti-βIII-tubulin (1:300, Abcam, ab18207), Rabbit anti-α-SMA (1:300, Abcam, ab5694), Goat anti-GATA4 (1:100, Santa Cruz Biotechnology, sc-1237), Rabbit anti-GATA4 (1:100, Abcam, ab84593), Goat anti-BRACHYURY (N-19) (1:50, Santa Cruz Biotechnology, sc-17743), Goat anti-Brachyury (1:300, R&D Systems, AF2085), Rabbit anti-Eomes (1:500, Abcam, ab23345), Goat anti-GSC (1:50, Santa Cruz Biotechnology,sc-22234)、Rabbit anti-GATA6(1:300,Abcam,ab22600)、Goat anti-HAND1(1:300,R&D Systems,AF3168)、Rabbit anti-3×Flag(1:1000,Easybio,BE2005)、Rabbit anti-NANOS3(1:50,Invitrogen,PA5-115615)、Rabbitanti-MKi67(1:1000,Abcam,ab15580)、Mouse anti-TNAP(1:50,Santa CruzBiotechnology,sc-137213)、Goat anti-SOX17(1:500,R&D Systems,AF1924)、Rabbitanti-SOX17(1:1000,Cell Signaling Technology,81778s)、Rabbit anti-PRDM1(1:50,Cell Signaling Technology,9115S)、Rat anti-PRDM1(1:50,eBioscience,14-5963-82)、Goat anti-AP2γ(1:300,R&D Systems,AF5059)、Rabbit anti-TET2(1:600,GeneTex,GTX124205)、Mouse anti-5-mC(1:300,GeneTex,GTX60803)、Rabbit anti-DAZL(1:300,Abcam,ab34139)、Rabbit anti-DDX4(1:300,Abcam,ab13840)、Rabbit anti-PLZF(1:300,Abcam,ab39354)、Rabbit anti-TurboGFP(1:100,Invitrogen,PA5-22688)、Mouse anti-GFP (B-2)(1:100,Santa Cruz Biotechnology,sc-9996)、Rabbit anti-Stra8(1:200,Abcam,ab49602)、Rabbit anti-Rad51(1:100,Abcam,ab133534)、Rabbit anti-gammaH2A.X(1:100,Abcam,ab11174)、Mouse anti-SYCP3(1:100,Novus,NBP2 - 23487), Mouse anti - Acrosin (1:100, Abcam, ab1900), Mouse anti - Acrosin (1:100, Santa Cruz Biotechnology, sc - 51504), Rabbit anti - Protamine 1 (1:100, R&D Systems, NBP2 - 30949), Peanut Agglutinin (PNA) Rhodamine (1:300, Vector laboratories, RL - 1072 - 5), Rabbit anti - CDX2 (1:200, Proteintech, 82659 - 1 - RR), Alexa Fluor 488 conjugated donkey anti - rabbit IgG (1:1000, Life Technologies, A21206), Alexa Fluor 488 conjugated donkey anti - rat IgG (1:1000, Life Technologies, A21208), Alexa Fluor 488 conjugated donkey anti - goat IgG (1:1000, Life Technologies, A11055), Alexa Fluor 488 conjugated donkey anti - mouse IgG (1:1000, Life Technologies, A21202), Alexa Fluor 594 conjugated donkey anti - rabbit IgG (1:1000, Life Technologies, A21207), Alexa Fluor 594 conjugated donkey anti - goat IgG (1:1000, Life Technologies, A11058), Alexa Fluor 594 conjugated donkey anti - mouse IgG (1:1000, Life Technologies, A21203), Alexa Fluor 594 conjugated goat anti - chicken IgY (1:1000, Life Technologies, A11042).

[0085] Example 1. Amplification and culture of pEpiSCs 1. Preparation of MEF feeder layer cells Neutralization medium composition: 10% fetal bovine serum (FBS, Thermo Fisher product, catalog number 16000044), the remainder being DMEM medium (Thermo Fisher product, catalog number 11995073), the percentages mentioned above are volume percentages.

[0086] MEF medium composition: 10% FBS, 1% non-essential amino acids (NEAAs, Thermo Fisher, 11140050), 1% sodium pyruvate (Thermo Fisher, 11360070), 1% glutathione (GlutaMax, Thermo Fisher, 35050061), and 1% penicillin-streptomycin (Thermo Fisher, 15140122), with the balance being DMEM medium. All percentages mentioned above are volume percentages, and all contents mentioned above are final contents of each substance.

[0087] Pregnant female CD1 mice, 12.5 days post-mating, were euthanized by cervical dislocation, and embryos were isolated from the uterus, designated E12.5 embryos. These E12.5 embryos were placed in cold DPBS (Thermo Fisher product, catalog number 14190144) containing 1% penicillin-streptomycin. The embryos were dissected under a stereomicroscope, and the fetal torsos were collected in cold DMEM medium. The torsos were placed in 50 mL tubes and repeatedly minced with fine scissors for 10 min. The cells were washed once with DPBS, and digested with 0.25% trypsin-EDTA (Thermo Fisher, 25200072) for 10 min, gently agitated in a 37°C water bath. The cells were then quenched with neutralization medium to obtain mouse embryonic fibroblasts (MEFs). The mouse embryonic fibroblasts (MEFs) were resuspended in MEF medium and seeded in 10 cm culture dishes, approximately one dish per fetus, labeled P0 generation. Once the cells have reached 90% confluence, they can be frozen as primary cells or passaged into 15cm culture dishes. Cells are expanded to passage P3 at a ratio of 1:2 or 1:3, treated with 13 μg / mL mitomycin C (Selleck, S8146) for 2.5 hours, washed twice with pre-warmed DPBS, digested with 0.125% trypsin to form a single-cell suspension, neutralized, and counted. Cells are counted at a ratio of 2.4 × 10⁻⁶. 6 Cryopreserved at a density of 1 cell / mL for use as feeder cells in subsequent stem cell cultures.

[0088] 2. Culture and sex determination of porcine epiblast stem cells (pEpiSCs) Composition of 3i / LAF medium: 5% knockout serum substitute (KSR, Thermo Fisher, 10828028), 1×N2 supplement (Thermo Fisher, 17502048, 100×), 1×B27 supplement (Thermo Fisher, 12587010, 50×), 1% NEAAs, 1% penicillin-streptomycin, 0.5% glutathione, 0.1mM β-mercaptoethanol (Thermo Fisher, 21985023), 50μg / mL ascorbic acid (Sigma-Aldrich, A4544), 10ng / mL human leukemia inhibitory factor (hLIF, PeproTech, 300-05), 25ng / mL activin A. A, PeproTech, 120-14E), 10 ng / mL recombinant human basic fibroblast growth factor (bFGF, PeproTech, 100-18B), 2.5 μM IWR-1 (WNTi, Selleckchem, S7086), 1 μM CHIR99021 (GSK3α / β, Selleckchem, S1263), 1 μM WH-4-023 (LCK / SRCi, Selleckchem, S7565) and 5 μM Y-27632 (ROCK inhibitor, Selleckchem, S1049), with the balance being DMEM / F12 (Thermo Fisher, 10565018) and Neurobasal medium (Thermo Fisher, 21103049). The volume ratio of medium is 1:1, all percentages mentioned above are volume percentages, and the contents of each substance mentioned above are the final contents of the substance in 3i / LAF medium.

[0089] Porcine gastrulation epiblast stem cell lines (referred to as pEpiSCs in this patent): described in the non-patent literature “Zhi, ML et al. Generation and characterization of stable pig pregastrulation epiblast stem cell lines. Cell Res. 32, 383-400 (2022)”, and named pgEpiSCs in the aforementioned literature, are available to the public from China Agricultural University. This biomaterial is only for repeating the relevant experiments of this invention and may not be used for other purposes.

[0090] Undifferentiated porcine epiblast stem cell lines (pEpiSCs) were seeded onto MEF feeder cells obtained in step 1, MEF feeder cell preparation, for maintenance growth. The culture medium used was 3i / LAF, which was replaced daily with fresh 3i / LAF. Enzymatic passages were performed every 2-3 days with Accutase (Thermo Fisher, A1110501) for 3-5 minutes at a 1:3 ratio. Small clumps or single cells were reseeded onto new feeder cells. Sex of each cell line was determined by sex-specific PCR, using the following method: Using genomic DNA as a template, cell lines that amplified a 400bp fragment with pig SRY-F and pig SRY-R (primer information as described above) and amplified a 506bp fragment with pig ZFX-F and pig ZFX-R (primer information as described above) were male; cell lines that could not amplify a 400bp fragment with pig SRY-F and pig SRY-R but amplified a 506bp fragment with pig ZFX-F and pig ZFX-R were female. The identification results are shown below. Figure 2 Lane 1 was used for DL2000 marker detection, lanes 2 and 4 for pig ZFX gene detection, and lanes 3 and 5 for pig SRY gene detection. Male porcine epiblast stem cell lines (pEpiSCs) were selected for subsequent experiments, and will be referred to as pEpiSCs in subsequent experiments.

[0091] 3. Direct induction of porcine primordial germ cell-like cells (pPGCLCs) in existing technologies aRB27 basal medium composition: 1×B27 supplement, 1% penicillin-streptomycin, 1% GlutaMax, and 0.1 mM β-mercaptoethanol, with the balance being Advanced RPMI 1640 medium (Thermo Fisher, 12633012). The percentages mentioned above are volume percentages.

[0092] The B27 supplement is a product of Thermo Fisher Scientific, catalog number 12587010, with a concentration of 50×.

[0093] The PGC induction medium consists of: 200-500 ng / mL BMP4, 10 ng / mL hLIF, 100 ng / mL stem cell factor (SCF, PeproTech product, catalog number 300-07), 50 ng / mL epidermal growth factor (EGF, PeproTech product, catalog number AF-100-15), and 10 μM Y-27632, with the remainder being aRB27 basal medium. All the contents of the aforementioned substances are final contents.

