Method for identifying pluripotent status of bovine epidermis

Abstract

The invention discloses a method for identifying a pluripotent state of a cattle epidermal layer, and achieves the technical effect of identifying the pluripotent state of a cattle embryo, especially the pluripotent state of the cattle epidermal layer. The invention specifically discloses gene markers for different pluripotent stages and different developmental stages, and the pluripotent and developmental stages of to-be-detected bovine embryonic cells or cell clusters can be rapidly identified by using the gene markers for different pluripotent stages and different developmental stages of bovine embryos.

Description

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on March 21, 2024, with application number 2024103257603 and application name “A bovine intermediate stem cell line and its in vitro preparation method and use”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention particularly relates to methods for identifying the pluripotent state of bovine epiblast. Background Art

[0003] Embryonic ectoderm cells can differentiate into a complete fetus and serve as an important source of pluripotent stem cell (PSC) lines. PSCs derived from the ectoderm at different developmental stages exhibit different pluripotent states and can be specifically divided into naive, formative, or primed states. Human and mouse PSCs have been widely used in embryonic development, directed differentiation, and 12 and disease modeling-related research.

[0004] Stable livestock PSCs not only help us understand the embryonic development and pluripotency of livestock, but are also the best cell source for animal breeding and cultured meat production. However, due to species specificity, progress in establishing stable pluripotent stem cell lines in large animals has been slow. Although some progress has been made in establishing bovine embryo-derived PSCs, the pluripotency of bovine PSCs has not been fully characterized, and our understanding of the mechanisms regulating pluripotency and maintaining stem cell stability remains limited. Recently, the establishment of porcine preimplantation epiblast stem cells (pgEpiSCs) has attracted widespread attention due to their potential applications in various fields. The use of the 3i / LAF culture system has been shown to support long-term passaging and maintenance of pluripotency of porcine pluripotent stem cells. 21 Therefore, the similarities between bovine and porcine embryonic development and the maintenance of stem cell pluripotency have become an important research topic.

[0005] Single-cell RNA sequencing enables precise mapping of embryonic development, helping to track early embryonic lineages and the transition from embryonic cells to stem cells. However, research on the molecular basis of bovine embryogenesis and PSCs lags far behind that of pigs, mice, humans, and non-human primates. Existing single-cell transcriptome studies of bovine embryos do not provide accurate and high-resolution transcriptome maps to dissect the alterations in bovine epiblast pluripotency that could guide the generation of stable bovine PSCs.

[0006] Because the molecular basis of bovine embryonic development and pluripotent stem cell self-renewal remains unclear, establishing bovine epiblast-derived pluripotent stem cells (PSCs) is challenging. Therefore, further tracking bovine embryonic stem cell lines at different developmental stages has important practical application value. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for identifying the pluripotency state of bovine epiblast. By using gene markers of different pluripotency stages and developmental stages of bovine embryos, the pluripotency and developmental stage of bovine embryo cells or cell clusters to be tested can be quickly identified.

[0008] In a first aspect, the present invention provides the use of a substance for identifying gene markers in the preparation of a product for identifying the developmental stage or pluripotency stage of bovine embryonic cells, wherein the gene markers are selected from one or more genes shown in Tables 4 to 13, or any combination thereof.

[0009] In certain embodiments, the bovine embryonic cell developmental stage comprises a pre-inner cell mass, a pre-trophoblast, an inner cell mass, a trophoblast, an epiblast, a hypoblast, an ectoderm, a mesoderm, an endoderm, or a primordial germ cell-like cell.

[0010] In certain embodiments, the anterior inner cell mass and / or the anterior trophoblast are selected from bovine embryo E5; or, the inner cell mass is selected from bovine embryo E6 and / or E7; or, the trophoblast is selected from bovine embryo E6-E14; or, the epiblast is selected from bovine embryo E10-E12; or, the hypoblast is selected from bovine embryo E10-E12; or, the ectoderm, mesoderm, endoderm and / or primordial germ cell-like cells are selected from bovine embryo E14;

[0011] In certain embodiments, the bovine embryos E10-E12 are selected from E10 or E12, or any combination thereof;

[0012] In certain embodiments, the self-derived bovine embryo E6-E14 is selected from E6, E7, E10, E12 or E14, or any combination thereof.