[0094] To clarify the germline differentiation capacity of pEpiSCs, they were first cultured in a medium containing cytokines such as BMP4 for 96 hours (h), and the differentiation of pPGCs was induced using a three-dimensional (3D) culture method. Figure 3 (a) and (b) were repeated three times, with each repetition as follows: pEpiSCs were digested into single cells, resuspended in PGC medium at a density of 3000 cells / 100 μL, and 100 μL was seeded per well. The cells were then cultured at 37°C with 5% CO2. During the culture process, the cells aggregated at the bottom of the U-shaped section of the 96-well plate, forming spherical structures. The results were observed at 24 hours (i.e.,...). Figure 3 D1 in c) 48h (i.e.) Figure 3 D2 in c) 72h (i.e.) Figure 3 D3 in c) and 96h (i.e. Figure 3 Cells are collected from D4 in c.

[0095] Total RNA was extracted from collected cells using the RNAprep Cell / Bacteria Kit (Tiangen, DP430) according to the manufacturer's instructions. cDNA was reverse transcribed using the 5×All-In-One RT Master Mix Kit (abm, G486). Quantitative PCR was performed using 2×RealStar Power SYBR Mixture (GeneStar, A311) on an Archimed X6 instrument (Kunpeng Gene). Changes in the expression levels of the genes pig NANOS3, pig SOX17, pig PRDM1, pig TFAP2C, and pig OCT4 in the collected cells were detected. Primer sequences were as described above. Each sample was tested three times, and normalization was performed using GAPDH and EEF1A1 as housekeeping genes.

[0096] The results showed that the cells obtained by the above three-dimensional (3D) culture method, SOX17 or NANOS3 The expression was not upregulated. Figure 3 c) indicates that pEpiSCs were not induced into porcine primordial germ cell-like cells by the aforementioned three-dimensional (3D) culture method.

[0097] Example 2: Method for inducing porcine primordial germ cell-like cells (pPGCLCs) provided by the present invention I. Targeted Integrated Fluorescence Reporting System 1. Construction of the cutting carrier Design three CRISPR-specific lines for pigs using the online design tool (http: / / crispr.mit.edu). SOX17 or NANOS3Single guide RNAs (sgRNAs) near the stop codon include sgRNA1, sgRNA2, and sgRNA3.

[0098] The primer pairs used for target vector construction mentioned above (Fw and Rv of pSOX17-sgRNA1, Fw and Rv of pSOX17-sgRNA2, Fw and Rv of pSOX17-sgRNA3, Fw and Rv of pNANOS3-sgRNA1, Fw and Rv of pNANOS3-sgRNA2, and Fw and Rv of pNANOS3-sgRNA3) were annealed to obtain six annealing products. The six annealing products were cloned into nucleotides 6853 to 6875 of pX330-puro (the sequence of which is described in "nucleotide sequence of pX330-puro plasmid" below) to obtain the cleavage plasmids pX330-sgRNA1, pX330-sgRNA2, pX330-sgRNA3, pX330-sgRNA4, pX330-sgRNA5 and pX330-sgRNA6.

[0099] The cleavage plasmid pX330-sgRNA1 is a cleavage expression vector obtained by replacing nucleotides 6853 to 6875 of pX330-puro with the Fw and Rv sequences of pSOX17-sgRNA1 through annealing. The other sequences of pX330-puro remain unchanged. The DNA molecule formed by annealing the Fw and Rv sequences of pSOX17-sgRNA1 encodes an sgRNA targeting the porcine SOX17 gene. The nucleotide sequence of this sgRNA is 5'-GUAACUACCCCGACGUCUGA-3', targeting nucleotides 76855151 to 76855170 of the porcine SOX17 gene (NCBI Reference Sequence: NC_010446.5, CON 12-JAN-2018).

[0100] The cleavage plasmid pX330-sgRNA2 is a cleavage expression vector obtained by replacing nucleotides 6853 to 6875 of pX330 with the Fw and Rv sequences of pSOX17-sgRNA2 through annealing. The DNA molecule formed by annealing the Fw and Rv sequences of pSOX17-sgRNA2 encodes an sgRNA targeting the porcine SOX17 gene. The nucleotide sequence of this sgRNA is 5'-UAACUACCCCGACGUCUGAC-3', targeting nucleotides 76855150 to 76855169 of the porcine SOX17 gene.

[0101] The cleavage plasmid pX330-sgRNA3 is a cleavage expression vector obtained by replacing nucleotides 6853 to 6875 of pX330 with the Fw and Rv sequences of pSOX17-sgRNA3 and annealing them. The DNA molecule formed by annealing the Fw and Rv sequences of pSOX17-sgRNA3 encodes an sgRNA targeting the porcine SOX17 gene. The nucleotide sequence of this sgRNA is 5'-GAUGGGCACCCGUCAGACGU-3', targeting nucleotides 76855139 to 76855158 of the porcine SOX17 gene.

[0102] The cleavage plasmid pX330-sgRNA4 is a cleavage expression vector obtained by replacing nucleotides 6853 to 6875 of pX330 with the Fw and Rv of pNANOS3-sgRNA1 through annealing. The DNA molecule formed by annealing the Fw and Rv of pX330 is kept unchanged. The DNA molecule formed by annealing the Fw and Rv of pNANOS3-sgRNA1 encodes an sgRNA targeting the porcine NANOS3 gene. The nucleotide sequence of this sgRNA is 5'-AAAGGUGCCGGGAAGUCUUC-3', targeting nucleotides 65275496 to 65275515 of the porcine NANOS3 gene (NCBI ReferenceSequence:NC_010444.4, REGION:65274249-65279289, CON 12-JAN-2018).

[0103] The cleavage plasmid pX330-sgRNA5 is a cleavage expression vector obtained by replacing nucleotides 6853 to 6875 of pX330 with the Fw and Rv of pNANOS3-sgRNA2 through annealing. The rest of the pX330 sequence remains unchanged. The DNA molecule formed by annealing the Fw and Rv of pNANOS3-sgRNA2 encodes an sgRNA targeting the porcine NANOS3 gene (NCBI ID: NC_010444, REGION: 65274249-65279289). The nucleotide sequence of this sgRNA is 5'-CCUAAGAGGCUGGCGCGAGC-3', targeting nucleotides 65275438 to 65275457 of the porcine NANOS3 gene.

[0104] The cleavage plasmid pX330-sgRNA6 is a cleavage expression vector obtained by replacing nucleotides 6853 to 6875 of pX330 with the Fw and Rv of pNANOS3-sgRNA3 through annealing. The rest of the pX330 sequence remains unchanged. The DNA molecule formed by annealing the Fw and Rv of pNANOS3-sgRNA3 encodes an sgRNA targeting the porcine NANOS3 gene (NCBI ID: NC_010444, REGION: 65274249-65279289). The nucleotide sequence of this sgRNA is 5'-UCCACUUCUGCCUAAGAGGC-3', targeting nucleotides 65275448 to 65275467 of the porcine NANOS3 gene.

[0105] 2. Cell transfection The aforementioned cleavage plasmids were transiently transfected into the male pEpiSCs cells prepared in Example 1 using Lipofectamine 3000 (Thermo Fisher, L3000150), with each plasmid replicated in two wells. 24 hours after transfection, the medium was replaced with fresh 3i / LAF medium, and puromycin (Puro, Thermo Fisher, A1113803) was added to the 3i / LAF medium at a final concentration of 0.5 μg / mL for selection. After culturing for 3 days, pEpiSCs cells transfected with the plasmids were obtained.

[0106] 3. Cutting efficiency verification According to the instructions, genomic DNA was extracted from pEpiSCs cells transfected with plasmids as described in "2. Cell Transfection" using the TIANamp Genomic DNA Extraction Kit (TIANGEN, DP316). PCR primers (pSOX17-sgRNA test, pNANOS3-sgRNA test, primers as described above) were designed to generate amplified fragments containing sgRNA target sites, and mutations were detected by Sanger sequencing. The PCR products were further digested with T7 endonuclease I (T7E1). The digestion system consisted of: 200-500 ng of gel-recovered DNA, 2 μL of 10×NEBuffer2, and 20 μL of deionized water. The annealing program was: 95℃ for 5 min, 95℃ for 5 sec, -2℃ / cycle, 85℃ for 5 sec, -0.1℃ / cycle, 25℃. After heterodimer formation, 0.5 μL of T7E1 was added, mixed thoroughly, and incubated at 37℃ for 1 h. Enzyme digestion products can be detected by 2% agarose gel electrophoresis or by the more sensitive 12% acrylamide gel electrophoresis. The gel electrophoresis bands are analyzed using ImageJ software to calculate the digestion efficiency.

[0107] Sequencing results showed that pSOX17-sgRNA1 and pNANOS3-sgRNA2 had higher sequencing peaks ( Figure 4 In the diagram, 'a' represents pSOX17-sgRNA1 to pSOX17-sgRNA3, and 'c' represents pNANOS3-sgRNA1 to pNANOS3-sgRNA3. Further enzyme digestion results confirmed that the cleavage efficiency of pSOX17-sgRNA1 was 51.6%, pSOX17-sgRNA2 was 28.2%, and pSOX17-sgRNA3 was 40.1%. Figure 4 (b); Therefore, pSOX17-sgRNA1 and pNANOS3-sgRNA2, which have high cleavage efficiency, were selected for further research.

[0108] 4. Donor plasmid construction Homologous arms of the SOX17 gene (5'arm: 1116bp, 3'arm: 842bp) and the NANOS3 gene (5'arm: 693bp, 3'arm: 699bp) were obtained by PCR amplification. Wild-type pEpiSCs genomic DNA was used as a template, and amplification was performed using PrimeSTAR GXL DNA polymerase (TaKaRa, R050A). The required primers included Fw and Rv of pSOX17-5'arm, Fw and Rv of pSOX17-3'arm, Fw and Rv of pNANOS3-5'arm, and Fw and Rv of pNANOS3-3'arm (as described above).

[0109] The nucleotide sequence of the amplified pSOX17-5'arm is nucleotides 76855154 to 76856269 of NCBI genbank number NC_010446 REGION (update: 12-JAN-2018).

[0110] The nucleotide sequence of the amplified pSOX17-3'arm is: nucleotides 76854312 to 76855153 from NCBI genbank number NC_010446.5 (update: 12-JAN-2018).

[0111] The nucleotide sequence of the amplified pNANOS3-5'arm is nucleotides 65275456 to 65276148 of NCBI GenBank number NC_010444.4 (update: 12-JAN-201).

[0112] The nucleotide sequence of the amplified pNANOS3-3'arm is nucleotides 65274749 to 65275447 in NCBI genbank number NC_010444.4 (update: CON 12-JAN-2018).

[0113] Subsequently, the 5'arm and 3'arm of the amplified pSOX17 from the genome were recombined into the plasmid donor OCT4-A5-3×Flag-PNP-tdtomato-3' (the sequence of which is described in "nucleotide sequence of donor OCT4-A5-3×Flag-PNP-tdtomato-3' plasmid" below) by enzyme digestion and ligation, thereby obtaining the donor plasmids (donor pSOX17-5'arm-3×Flag-PNP-tdtomato-3'arm and donor pNANOS3-5'arm-3×Flag-PNP-tdtomato-3'arm).

[0114] The donor plasmid donor pSOX17-5'arm-3×Flag-PNP-tdtomato-3'arm is a recombinant vector obtained by replacing nucleotides 17-1183 and 4192-4220 of the plasmid donor OCT4-A5-3×Flag-PNP-tdtomato-3' with pSOX17-5'arm and pSOX17-3'arm respectively, while keeping the other nucleotides of the donor OCT4-A5-3×Flag-PNP-tdtomato-3' plasmid unchanged.

[0115] Donor plasmid pNANOS3-5'arm-3×Flag-PNP-tdtomato-3'arm: This is a recombinant vector obtained by replacing nucleotides 17-1183 and 4192-4220 of plasmid donor OCT4-A5-3×Flag-PNP-tdtomato-3' with pNANOS3-5'arm and pNANOS3-3'arm respectively, while keeping the other nucleotides of donor OCT4-A5-3×Flag-PNP-tdtomato-3' plasmid unchanged.

[0116] 5. Construction of the pEpiSC system for SOX17-tdTomato and NANOS3-tdTomato key-in reports The aforementioned pEpiSCs identified as male were washed with DPBS and then dissociated using Accutase at 37°C for 5 min. Dispersed single cells were seeded onto DR4 MEF feeder cells (purchased from Thermo Fisher, catalog number A34966) for 6-8 hours (DR4 MEF feeder cells were pre-cultured in MEF medium for 12 hours). The medium was then replaced with antibiotic-free medium. Lipofectamine 3000 and 2 μg DNA (1 μg pX330-sgRNA plasmid (cutting plasmid pSOX17-sgRNA1 or cutting plasmid pNANOS3-sgRNA2) were gradually mixed with OptiMEM (Thermo Fisher, 31985070) and then added to pEpiSCs. 24 hours post-transfection, cells were screened with 0.5 μg / mL puromycin for 7 consecutive days. Viable single clones of pEpiSCs were manually picked using pipettes and amplified for 10-14 days. Genomic DNA was extracted from the gene-edited cells, and site-specific integration of the 5' and 3' ends of the targeting vector was examined. Using 1 μL of DNA as a template, 0.5 μL PrimeSTAR GXL DNA polymerase (TaKaRa, R050A), 5 μL 5×PrimeSTAR GXL buffer, 2 μL dNTP mixture, 0.5 μL 10 μM forward primer, 0.5 μL 10 μM reverse primer, and 12 μL nuclease-free water were added for PCR. The reaction program was as follows: 98℃ for 10 s, 55℃ or 60℃ for 15 s, and 68℃ for 1 min / kb for 35 cycles. Gel electrophoresis confirmed that the size of the PCR product matched the expected amplicon size. The primer pairs in the aforementioned system include the Fw and Rv of pSOX17-3×Flag-P2A-NLS-tdTomato-puro and the Fw and Rv of pNANOS3-3×Flag-P2A-NLS-tdTomato-puro (primer information as described above).

[0117] Two homozygous SOX17 knock-in pEpiSCs clones were obtained (numbered 5 and 6). Figure 5 a) and eight NANOS3 homozygous knock-in pEpiSCs clones (numbered 2, 4, 7, 8, 10, 15, 16 and 17, Figure 5 (b) (respectively named) SOX17 - P2A - tdTomato ( STpEpiSCs cells and NANOS3 - P2A - tdTomato ( N3T pEpiSCs cells.

[0118] Compared to male pEpiSCs, SOX17 - P2A - tdTomato ( ST The pEpiSCs cells have a 3×Flag-P2A-NLS-tdTomato-loxP-pgk-puro-loxP fragment inserted before the SOX17 gene stop codon in the male pEpiSCs genome (nucleotides 76855153 to 76855154 of plasmid donor OCT4-A5-3×Flag-PNP-tdtomato-3', nucleotides 1179-4196). This will be discussed later. SOX17 - P2A - tdTomato ( ST pEpiSCs cells are abbreviated as pEpiSCs. ST pEpiSCs cells.

[0119] Compared to male pEpiSCs, NANOS3 - P2A - tdTomato ( N3T The pEpiSCs cells have a 3×Flag-P2A-NLS-tdTomato-loxP-pgk-puro-loxP fragment inserted before the NANOS3 gene stop codon (nucleotides 65275455 to 65275456 of plasmid donor OCT4-A5-3×Flag-PNP-tdtomato-3') in the male pEpiSCs genome (NCBI number: NC_010444.4).

[0120] 6. SOX17 - P2A - tdTomato ( ST Functional identification of pEpiSCs (1) Alkaline phosphatase staining Logarithmic growth phase ST pEpiSCs cells were stained using the alkaline phosphatase staining method described above, and the results are as follows: Figure 6 As shown in Figure a, ST pEpiSCs cell clones maintain alkaline phosphatase (ALP) positivity.

[0121] (2) Karyotype analysis Analysis using the karyotype analysis method described above ST The karyotype of pEpiSCs cells showed that... ST pEpiSCs cells possess a normal male karyotype (37+XY). Figure 6 (b)

[0122] (3) Gene expression level analysis The detection was performed according to the method described in "3. Direct induction of porcine primordial germ cell-like cells (pPGCLCs) in the prior art" of this embodiment. ST pEpiSCs cells NANOG Gene, OCT4 Gene, SOX2 The relative expression levels of genes were detected using primers including pig NANOG-Fw and pig NANOG-Rv, pig OCT4-Fw and pig OCT4-Rv, and pig SOX2-Fw and pig SOX2-Rv.

[0123] The results are as follows Figure 6 As shown in c, SOX17 - P2A - tdTomato ( ST pEpiSCs NANOG Gene, OCT4 Gene, SOX2 The genes remained unchanged compared to wild-type cells and can be used for subsequent analysis.

[0124] (4) Immunofluorescence The immunofluorescence method described above was used to... ST pEpiSCs cells were stained with primary antibodies NANOG, OCT4, and SOX2. Results are as follows: Figure 6 As shown in d, ST pEpiSCs cells express the core pluripotency transcription factors NANOG, OCT4, and SOX2, indicating that they maintain their pluripotency characteristics.

[0125] (5) Verification of endoderm differentiation The composition of the endoderm (DE) induction medium was: 10 ng / mL bone morphogenetic protein 4 (BMP4, PeproTech, 120-05), 5 μM SB431542 (Selleckchem, S1067) and 10 ng / mL bFGF, with the remainder being the aforementioned aRB27 basal medium.

[0126] Will STAfter culturing pEpiSCs cells in 3i / LAF medium for 48 hours, the medium was aspirated, the cells were washed with DPBS, and the medium was replaced with endoderm induction medium for 2-3 more days.

[0127] The results showed that, ST tdTomato fluorescence was detected in pEpiSC-derived cells that expressed GATA4 but not OCT4. Figure 6 (e). Immunofluorescence (IF) experiments further confirmed the co-localization expression of tdTomato and SOX17 or 3×Flag in the nuclei of DE cells, indicating that SOX17-P2A-tdTomato fluorescence accurately reflects the endogenous expression pattern of the SOX17 gene. Figure 6 (f)

[0128] two, ST pEpiSCs differentiate into mesoendothelial progenitor cells (pMEPCs) in vitro. The composition of the mesendoderm (ME) induction medium was as follows: 50 ng / mL Activin A (50-100 ng / mL is acceptable, 50 ng / mL in this example), 0.3 μM WNT agonist (CHIR99021) (0.3-1 μM is acceptable, 0.3 μM in this example), and 10 μM ROCK inhibitor (Y-27632) (5-10 μM is acceptable, 10 μM in this example), with the remainder being the aforementioned aRB27 basal medium.