[0013] In certain embodiments, the gene markers for identifying the anterior inner cell mass are the gene markers shown in Table 4, or any combination thereof; or, the gene markers for identifying the anterior trophoblast are the gene markers shown in Table 5, or any combination thereof; or, the gene markers for identifying the inner cell mass are the gene markers shown in Table 6, or any combination thereof; or, the gene markers for identifying the trophoblast are the gene markers shown in Table 7, or any combination thereof; or, the gene markers for identifying the epiblast are the gene markers shown in Table 8, or any combination thereof; or, the gene markers for identifying the hypoblast are the gene markers shown in Table 9, or any combination thereof; or, the gene markers for identifying the ectoderm are the gene markers shown in Table 10, or any combination thereof; or, the gene markers for identifying the mesoderm are the gene markers shown in Table 11, or any combination thereof; or, the gene markers for identifying the endoderm are the gene markers shown in Table 12, or any combination thereof; or, the gene markers for identifying primordial germ cell-like cells are the gene markers shown in Table 13, or any combination thereof.

[0014] In certain embodiments, the bovine embryonic cell developmental stages include the naive pluripotency stage, the intermediate pluripotency stage, and the initiated pluripotency stage.

[0015] In certain embodiments, the gene markers for identifying the naive pluripotency stage are gene markers for identifying the pre-inner cell mass, gene markers for identifying the pre-trophoblast, gene markers for identifying the inner cell mass and / or gene markers for identifying the trophoblast; or the gene markers for identifying the intermediate pluripotency stage are gene markers for identifying the epiblast and / or gene markers for identifying the hypoblast; or the gene markers for identifying the naive pluripotency stage are gene markers for identifying the ectoderm, gene markers for identifying the mesoderm, gene markers for identifying the endoderm and / or gene markers for identifying primordial germ cell-like cells.

[0016] In certain embodiments, the substance used to identify the primitive stage of bovine embryonic cells is a substance for detecting the gene marker for identifying the pluripotency stage of bovine embryonic cells is a substance for detecting the JAK / STAT3 signaling pathway, or, the substance used to identify the intermediate pluripotency stage of bovine embryonic cells is a substance for detecting the FGF / ERK signaling pathway and / or a substance for detecting the TGFβ / SMAD signaling pathway, or, the substance used to identify the initial stage of bovine embryonic cells is a substance for detecting the JWNT / β-catenin signaling pathway.

[0017] In certain embodiments, the substance for detecting the JAK / STAT3 signaling pathway comprises a substance for detecting the IL6ST gene, STAT3 gene, PIK3R2 gene, PIK3R1 gene, LIFR gene, KLF4 gene, TBX3 gene, IL6R gene, PIK3CA gene or JAK2 gene, or any combination thereof;

[0018] Or, the substance for detecting the TGFβ / SMADs signaling pathway includes a substance for detecting ID1 gene, BMP1 gene, ACVR1 gene, TGFBR2 gene, SMAD5 gene, ID3 gene, BMP7 gene, BMPR2 gene, TGFB1 gene, BMPR1B gene, ACVR1B gene, INGBA gene, NODAL gene, ACVR2A gene, ACVR2B gene, SMAD9 gene, TDGF1 gene, BMP4 gene, SMAD4 gene or BMPR1A gene, or any combination thereof;

[0019] Or, the substance for detecting the FGF / ERK signaling pathway includes a substance for detecting the MAPK14 gene, the MAPK1 gene, the ETS1 gene, the FGF2 gene, the MAPK3 gene, the FGFR2 gene, the JUN gene, the KDR gene, the MAP2K2 gene, the PDGFA gene, the FGFR1 gene, the ITGA6 gene, the ETS2 gene or the ITGA3 gene, or any combination thereof;

[0020] Or, the substance for detecting the WNT / β-catenin signaling pathway includes a substance for detecting the GSK3B gene, TCF7 gene, CDH12 gene, APC gene, WNT11 gene, FZD1 gene, FZD10 gene, CTNNB1 gene, FZD2 gene, WNT3A gene, WNT5B gene or WNT7B gene, or any combination thereof;

[0021] In certain embodiments, the bovine embryonic cell is selected from the group consisting of anterior inner cell mass, inner cell mass, epiblast, and ectoderm;

[0022] In certain embodiments, the bovine embryo is a bovine embryo E5-E14;

[0023] In certain embodiments, the bovine embryo E5-E14 is selected from E5, E6, E7, E10, E12 or E14, or any combination thereof.