[0129] The experiment was repeated 3 times, with each repetition as follows: Will ST After washing with DPBS, pEpiSCs were dissociated using Accutase at 37°C for 5 min. The dispersed single cells were seeded onto CD1 MEF feeder cells (CD1 MEF feeder cells were pre-cultured in MEF medium for 12 h), dissociated into small clumps using Accutase, centrifuged at 1000 rpm for 3 min, and then seeded into gelatin-coated culture dishes. Feeder cells were removed by differential adhesion. The collected cells were... ST pEpiSCs were re-inoculated into gelatin-coated culture dishes and cultured as a monolayer in mesendoderm (ME) induction medium. Mesendoderm (ME) differentiation was monitored every 6 hours. Figure 7 (a) The specific methods for monitoring the differentiation of the mesoderm are as follows: 1. ST Bright-field images and real-time ST fluorescence images of pEpiSCs differentiated through monolayer culture. Figure 7 b is ST Bright-field images and real-time ST fluorescence images of pEpiSCs during monolayer culture differentiation into ME were captured. Analysis was performed using software. Figure 7 The fluorescence intensity of SOX17 in b is shown in the results. Figure 7 As shown in Figure c, the relative strength of SOX17 increases significantly with increasing differentiation time.

[0130] 2. Flow cytometry analysis of pEpiSCs and STs during differentiation + Cell ratio After differentiation ST pEpiSCs cells were washed with DPBS and dissociated with 0.25% trypsin-EDTA. Single cells were resuspended in pre-chilled DPBS containing 1% BSA and passed through a 40 μm filter. The cell suspension was sorted and analyzed using a MoFlo® high-performance cell sorter (Beckman Coulter). FACS data were analyzed using Summit 5.2 software (Dako Cytomation, Copenhagen, Denmark) and Flowjo 10.1r5 (BD Biosciences). All experimental replicates used the same gating parameters.

[0131] The results are as follows Figure 7 As shown in d, with the increase of differentiation time, ST The proportion of SOX17-tdTomato-positive cells was significantly increased in pEpiSCs cells.

[0132] 3. Analyze the mRNA expression levels of SOX17, core pluripotency markers, and original streak-related genes during ME differentiation. The experiment was repeated three times, with the specific method for each instance as follows: Referring to the method shown in "3. Direct induction of porcine primordial germ cell-like cells (pPGCLCs) in the prior art" in Example 1, the ME differentiation process was detected. SOX17 , SOX2 , OCT4 , NANOG , EOMES , GSC , TBXT (Brachyury) , GATA4 , FOXA2 , PDGFRA , PRDM1 , TET2 and DNMT3B The relative expression level changes.

[0133] The results are as follows Figure 7 As shown in Figure e, with the increase of differentiation time, SOX17 The relative expression level gradually increased. SOX2 , OCT4 and NANOG The relative expression level first increases and then gradually decreases. Figure 7 The results showed that the original marker gene EOMES and GSC The expression was moderately adjusted upwards, while GATA4 , FOXA2 , PDGFRA , PRDM1 and TET2 The expression of methyltransferases is induced in the later stages of ME differentiation. DNMT3B The expression is then downgraded. It is known that... TBXT (Brachyury) TBXT expression is a common characteristic that determines the fate of germ cells (GCs). The inventors observed that TBXT expression peaked at approximately 36 hours during ME differentiation, after which it gradually decreased. Figure 7 (e).

[0134] 4. Sequential co-immunofluorescence staining The experiment was repeated 3 times, with the following steps repeated each time: Refer to the cell immunofluorescence analysis method described above to detect ST-positive cells during ME differentiation ( SOX17 - P2A - tdTomato ( ST pEpiSCs gradually express SOX17 during differentiation, leading to tdTomato Cells that test positive at this stage are called ST-positive cells, or simply ST cells. + The expression of OCT4, SOX2, and PRDM1 in ST-positive cells was analyzed, and the expression of NANOG and TBXT was detected separately. Furthermore, the expression of EOMES, GSC, GATA4, HAND1, and NANOG in ST-positive cells at 36 hours was examined.

[0135] The results are as follows Figure 7 As shown in gk, relative to male pEpiSCs cells, starting from 24h, OCT4 + SOX17 expression in cells gradually increases ( Figure 7 (g and h), while SOX2 expression decreased sharply ( Figure 7 The expressions for i and j in the NANOOG expression remain largely unchanged. Figure 7 The "l" and "m" in the text indicate that differentiated cells, over time, exit pluripotency and enter a mesoendodermal fate rather than an ectoderm fate (e.g., ...). Figure 7 (As shown in Figure k). Notably, the proportion of cells co-localized with SOX17 and OCT4 reached its highest level at 36 h, at which time TBXT was also highly expressed ( Figure 7 (l and m in the text), and also, with ST +As the number of cells gradually increased, PRDM1 expression peaked at 36 hours. Figure 7 (l and m in the text).

[0136] Further staining at 36 hours revealed ST + The cells were positive for OCT4, NANOG, PRDM1, EOMES, TBXT, GSC, and GATA4; therefore, the cells cultured for 36 hours were... SOX17 - P2A - tdTomato ( ST pEpiSCs cells are called pMEPCs cells ( Figure 7 (n) in it has the ability to induce PGC.

[0137] 5. Conventional transcriptome sequencing collect ST pEpiSCs and their differentiated pMEPCs were sent to Beijing Novogene Technology Co., Ltd. for routine transcriptome sequencing analysis. Results are as follows: Figure 7 As shown in the middle (o), which is a scatter plot, the average gene expression levels of pEpiSCs (left) and pMEPCs (right) are compared. (Adjusted) p Differentially expressed genes (DEGs) were identified using thresholds padj < 0.05 and absolute log2-fold change ≥ 1. Red indicates upregulated genes (log2-fold change ≥ 1), blue indicates downregulated genes (log2-fold change ≤ -1), and gray indicates insignificant genes. Results showed that pMEPCs upregulated the expression of 1112 genes (119 transcription factors), including... SOX17 , EOMES , MIXL1 and BMP4 They play a central role in early mesodermal differentiation; meanwhile, the expression of 1086 genes (101 transcription factors) is specifically downregulated. Figure 7 (middle o). Figure 7 As shown in p, these genes are enriched with genes related to "epithelial-mesenchymal transition", "mesoderm formation", "anteroposterior axis pattern specialization", "tubular morphogenesis", "gastrulism", "endoderm development", and "focal adhesion". The results indicate that the genes cultured for 36 hours... SOX17 - P2A - tdTomato ( ST pEpiSCs cells are similar to SOX17-positive pMEPCs in E12-E13 EPIs in vivo.

[0138] III. pMEPCs are further induced into pPGCLCs Includes the following steps: Step 1, ST pEpiSCs cells follow the "two, SOX17 - P2A - tdTomato ( ST pEpiSCs were cultured for 36 hours using the method described above for in vitro differentiation into mesoendothelial precursor cells (pMEPCs). (During this stage, the cells were identified as pMEPCs according to the aforementioned description.) Step 2: Replace the culture medium of the cells from Step 1 with PGC induction medium (see Example 1), and culture for 48 hours. During this period, every 6 hours, identify whether the aforementioned pMEPCs cells have differentiated into pPGCLCs using the following method: 1. Temporal analysis of mRNA expression levels of pluripotency markers and early PGCs markers during pPGCLC differentiation. like Figure 8 As shown in Figures a and b, the expression of red fluorescence was observed every 6 hours, and cells were collected once. The expression of the genes pig OCT4, pig NANOG, pig STELLA, pig KLF4, pig NANOS3, pig SOX17, pig PRDM1, pig TFAP2C, pig PRDM14, pig DNMT3B, pig TET2, and pig TFCP2L1 was detected according to the method described in "3. Direct Induction of Porcine Primitive Germ Cell-like Cells (pPGCLCs) in the Prior Art" in Example 1. Primers were as described above.

[0139] The results showed that starting at 6 hours, STELLA and TET2 The expression of pluripotency markers gradually increased, while pluripotency markers such as NANOG , OCT4 , KLF4 , PRDM14 and DBNT3B The expression of these genes decreased significantly. However, the expression of primordial pluripotency genes... TFCP2L1 The expression of this substance can only be detected in cells 24 hours after differentiation. Figure 8 (c). Simultaneously, starting from 6 hours, cells express early germ cell-specific genes ( NANOS3 , SOX17 , PRDM1 and TFAP2C () Figure 8 (c)

[0140] 2. Flow cytometry analysis of ST in 24-hour pPGCLCs + Cell ratio Will STpMEPCs differentiated for 24 h were washed with DPBS and dissociated with 0.25% trypsin-EDTA. Single cells were resuspended in pre-chilled DPBS containing 1% BSA and passed through a 40 μm filter. The cell suspension was sorted and analyzed using a MoFlo® high-performance cell sorter (Beckman Coulter). FACS data analysis was performed using Summit 5.2 software (Dako Cytomation, Copenhagen, Denmark) and Flowjo 10.1r5 (BD Biosciences). All experimental replicates used the same gating parameters.

[0141] The results are as follows Figure 8 As shown in d and e, after 24 hours of treatment, the proportion of SOX17-tdTomato positive cells remained at a high level of 50.5%.

[0142] 3. Protein expression level detection First use ST Time-series analysis of pMEPCs during culture and differentiation process, ST + The co-expression of cells with OCT4, PRDM1, and TFAP2C proteins was used to identify true pPGCLCs.

[0143] like Figure 8 As shown in f and g, although the expression level of OCT4 decreased during differentiation, most OCT4-positive cells remained consistent with ST. + Cell colocalization, and simultaneously, dual fluorescence detection results based on ST / PRDM1 (fluorescence results of tmotdo and PRDM1) and ST / TFAP2C (fluorescence results of tmotdo and TFAP2C). Figure 8 (h and i in the text), the inventors believe that the ST differentiates 24h. + The cells should be assumed to be pPGCLCs.

[0144] Secondly, ST cultured for 24 hours after replacing the PGC induction medium... + The expression of TNAP, NANOS3, and SOX17 in the cells (i.e., ST pPGCLCs cultured for 60 h) was analyzed using the aforementioned immunofluorescence assay. Simultaneously, the previously constructed NANOS3-tdTomato cell line was used for replication and validation experiments.

[0145] Most ST + pPGCLCs were positive for NANOS3, the mitotic activity marker (MKI67), and TNAP. Figure 8 Using the same conversion method, the inventors obtained NANOS3-3×Flag-P2A-NLS-tdTomato+ (N3T)pPGCLCs, these cells exhibit ALP activity ( Figure 8 In addition, SOX17, OCT4, NANOOG, and PRDM1 are co-expressed, but SOX2 expression is low or absent. Figure 8 (l).

[0146] 4. Epigenetic level testing Experimental methods: Bisulfite sequencing: Genomic DNA was extracted from pEpiSCs and pPGCLCs using the TIANamp Genomic DNA Kit (Tiangen, DP304). DNA was bisulfite-treated using the EZ DNA Methylation Kit (Zymo Research, D5005). Nested PCR amplification was performed using HotStart DNA polymerase (TAKARA, R007Q). Specific primers for the H19 / IGF2 and SNRPN DMR imprinted regions were as described previously. The PCR products were cloned into the pMD19T vector (TAKARA, 6013) and transformed into *E. coli*, with at least 20 insertion-positive clones sequenced. The bisulfite cytosine conversion rate was greater than 98%. The obtained data were analyzed using an online methylation analysis and quantification tool (QUMA, http: / / quma.cdb.riken.jp / ).

[0147] The results showed that the inventors compared pEpiSCs and pPGCLCs in terms of imprinted genes. H19 / IGF2 and SNRPN Methylation status of differentially methylated regions (DMRs). Bisulfite sequencing results showed that the methylation level of pEpiSCs was approximately 70% to 80%, while the level of pPGCLCs dropped sharply to half of that, indicating a significant DNA demethylation process. Figure 8 (m).

[0148] The fluorescence of 5-methylcytosine (5mC), methylcytosine dioxygenase 2 (TET2), H3K27me3, and H3K9me2 was detected using the aforementioned immunofluorescence assay. Immunofluorescence analysis of methylcytosine dioxygenase 2 (TET2) and its product 5-methylcytosine (5mC) showed that differentiated pPGCLCs exhibited low levels of overall DNA methylation. Figure 8 (n, o, p, and q). Furthermore, these cells also showed significant changes in histone modification levels, manifested as a decrease in H3K9me2 levels and an increase in H3K27me3 levels (n, o, p, and q). Figure 8(r, s, t, and u). In summary, the inventors' results demonstrate that pEpiSC-derived pPGCLCs undergo genome-wide epigenetic reprogramming.

[0149] 5. Conventional transcriptome and single-cell transcriptome sequencing Experimental Methods: pEpiSCs and their differentiated pMEPCs and pPGCLCs were collected and sent to Beijing Novogene Technology Co., Ltd. for routine transcriptome sequencing analysis. Single-cell RNA sequencing libraries were prepared using a modified Smart-seq2 method. Briefly, single cells were transferred via pipette to lysis buffer containing 8-base barcodes. Subsequently, first-strand cDNA was synthesized and amplified in reverse transcription (RT) using a mixture containing 4U RNase inhibitor, 100U SuperScript II reverse transcriptase (Invitrogen, 18064071), 1M mdNTPs (TAKARA, 4019), 60mM magnesium chloride, 3μM RT primers, and 10μM TSO primers. After PCR amplification, the products were purified using 0.8×AMPure XP magnetic beads (Beckman, A63882). Biotin PCR was then performed for enrichment. Finally, a single-cell RNA sequencing library was constructed according to the instructions for the KAPA Hyper PrepKit series (KAPA, KK8054). The high-quality library was sequenced at 150 bp paired ends using Illumina NovaSeq X Plus (Novogene).

[0150] Plain transcriptome analysis showed that differential gene heatmaps and correlation coefficient heatmaps indicated that pPGCLCs and pMEPCs were more similar than pEpiSCs. Figure 9 (a and b in the text). Figure 9 The c-analysis results showed that differentially expressed genes that were significantly upregulated during pPGC specialization included SOX17 , PRDM1 , CXCR4 , ITGB3 and DAB2 These genes are significantly enriched in the extracellular space, calcium ion binding, extracellular matrix, and cell migration involved in gastrulation. Figure 9 (d). To further elucidate the differentiation trajectory of pPGCLCs, the cell types involved in the differentiation process of pPGCLCs (pEpiSCs, ST) were analyzed. + pMEPCs, D2.5 somatic cells and N3T +Single-cell RNA sequencing analysis was performed on pPGCLCs. The results showed that in the UMAP diagram, pEpiSCs roughly corresponded to porcine epiblast cells (pEPIs) at E10-E11 stages, pMEPCs clustered with pEPIs at E12-E13 stages, somatic cells at D2.5 stage were similar to somatic cells at E14 stage, while pPGCLCs were equivalent to early PGCs at E14 stage in vivo. Figure 9 (e). Furthermore, the expression of key marker genes was consistent with the inventors' cell annotation results ( Figure 9 (f)

[0151] In conclusion, SOX17 - P2A - tdTomato pEpiSCs cells ( ST pEpiSCs cells differentiated into pMEPCs after being induced and cultured in mesoendothelial induction medium for 36 hours. pMEPCs then differentiated into pPGCLCs after being induced and cultured in PGC induction medium for 24 hours.

[0152] It should be understood that constructing male pEpiSCs cells as SOX17 - P2A - tdTomato ( ST pEpiSCs or NANOS3 - P2A - tdTomato ( N3T The pEpiSCs cells are used to determine or assist in determining the cell differentiation stage by using the expression or expression level of the marker proteins SOX17 and NANOS3. However, this should not be used to limit the methods provided in this invention for inducing pMEPCs into pPGCLCs, pEpiSCs into pPGCLCs, or pEpiSCs into pMEPCs.

[0153] pMEPCs have at least the following characteristics: high expression of SOX17 protein and ability to differentiate into primordial germ cells.

[0154] pPGCLCs possess at least the following characteristics: morphology, gene expression, epigenetic features, and developmental potential similar to primordial germ cells in an in vivo day 14 embryo.

[0155] Example 3: Transplanting pPGCLCs into the testes of a NANOS3-deficient pig model resulted in the production of sperm-like cells. 1. NANOS3 - P2A - tdTomato ( N3T pEpiSCs induce pPGCLCs Will NANOS3 - P2A - tdTomato ( N3T After washing with DPBS, pEpiSCs were dissociated using Accutase at 37°C for 5 min. The dispersed single cells were seeded onto CD1 MEF feeder cells (CD1 MEF feeder cells were pre-cultured in MEF medium for 12 h), dissociated into small clumps using Accutase, centrifuged at 1000 rpm for 3 min, and then seeded into gelatin-coated culture dishes. Feeder cells were removed by differential adhesion. The collected cells were... NANOS3 - P2A - tdTomato ( N3T pEpiSCs were re-inoculated into gelatin-coated culture dishes and cultured in a monolayer in mesendoderm (ME) induction medium. After 36 h of culture, the medium was replaced with PGC induction medium and cultured for 24 h to obtain pPGCLCs.

[0156] 2. Transplanting pPGCLCs into the testes of NANOS3-deficient pig models. Preparation for one transplant experiment (bilateral testes from recipient pigs) requires approximately 1-2 × 10⁻⁶ testes. 8 In this embodiment, "1, NANOS3 - P2A - tdTomato ( N3T pEpiSCs induced to become pPGCLCs were the pPGCLCs obtained.

[0157] 1. NANOS3 - P2A - tdTomato ( N3T pEpiSCs were induced into pPGCLCs. The resulting pPGCLCs were treated with 0.125% trypsin-EDTA at 37°C for 5 min, and the reaction was terminated by adding an equal volume of neutralizing medium. Single cells were collected and resuspended in aRB27 basal medium (see Example 1) to a final concentration of 3-5 × 10⁻⁵ cells / mL. 7 1 cell / mL (i.e., pPGCLCs suspension), store on ice until the start of the injection experiment.

[0158] NANOS3 knockout Bama boars aged 1-3 months before puberty ( NANOS3 KO pigs were anesthetized with isoflurane and placed in a supine position. Ultrasonic transmission gel was applied to the scrotum. Using a 20-gauge needle, the aforementioned pPGCLCs suspension was aspirated and carefully inserted through the scrotum into the rete testis under the guidance of 7.5 MHz linear array ultrasound (Philips, CX50). Figure 10(a) and slowly inject the suspension. The infusion rate is approximately 0.5-1 mL / min, and 2-3 mL of suspension can be injected into each testis. After injection, the recipient pigs recover in a quiet and comfortable environment. Semen collection training and induced sperm testing are performed approximately 3 months post-transplantation. Figure 10 (ac in the text). A total of 7 pigs were transplanted, hereinafter referred to as recipient pigs. In addition, wild-type pigs that did not undergo NANOS3 knockout were used as controls.

[0159] 3. Recipient pig testing (1) Histological analysis of the testes of the recipient pig Testes and epididymis from recipient pigs were collected, washed twice with DPBS, cut into appropriate sizes, and fixed overnight at 4°C with 4% PFA. See the histological analysis methods described above for the analysis of testes and epididymis from recipient pigs.

[0160] The results are as follows Figure 10 As shown, e represents the porting of pPGCLCs to... NANOS3 Histological analysis of the testes of recipient pigs 4 months after KO. White arrow clusters indicate germ cells, while black arrows indicate supporting cells. Scale bar: left image 100 μm, right image 25 μm. Figure 10 As shown in d, h, and e, germ cells (GCs) are present in the seminiferous tubules of the recipient pig's testes, and no teratomas are found. Sperm-like cells can also be observed in the epididymis. Figure 10 (f)

[0161] Four months after pPGCLC transplantation, testicular tissue from recipient pigs was collected. Genomic DNA was extracted according to the method described in "Example 2, Section 3: Verification of Cutting Efficiency". Specific primers (Fw and Rv of tdTomato-test) were used to identify the genotyping results of NANOS3-tdTomato, where PC was the positive control (N3T+ pPGCLCs) and NC was the negative control (WT pEpiSCs). PCR results showed the presence of NANOS3-KI (full name: KI) in the recipient pig testes. NANOS3 - P2A - tdTomato ( N3T Positive band ( Figure 10 (g) demonstrates that transplanted pPGCLCs can survive in the testes and may even develop.