[0024] In another aspect, the present invention provides a method for identifying the developmental stage of bovine embryonic cells, the method comprising detecting the bovine embryonic cells to be detected by any one or more methods selected from the following:

[0025] (1) Identification using genetic markers;

[0026] (2) using signaling pathway identification;

[0027] (3) using signaling pathway-related genes for identification;

[0028] The gene marker is a gene marker in any of the above applications, the signal pathway is a signal pathway in the above applications, and the signal pathway-related gene is the above gene;

[0029] If the results are as follows, the bovine embryonic cell to be tested is the anterior inner cell mass:

[0030] expressing one or more of the genes described in Table 4;

[0031] If the results are as follows, the bovine embryonic cell to be tested is the pretrophoblast:

[0032] expressing one or more of the genes described in Table 5;

[0033] If the results are as follows, the bovine embryonic cell to be tested is the inner cell mass:

[0034] expressing one or more of the genes described in Table 6;

[0035] If the results are as follows, the bovine embryonic cells to be tested are trophoblasts:

[0036] expressing one or more of the genes described in Table 7;

[0037] If the results are as follows, the bovine embryonic cell to be tested is epiblast:

[0038] express one or more of the genes described in Table 8;

[0039] If the results are as follows, the bovine embryonic cell to be tested is hypoblast:

[0040] express one or more of the genes described in Table 9;

[0041] If the results are as follows, the bovine embryonic cell to be tested is ectoderm:

[0042] expressing one or more of the genes described in Table 10;

[0043] If the results are as follows, the bovine embryonic cells to be tested are mesoderm:

[0044] expressing one or more of the genes described in Table 11;

[0045] If the results are as follows, the bovine embryonic cell to be tested is endoderm:

[0046] express one or more of the genes described in Table 12;

[0047] If the results are as follows, the bovine embryonic cells to be tested are primordial germ cell-like cells:

[0048] Express one or more of the genes described in Table 13.

[0049] In another aspect, the present invention provides a method for identifying the pluripotency stage of bovine embryonic cells, the method comprising detecting the bovine embryonic cells to be detected by any one or more methods selected from the following:

[0050] (1) Identification using genetic markers;

[0051] (2) using signaling pathway identification;

[0052] (3) using signaling pathway-related genes for identification;

[0053] The gene marker is a gene marker in any of the above applications, the signal pathway is a signal pathway in the above applications, and the signal pathway-related gene is the above gene;

[0054] If the results are as follows, the bovine embryonic cells to be tested are at the naive pluripotency stage:

[0055] The test result is that the bovine embryonic cell to be tested is a pre-inner cell mass, a pre-trophoblast, an inner cell mass, a trophoblast, or any combination thereof;

[0056] If the results are as follows, the bovine embryonic cells to be tested are at the intermediate pluripotency stage:

[0057] If the test result is positive, the bovine embryonic cell to be tested is epiblast, hypoblast, or any combination thereof;

[0058] If the results are as follows, the bovine embryonic cells to be tested are at the primed pluripotent stage:

[0059] If the test result is yes, the bovine embryonic cells to be tested are ectoderm, mesoderm, endoderm, or primordial germ cell-like cells;

[0060] In certain embodiments, the detection is expression level detection;

[0061] In certain embodiments, the detection is single-cell or multi-cell transcriptome analysis;

[0062] In certain embodiments, the bovine embryonic cell to be tested is selected from bovine embryos E5-E14;

[0063] In certain embodiments, the bovine embryo E5-E14 is selected from E5, E6, E7, E10, E12 or E14, or any combination thereof.

[0064] In certain embodiments, the method further comprises comparing the test results of the bovine embryonic cell to be tested with the embryonic lineage and pluripotency stage characteristics established for pigs to determine the bovine embryonic cell developmental characteristics of the bovine embryonic cell to be tested;

[0065] In certain embodiments, the bovine embryonic cell is selected from the group consisting of anterior inner cell mass, inner cell mass, epiblast, and / or ectoderm;

[0066] In certain embodiments, the bovine embryo is a bovine embryo E1-E14;

[0067] In certain embodiments, the bovine embryos E1-E14 are selected from E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, E12, E13 or E14, or any combination thereof.