[0162] (2) Immunostaining of tissues Testicular and epididymal tissues from recipient pigs 4 months after pPGCLC transplantation were collected and treated overnight at 4°C in 4% PFA. The fixed tissues were rinsed twice with DPBS to remove excess paraformaldehyde and incubated overnight at 4°C in 30% sucrose / DPBS. The samples were blotted dry with paper towels, placed in disposable molds, and directly covered with the OCT compound (Sakura brand, Tissue-Tek® OCT Compound), avoiding air bubbles. The molds were partially immersed in liquid nitrogen until the OCT compound solidified. If the blocks were not used immediately, they were wrapped in aluminum foil and stored at -80°C to prevent evaporation. The blocks were cut into 8μm thick pieces using a cryostat, mounted on glass slides, air-dried for 1 hour, and then subjected to immunofluorescence staining. The tissues were transferred to DPBS for 5 minutes and permeabilized with 1% Triton X-100 for 30 minutes. Wash the slides three times, 2 min each time, block with 3% BSA / 10% donkey serum for 40 min, and then incubate overnight at 4°C with the required primary antibody (DDX4, ACROSIN (a sperm-specific marker) or PNA). After thorough washing, add the appropriate secondary antibody and incubate in the dark for 30 min. After washing the tissue again, mount with a DAPI-containing antifluorescence quencher and seal with nail polish. The slides can be stored at 4°C until observation.

[0163] The results are as follows Figure 10 Figures h, i, and j are shown in the middle. h is a representative IF image of a testicular section from the recipient pig 4 months post-transplantation, showing the expression of designated key GC markers (red: DDX4, ACROSIN) and DAPI (blue). Scale bar: left image 50 μm, right image... Figure 10 μm. i represents 4 months of age. NANOS3 Representative IF images of KO pig testicular sections, showing no expression of key GC markers (DDX4, ACROSIN). Scale bar, 50 μm. j: Representative IF images of recipient pig epididymal sections 4 months post-transplantation, showing expression of key sperm markers (red: PNA) and DAPI (blue). Scale bar, 50 μm.

[0164] The results showed that DDX4 and ACROSIN-positive GCs could be detected in some seminiferous tubules of the recipient porcine testicular tissue 4 months after transplantation. Figure 10 (h) was not detected in most of the small tubes. Figure 10 i). PNA-positive sperm-like cells were observed in cross-section of the epididymal duct of the recipient pig. Figure 10 (j). This indicates that pPGCLCs home to the seminiferous tubules in the testes and can further undergo meiosis to produce haploid cells.

[0165] (3) Semen collection and analysis Approximately 3-4 months after pPGCLC transplantation, recipient boars undergo semen collection training using an artificial vagina (AV). Through repeated training and reinforcement, the boars are trained to mount the sow or dummy sow. All semen collection training procedures are performed by the same experienced technician. The collected semen is diluted with an equal volume of preheated sperm diluent at 37°C and transported to the laboratory at 17°C for microscopic examination to detect the presence of sperm or sperm cells. Simultaneously, various sperm parameters are measured and counted. Subsequently, immunostaining, DNA or RNA extraction, single-cell analysis, or cryopreservation are performed.

[0166] (4) Semen immunofluorescence staining The experiment was repeated 3 times, with each repetition as follows: See the aforementioned cell immunofluorescence analysis method for NANOS3 Sperm cells were collected from the semen of KO recipient pigs and stained.

[0167] The results are as follows Figure 11 As shown in Figure a (scale bar 25 μm), semen was collected regularly from recipient pigs starting about 100 days after transplantation. Continuous observation and staining revealed that the recipient pig semen contained sperm-like cells that were positive for ACROSIN staining and had small tails, resembling sperm.

[0168] (5) Expression analysis of sperm-related genes in the semen of NANOS3 knockout recipient pigs Total RNA was extracted according to the method described in "3. Direct induction of porcine primordial germ cell-like cells (pPGCLCs) in the prior art" in Example 1, and then reverse transcribed into cDNA. Sperm-related genes in the recipient porcine semen were then detected. PRM1 , PRM2 , ACROSIN and TNP1 The expression of ) is described above, and the primers are mentioned earlier.

[0169] The results are as follows Figure 11 As shown in Figure b, the semen of the recipient pigs expresses genes related to sperm cells ( PRM1 , PRM2 , ACROSIN and TNP1 ).

[0170] (6) Haplotype analysis For haploid analysis, approximately 20 mL of recipient Bama pig semen was obtained via straddling and in vitro semen collection. The semen was mixed with sperm dilution buffer in an equal ratio, filtered through a 40 μm cell filter, and sperm or spermatocyte counts were performed. After centrifugation, the semen was resuspended in 1% BSA / DPBS and Hoechst 33342 staining solution (Thermo Fisher, R37605) was added. The mixture was incubated at room temperature in the dark for 5 min. The staining solution was discarded, and the sample was washed with ice-cold 1% BSA / DPBS. After resuspending, flow cytometry was performed for detection and analysis.

[0171] The results are as follows Figure 11 In the case of c, the collected semen from the recipient pig did indeed contain haploid cells.

[0172] (7) Origin and sex identification of haploid cells in recipient pig semen Microsatellite DNA fingerprinting analysis method: Genomic DNA was extracted from pPGCLCs derived from pEpiSCs, recipient pig blood (#2205902), and recipient pig semen (#2205902) for microsatellite repeat fingerprinting analysis. Microsatellite primers (Fw and Rv of S0090) jointly recommended by the Food and Agriculture Organization of the United Nations (FAO) and the International Society for Animal Genetics (ISAG) were used. The forward primer was labeled with FAM fluorescence at the 5' end. The PCR reaction consisted of 20 ng template DNA, 0.5 μM of each primer, and 10 μL of TaqMix. Pre-denaturation was performed at 95 °C for 3 min, followed by 35 cycles, each consisting of 95 °C denaturation for 30 s, 60 °C annealing for 30 s, 72 °C extension for 1 min, and 72 °C cooling for 5 min. The fluorescently labeled PCR products were identified by 1% agarose gel electrophoresis, and then PCR-STR detection was performed by Beijing Zhongke Gene Research Center Co., Ltd. The sequencing results were read using Peak Scanner software v1.0 (Applied Biosystems) to determine and label the genotypes.

[0173] The results are as follows Figure 11 As shown in d, donor-specific signals were detected in the semen of fertilized boars.

[0174] Methods for identifying the cell origin in recipient pig semen: PCR was performed using the pig NANOS3-KO test as amplification primers and genomic DNA extracted from recipient pig semen as a template. The obtained PCR products were then subjected to agarose gel electrophoresis. Controls included N3T pEpiSCs (positive control) and blood from recipient pigs (negative control). The molecular weight (MW) was D2000bp DNA gradient. Fragments of 872bp in the amplification results were considered the cellular origin. NANOS3 - P2A - tdTomato ( N3TSperm derived from pEpiSCs.

[0175] The results are as follows Figure 11 As shown in Figure e, the semen of recipient pigs at 4, 5, and 10 months post-transplantation contained... NANOS3 - P2A - tdTomato ( N3T Sperm derived from pEpiSCs. During the evaluation period of over 400 days, round sperm cells or sperm were never observed in semen collected from knockout pigs.

[0176] The aforementioned PCR products were sequenced, and the results are as follows: Figure 11 As shown in figure f, Allele1 is the full-length NANOS3 gene, while Allele2 is the NANOS3 gene with 679 bp knocked out, leaving only 193 bp. The results indicate that the recipient pig semen contains... N3T Sperm derived from pEpiSCs.

[0177] Sex determination of recipient pig sperm cells: The sex of individual sperm cells was determined by referring to the method in "2. Culture and sex determination of porcine epiblast stem cells (pEpiSCs)" in Example 1.

[0178] Figure 11 The g and h results showed that the recipient pig semen contained both X and Y sperm, consistent with the results of wild-type pig sperm identification.

[0179] (8) Genome sequencing of single sperm cells Selected single N3T homozygous pEpiSCs cells, N3T cells derived from single N3T pEpiSCs + pPGCLCs and sperm cells derived from a single recipient pig were used to analyze genomic insertions near the NANOS3 stop codon site. 3×Flag - P2A - NLS - tdTomatoThe genome sequencing of single sperm cells was performed as follows: Single cells were lysed in a buffer containing 0.1 M DTT, 100 mM Tris-EDTA (pH 8.0), 20 mg / ml Qiagen proteinase K, and 10% Triton X-100 to release genomic DNA. A limited number of linear pre-amplifications were then performed using random primers (GAT3G primers) to generate DNA fragments with universal sequence tags. The amplified products were then circularized using primer complementarity, a crucial step in reducing amplification bias. Next, the circularized products were amplified by PCR using universal primers to enrich the DNA content. After purification, the amplified products underwent end repair, adapter ligation, and library amplification to construct the sequencing library. After quality control and quantification, paired-end sequencing (PE150) was performed using the Illumina NovaSeq XPlus platform. By introducing a cyclic step, MALBAC improves the uniformity of amplification, making it ideal for whole-genome analysis at the single-cell level.

[0180] The results are as follows Figure 11 As shown in Figure i, the collected recipient pig sperm and NANOS3 - P2A - tdTomato ( N3T The genome sequencing results of pEpiSCs and their derived pPGCLCs are identical, both containing [missing information - likely a specific gene term or marker] near the stop codon of the NANOS3 gene. 3×Flag Precise insertion of segments.

[0181] (9) In sperm cells derived from pPGCLCs and wild-type (WT) sperm cells H19 / IGF2 and SNRPN Methylation status of differentially methylated regions (DMRs) Sperm DNA was isolated using the TIANamp Micro DNA Kit (Tiangen, DP316) according to the manufacturer's instructions, and methylation-related genes in the recipient porcine semen were detected using the bisulfite sequencing method described in "3. Epigenetic Detection" of Example 2. H19 / IGF2 and SNRPN DMR The expression of the primers is as described above.

[0182] The results are as follows Figure 11 As shown in Figure j, each row represents a single allele. Black circles indicate methylated CpG islands, and white circles indicate unmethylated CpG islands. All representative data were obtained from at least three independent experiments, demonstrating that recipient sperm cells imprint genes... H19 / IGF2 and SNRPN DMR The methylation level was comparable to that of wild-type ejaculated sperm. Figure 10 (middle k).

[0183] In summary, the transplanted pPGCLCs not only survived, but also completed meiosis in the testes and produced haploid sperm cells.

[0184] Example 4: Functional Verification of Sperm-like Cells 1. Intracytoplasmic sperm injection (ICSI) See diagram Figure 12 In section a, sperm cells obtained fresh or after freeze-thaw cycles ("1. Semen Collection and Analysis") were resuspended at room temperature in fertilization medium (embryo culture medium containing 7.5 mM calcium chloride, 3.0 mM potassium chloride, 11 mM glucose, 113.1 mM sodium chloride, 20 mM Tris, 5.0 mM sodium pyruvate, 2 mM caffeine, and 0.6% bovine serum albumin). Using an injection needle with an inner diameter of 15-20 μm, and under an inverted microscope equipped with a micromanipulation system (Narishige, Japan), round sperm cells were selected based on their appearance.

[0185] Oocytes collected from the ovaries of Large White pigs were matured in vitro to metaphase II oocytes in HEPES-M199 medium and the cumulus oophorus was removed with 0.2% hyaluronidase. The oocytes were incubated in HEPES-M199 containing 7.5 μg / mL cytochalasin B for 10 min, followed by microinjection using a TransferMan NK2 micromanipulation system (Eppendorf, Germany). The polar body was positioned at the 6 or 12 o'clock position, and the injection site was at the 3 o'clock position. The aforementioned round sperm cells were injected into metaphase II oocytes and incubated for 30 min in PZM-3 medium (108.00 mM NaCl, 10.00 mM KCl, 0.35 mM KH2PO4, 0.40 mM MgSO4·7H2O, 25.07 mM NaHCO3, 0.20 mM sodium pyruvate, 2.00 mM Calcium Lactate, 1.00 mM L-Glutamine, 5.00 mM Hypotaurine, 20 mL / L BME amino acid, 10 mL / L MEM non-essential amino acid, 0.3% BSA and MilliQ-water). Then, the cells were transferred to activation medium (0.25 mM mannitol, 0.1 mM calcium chloride, 0.1 mM magnesium chloride, 0.5 mM HEPES, 0.01% PVA) for activation. Oocytes were activated for 80 μs using a cell electrofusion instrument (BLS, CF-150B, Hungary) at a pulse intensity of 80 V / mm. Embryos were washed with PZM-3 medium and cultured in a humidified environment at 38.5°C and 5% CO2. The following day, embryos that formed double pronuclei (2PN) and two cells were transferred to the oviducts of 0.5 dpc pseudopregnant sows (Large White sows on the second day after standing estrus), with 100-200 embryos transferred per sow, for a total of 20 sows. The remaining embryos were transferred to fresh PZM-3 medium for further culture, followed by parentage analysis. Pregnancy was monitored by ultrasound 26 days after transfer.

[0186] 2. Intracytoplasmic sperm injection (ICSI) embryo testing (1) Fusion of male and female pronuclei Experimental methods: After intracytoplasmic sperm injection (ICSI), oocytes were washed with DPBS (Gibco, C14190500BT), fixed with 4% paraformaldehyde for 30 min at room temperature, washed again with DPBS, permeabilized in 0.1% Triton X-100 for 20 min, washed three times with washing buffer (DPBS containing 0.1% Triton X-100 and 0.1% Tween 20) for 5 min each time, and stained with DAPI to label the nucleus and polar body.

[0187] Representative images of oocytes with different chromatin types, such as Figure 12 As shown in Figure b (scale bar 50 μm), statistical results indicate that most oocytes injected at 0 h were in the MII stage, with only one (10%) remaining unfertilized. At 6 h, a significant number of oocytes clearly developed to the AII-TII stage (24%) or PNE stage (37.5%). At 9 h, of the 24 fertilized oocytes, 5 (20.8%) were in the metaphase to PN stage, and 10 (41.7%) were in the MIT stage. At 18 h, 50% of the oocytes were in the MIT stage, and 31.25% were in the cleavage stage. This demonstrates that the obtained haploid sperm cells possess fertilization capacity.

[0188] Table 1. Statistical Analysis of Pronucleation Data

[0189] MII: Metaphase II, AII-TII: Late Phase II to Telophase II, PNE: Early Prokaryote, PNM: Metaphase Prokaryote, PNL: Late Prokaryote, MIT: First Mitosis, ND: Not Detected.

[0190] (2) Statistics on the in vitro development rate of embryos produced by intracytoplasmic sperm injection from pPGCLCs-derived sperm cells The number of two-cell stage embryos and E7 blastocysts after intracytoplasmic sperm injection (ICSI) was counted. Results are as follows: Figure 12 As shown in Figure c, in five replicate ICSI experiments, the majority (87.26% ± 7.57%) of the injected oocytes underwent cleavage, and the proportion of in vitro cultured embryos developing to the blastocyst stage was 38.98% ± 9.61%, with blastocysts hatching on day 7 accounting for 21.51% ± 8.67. The results indicate that the obtained haploid sperm cells can support embryonic development.

[0191] (3) Immunofluorescence staining of embryos The expression of inner cell mass (SOX2) and trophoblast (CDX2) in intracytoplasmic sperm injection (ICSI) blastocysts was detected. The experimental method was as follows: Embryos were washed with DPBS (Gibco, C14190500BT), fixed with 4% paraformaldehyde at room temperature for 30 min, washed again with DPBS, permeabilized in 0.1% Triton X-100 for 20 min, and blocked with 3% BSA for 1 hour. Embryos were incubated overnight at 4°C with primary antibody diluted in 3% BSA. Subsequently, embryos were washed three times with washing buffer (DPBS containing 0.1% Triton X-100 and 0.1% Tween 20) for 5 min each time. Secondary antibody was diluted and incubated with washing buffer at room temperature for 1 hour, then washed three times with washing buffer for 5 min each time. Finally, the nuclei were stained with DAPI (Thermo Fisher, 62248) for 3 min. Images were captured using a confocal fluorescence microscope (Nikon, A1-Sim).

[0192] The results are as follows Figure 12 As shown in image d, d represents a representative IF image of an E7 blastocyst produced by ICSI. The inner cell mass (SOX2 positive) is shown in green, the trophoblast (CDX2 positive) in red, and the nucleus (DAPI) in blue. Scale bar, 50 μm. Figure 12 The middle d-figure shows the statistical analysis of the number and proportion of positive cells in the inner cell mass and trophoblast of E7 blastocysts produced by ICSI. The results are as follows: Figure 12 As shown in Figure e, the average cell count of morphologically normal blastocysts was 58.9, indicating good developmental quality. The cell ratio of the inner cell mass (ICM, SOX2 positive) to the trophectoderm (TE, CDX2 positive) in ICSI-derived blastocysts was approximately 0.1–0.41. Figure 12 (e) This shows that it is close to the level of blastocysts derived from in vitro fertilization, but still lower than the level of in vivo derived embryos.

[0193] (4) Images of pig fetuses derived from pPGCLCs-derived sperm cells. One- or two-cell stage embryos cultured in vitro were transferred into the fallopian tubes of twenty surrogate sows. Twenty-six days after transfer, ultrasound examinations were performed on the sows, revealing that twelve sows (60%) had successfully conceived, with six fetuses detected. Figure 12 (See figure f, scale bar 5mm). The results show that embryos derived from haploid sperm cells can establish pregnancy and develop into a complete fetus.

[0194] (5) Isolation, establishment, and tissue origin identification of the E30 fetal fibroblast cell line generated by intracytoplasmic sperm injection. Experimental Methods: Fibroblasts were isolated from the trunk tissue of ICSI-derived E30 fetuses according to the method described in "1. MEF Feeder Cell Preparation" of Example 1. Simultaneously, fibroblasts were isolated from the trunk tissue of ICSI-derived E30 fetuses according to "I. Site-Specific Integration Fluorescence Reporter System" of Example 2. SOX17 - P2A - tdTomato ( ST The method of “(2) Karyotype analysis” in “Construction and functional identification of pEpiSCs” is used to identify the karyotype of isolated cells.

[0195] The results are as follows Figure 12 As shown in g and h, two fetal fibroblast cell lines (37+XX) with normal chromosomal karyotypes were successfully established from the obtained E30 fetuses.

[0196] Experimental method: Using E30 fetal tissue DNA as an amplification template, PCR was used to detect whether N3T and ACROSIN-EGFP were expressed.

[0197] The results are as follows Figure 12 As shown in Figure i, reporter genes are expressed in all tissues of the fetus, indicating that the E30 fetus was produced by intracytoplasmic sperm injection of recipient pig sperm, and did not develop from a parthenogenetic embryo.

[0198] Table 2. Embryo transfer results after intracytoplasmic sperm injection (ICSI)

[0199] (6) Bisulfite sequencing of ICSI-derived and wild-type E30 fetuses Experimental methods: Bisulfite sequencing was performed on ICSI-derived and wild-type E30 fetuses according to the method shown in "3. Epigenetic level detection" in Example 2.