[0068] On the other hand, the present invention provides gene markers for identifying the pluripotency stage and / or developmental stage of bovine embryonic cells, wherein the genes include PDGFRA gene, NANOG gene, POU5F1 gene, DAB2 gene, TDGF1 gene, GATA4 gene, GATA6 gene, SOX17 gene, CDX2 gene, MESP1 gene, KDR gene, TBX6 gene, FOXF1 gene, FGFR1 gene, PMP22 gene, LAMA1 gene, FN1 gene, IHH gene, FOXA2 gene, NANOS3 gene, PRDM1 gene, or any combination thereof.

[0069] In certain embodiments, the gene marker is used to identify the developmental stage of bovine embryonic cells or cell clusters;

[0070] In certain embodiments, the PDGFRA gene, NANOG gene, POU5F1 gene, DAB2 gene or CDX2 gene, or any combination thereof, is used to identify bovine embryo E5;

[0071] In certain embodiments, the PDGFRA gene, NANOG gene, POU5F1 gene, DAB2 gene or CDX2 gene, or any combination thereof, is used to identify bovine embryo E6;

[0072] In certain embodiments, the PDGFRA gene, the NANOG gene, the POU5F1 gene, the DAB2 gene, or the CDX2 gene, or any combination thereof, is used to identify bovine embryo E7;

[0073] In certain embodiments, the NANOG gene, the POU5F1 gene, the TDGF1 gene, the GATA4 gene, the GATA6 gene, the PDGFRA gene, the SOX17 gene, the DAB2 gene, or the CDX2 gene, or any combination thereof, is used to identify bovine embryo E10;

[0074] In certain embodiments, the NANOG gene, the POU5F1 gene, the TDGF1 gene, the GATA4 gene, the GATA6 gene, the PDGFRA gene, the SOX17 gene, the DAB2 gene, or the CDX2 gene, or any combination thereof, is used to identify bovine embryo E12;

[0075] In certain embodiments, the NANOG gene, the POU5F1 gene, the TDGF1 gene, the MESP1 gene, the KDR gene, the TBX6 gene, the FOXF1 gene, the FGFR1 gene, the PMP22 gene, the LAMA1 gene, the FN1 gene, the IHH gene, the FOXA2 gene, the PDGFRA gene, the SOX17 gene, the NANOS3 gene, the PRDM1 gene, the DAB2 gene, or the CDX2 gene, or any combination thereof, is used to identify bovine embryo E14;

[0076] In certain embodiments, the bovine embryonic E5 is selected from anterior inner mass cells and / or pretrophoblast cells;

[0077] In certain embodiments, the bovine embryonic E6 is selected from inner mass cells and / or trophoblast cells;

[0078] In certain embodiments, the bovine embryonic E7 is selected from inner mass cells and / or trophoblast cells;

[0079] In certain embodiments, the bovine embryo E10 is selected from epiblast cells, hypoblast cells and / or trophoblast cells;

[0080] In certain embodiments, the bovine embryo E12 is selected from epiblast cells, hypoblast cells and / or trophoblast cells;

[0081] In certain embodiments, the bovine embryo E14 cells are selected from ectoderm cells, mesoderm cells, endoderm cells, pluripotent stem cells and / or trophoblast cells.

[0082] In certain embodiments, the gene marker is used to identify the pluripotent stage of bovine embryonic cells or cell clusters;

[0083] Preferably, the gene for identifying bovine embryo E5, the gene for identifying bovine embryo E6 and / or the gene for identifying bovine embryo E7 are used to identify the naive pluripotency stage;

[0084] In certain embodiments, the gene for identifying bovine embryo E10 and / or the gene for identifying bovine embryo E12 is used to identify an intermediate pluripotency stage;

[0085] In certain embodiments, the gene used to identify bovine embryo E14 is used to identify the primed pluripotency stage.

[0086] In certain embodiments, the bovine embryo is a bovine embryo E5-E14;

[0087] In certain embodiments, the bovine embryo E5-E14 is selected from E5, E6, E7, E10, E12 or E14, or any combination thereof.