[0200] The results are as follows Figure 12 As shown in the diagram, each row represents a single allele. Black circles indicate methylated CpG islands, and white circles indicate unmethylated CpG islands. This demonstrates that these fetuses... SNRPN The methylation level of the imprinted genes was similar to that of normal fetuses, a result further supported by whole-genome bisulfite sequencing (WGBS). Figure 12 (k and l).

[0201] (7) Establish porcine embryonic stem cell lines from blastocysts produced at E7 ICSI (day 7 after ICSI). Experimental Methods: To establish next-generation porcine embryonic stem cell (pESC) lines from porcine ICSI-derived blastocysts, the E7 inner cell mass (ICM) was dissected under a microscope using an insulin needle. Intact ICMs were seeded into pre-coated feeder layers in four-well plates and cultured in 3i / LAF medium at 38.5℃, 5% O2, and 5% CO2. After 8-10 days of continuous culture, outgrowths were dissociated with Accutase for 2-3 minutes, then gently separated and broken into small clumps using a pipette. These clumps were then reseeded into new feeder layers using fresh 3i / LAF medium. Once a tightly packed epithelial-like cell population was formed, the cells could be further passaged and expanded into a continuously passaged cell line. Cell lines capable of more than 20 passages were obtained, and the biological characteristics of ICSI blastocyst-derived cell lines were analyzed.

[0202] The results are as follows Figure 13 Figures a and b show the establishment of pig embryonic stem cell (pESC) lines from blastocysts produced by E7 ICSI. Scale bar: 400 μm. Twenty-four derivatives (outgrowths) were obtained from 28 hatched blastocysts, and 12 stable cell lines (passaged more than 30 times) were established (see Figure 1). Figure 13 (a)

[0203] (8) Alkaline phosphatase (ALP) staining in pESCs derived from ICSI blastocysts Experimental method: Refer to "6" in Example 2. SOX17 - P2A - tdTomato ( ST The method shown in “(1) Alkaline phosphatase staining” of pESCs from ICSI blastocysts was used to stain pESCs for alkaline phosphatase (ALP) in “Functional identification of pEpiSCs”.

[0204] The results are as follows Figure 13 As shown in image c, this is a representative image of alkaline phosphatase (ALP) staining in pESCs from ICSI blastocysts. Scale bar: 400 μm. This demonstrates that pESCs are positive for alkaline phosphatase staining, exhibiting pluripotency.

[0205] (9) Karyotype analysis Experimental method: See "6" in Example 2. SOX17 - P2A - tdTomato ( ST The karyotype analysis of pESCs derived from ICSI blastocysts was performed in the "(2) Karyotype analysis" section of the "pEpiSCs functional identification".

[0206] The results showed that pESCs derived from ICSI blastocysts had a normal karyotype and were normal diploids. Figure 13 (d).

[0207] (10) Immunofluorescence staining and routine transcriptome analysis of embryonic stem cells Referring to the immunofluorescence staining analysis described above: the expression of pluripotency-related markers in pESCs derived from ICSI blastocysts was detected. The pluripotency markers included OCT4, SOX2, NANOG, OTX2, CDH1, PRDM14, SSEA1, SSEA4, TRA1-60, and TRA1-81.

[0208] The results are as follows Figure 13 As shown in e and f (scale bar 50 μm), pESCs express core pluripotency markers and possess pluripotency characteristics.

[0209] pESCs were collected and sent to Beijing Novogene Technology Co., Ltd. for routine transcriptome sequencing analysis.

[0210] The results are as follows Figure 13 As shown in g, pESCs and pEpiSCs have similar transcriptomic characteristics.

[0211] (11) Embryomorphic body differentiation experiment Experimental methods: pESCs were treated with Accutase at 37℃ for 5 min, dispersed into single cells by repeated pipetting, and then suspended and vortexed for 3 days to self-aggregate into embryoid bodies (EBs). Subsequently, the EBs were transferred to pre-gelatin-coated culture dishes, replaced with MEF medium, and cultured for 1 week. Figure 13 (h in the text). Spontaneous differentiation of the three germ layers was assessed by morphological observation (DP26 inverted microscope, Olympus) and immunofluorescence staining.

[0212] The results showed that pESCs could aggregate in vitro to form embryoid bodies ( Figure 13 In the i), the resulting embryoid body can randomly differentiate into three germ layers. The differentiated cells are positive for markers of the three germ layers, including the ectoderm marker β-IIItubulin, the mesodermal marker α-SMA, and the endoderm marker GATA6 (…). Figure 13 j in the middle.

[0213] (12) Primordial germ cell induction Experimental method: According to "II. SOX17 - P2A - tdTomato ( STThe method described in “III. Further induction into pPGCLCs” involves culturing pESCs derived from ICSI blastocysts for 36 hours (i.e., the aforementioned pMEPCs cells), replacing the PGC induction medium, and culturing for 24 hours to obtain primitive germ cell-like cells.

[0214] The results are as follows Figure 13 As shown in Figure k, k represents the differentiation of pESCs derived from ICSI blastocysts into N3T cells. + pPGCLCs. Scale bar, 100 μm.

[0215] (13) Immunofluorescence staining of primordial germ cells Referring to the immunofluorescence staining analysis described above: the expression of NANOS3, SOX17, PRDM1, and TFAP2C in pPGCLCs induced by ICSI blastocyst-derived pESCs was detected.

[0216] The results are as follows Figure 13 As shown in Figure l, l represents the differentiated N3T + Representative IF images of NANOS3, SOX17, PRDM1, and TFAP2C proteins in pPGCLCs. Scale bar, 50 μm. Representative data from at least three independent experiments. The results indicate that primordial germ cell-like cells induced by ICSI blastocyst-derived pESCs express germ cell core proteins.

[0217] In summary, the inventors have developed a closed-loop spermatogenesis system in pigs, a domestic animal, from which porcine gastrulated epiblast stem cells differentiate into functional gametes. Therefore, the sperm obtained from the differentiation of porcine gastrulated epiblast stem cells into mesoderm precursor cells, and further into primordial germ cell-like cells, possesses fertilization capacity, the ability to produce high-quality blastocysts in vitro, stem cell regeneration capacity, and the ability to develop into a complete fetus in vivo, demonstrating the potential of the mesoderm precursor cells provided by this invention to differentiate into primordial germ cells.

[0218] The nucleotide sequence of the pX330-puro plasmid is as follows:

[0219] The nucleotide sequence of the donor OCT4-A5-3×Flag-PNP-tdtomato-3' plasmid is as follows:

[0220] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for inducing porcine gastrulation epiblast stem cells into mesoendothelial precursor cells, comprising the following steps: inducing isolated porcine gastrulation epiblast stem cells into mesoendothelial precursor cells, wherein the mesoendothelial precursor cells express OCT4 protein, NANOG protein, SOX17 protein, PRDM1 protein, EOMES protein and TBXT protein.

2. The method according to claim 1, characterized in that, The induction time for inducing isolated porcine gastrulated epiblast stem cells into mesoendothelial precursor cells is 30-42 hours.

3. The method according to claim 1 or 2, characterized in that, The induction is carried out via composition one, which comprises an activator of the Activin / NODAL cell signaling pathway and an agonist of the Wnt / β-catenin cell signaling pathway.

4. A method for inducing mesoendodermal precursor cells into primordial germ cell-like cells, comprising the following steps: preparing mesoendodermal precursor cells according to any one of claims 1-3; inducing the mesoendodermal precursor cells into primordial germ cell-like cells; wherein the primordial germ cell-like cells express marker proteins, the marker proteins including NANOS3 protein, SOX17 protein, OCT4 protein, NANOG protein, PRDM1 protein, and TFAP2C protein.

5. The method according to claim 4, characterized in that, The induction time for the mesoderm precursor cells to be induced into primordial germ cell-like cells is 12-24 hours.

6. The method according to claim 4 or 5, characterized in that, The induction is carried out using composition two, which includes bone morphogenetic protein 4, human leukemia inhibitory factor, stem cell factor, and epidermal growth factor.

7. A method for inducing porcine embryonic stem cells into primordial germ cell-like cells, comprising the following steps: first preparing mesoderm precursor cells according to any one of claims 1-3, and then preparing primordial germ cell-like cells according to any one of claims 4-6.

8. A product for generating sperm-like cells in the testicular reticulum of a recipient animal, said product comprising primordial germ cell-like cells prepared according to the method of any one of claims 4-7.

9. A composition, wherein the composition is any one of the following: M1) A composition for inducing porcine gastrulated epiblast stem cells into mesoderm precursor cells, said composition comprising composition one of claim 3 and isolated porcine gastrulated epiblast stem cells; M2) A composition for inducing mesoendodermal precursor cells into primordial germ cell-like cells, said composition comprising composition two as described in claim 6 and mesoendodermal precursor cells prepared according to any one of claims 1-3; M3) A composition for inducing porcine gastrulation epiblast stem cells into primordial germ cell-like cells, said composition comprising composition one of claim 3, composition two of claim 6, and isolated porcine gastrulation epiblast stem cells; M4) is a composition consisting of composition one and composition two.

10. The following applications of the product of claim 8, or the mesoendothelial precursor cells or primordial germ cell-like cells of claim 9: A1) Construct mesoendothelial precursor cells with SOX17 or NANOS3 gene knock-in; A2) Prepare products for constructing mesoendodermal precursor cells with SOX17 or NANOS3 gene knock-in; A3) Produces mesoendothelial precursor cells; A4) Products prepared from mesoderm precursor cells; A5) Produces primordial germ cell-like cells; A6) Prepare products that produce primordial germ cell-like cells; A7) Produces sperm-like cells; A8) Prepare products that produce sperm-like cells; A9) Prepare products for treating diseases caused by a lack of sperm cells or insufficient sperm activity.

Images