[0088] In another aspect, the present invention provides a composition for identifying the pluripotency stage of bovine epiblast, the composition comprising a substance for detecting the JAK / STAT3 signaling pathway, a substance for detecting the FGF / ERK signaling pathway, the substance for detecting the TGFβ / SMAD signaling pathway, and / or a substance for detecting the JWNT / β-catenin signaling pathway;

[0089] The substances for detecting the JAK / STAT3 signaling pathway include substances for detecting the IL6ST gene, STAT3 gene, PIK3R2 gene, PIK3R1 gene, LIFR gene, KLF4 gene, TBX3 gene, IL6R gene, PIK3CA gene or JAK2 gene, or any combination thereof;

[0090] Or, the substance for detecting the TGFβ / SMADs signaling pathway includes a substance for detecting ID1 gene, BMP1 gene, ACVR1 gene, TGFBR2 gene, SMAD5 gene, ID3 gene, BMP7 gene, BMPR2 gene, TGFB1 gene, BMPR1B gene, ACVR1B gene, INGBA gene, NODAL gene, ACVR2A gene, ACVR2B gene, SMAD9 gene, TDGF1 gene, BMP4 gene, SMAD4 gene or BMPR1A gene, or any combination thereof;

[0091] Or, the substance for detecting the FGF / ERK signaling pathway includes a substance for detecting the MAPK14 gene, the MAPK1 gene, the ETS1 gene, the FGF2 gene, the MAPK3 gene, the FGFR2 gene, the JUN gene, the KDR gene, the MAP2K2 gene, the PDGFA gene, the FGFR1 gene, the ITGA6 gene, the ETS2 gene or the ITGA3 gene, or any combination thereof;

[0092] Alternatively, the substance for detecting the WNT / β-catenin signaling pathway includes a substance for detecting the GSK3B gene, TCF7 gene, CDH12 gene, APC gene, WNT11 gene, FZD1 gene, FZD10 gene, CTNNB1 gene, FZD2 gene, WNT3A gene, WNT5B gene or WNT7B gene, or any combination thereof.

[0093] In certain embodiments, the substance detecting the JAK / STAT3 signaling pathway is used to identify the naive pluripotency stage;

[0094] Alternatively, the substance for detecting the FGF / ERK signaling pathway and / or the substance for detecting the TGFβ / SMAD signaling pathway is used to identify an intermediate pluripotency stage;

[0095] Alternatively, the detection of the JWNT / β-catenin signaling pathway is used to identify the primed pluripotency stage;

[0096] In certain embodiments, the composition is used to detect bovine embryonic cells;

[0097] In certain embodiments, the bovine embryonic cell is selected from the group consisting of anterior inner cell mass, inner cell mass, epiblast, and ectoderm.

[0098] In certain embodiments, the bovine embryo is a bovine embryo E5-E14;

[0099] In certain embodiments, the bovine embryo E5-E14 is selected from E5, E6, E7, E10, E12 or E14, or any combination thereof.

[0100] In certain embodiments, the composition is used to detect the degree of activation of related signaling pathways;

[0101] In certain embodiments, the composition is used to detect the expression levels of genes related to related signaling pathways.

[0102] In another aspect, the present invention provides a gene marker for identifying the developmental stage of the bovine epiblast, wherein the gene marker comprises an E5 gene marker, an E6 gene marker, an E7 gene marker, an E10 gene marker, an E12 gene marker or an E14 gene marker, or any combination thereof;

[0103] In certain embodiments, the E5 gene includes the PDGFRA gene, the NANOG gene, the OU5F1 gene, the DAB2 gene, or the CDX2 gene, or any combination thereof;

[0104] In certain embodiments, the E6 gene comprises the PDGFRA gene, the NANOG gene, the OU5F1 gene, the DAB2 gene, or the CDX2 gene, or any combination thereof;

[0105] In certain embodiments, the E7 gene comprises the PDGFRA gene, the NANOG gene, the OU5F1 gene, the DAB2 gene, or the CDX2 gene, or any combination thereof;

[0106] In certain embodiments, the E10 gene includes the NANOG gene, the POU5F1 gene, the TDGF1 gene, the GATA4 gene, the GATA6 gene, the PDGFRA gene, the SOX17 gene, the DAB2 gene, or the CDX2 gene, or any combination thereof;

[0107] Preferably, the E12 gene includes NANOG gene, POU5F1 gene, TDGF1 gene, GATA4 gene, GATA6 gene, PDGFRA gene, SOX17 gene, DAB2 gene or CDX2 gene, or any combination thereof;

[0108] In certain embodiments, the E14 gene comprises the NANOG gene, the POU5F1 gene, the TDGF1 gene, the MESP1 gene, the KDR gene, the TBX6 gene, the FOXF1 gene, the FGFR1 gene, the PMP22 gene, the LAMA1 gene, the FN1 gene, the IHH gene, the FOXA2 gene, the PDGFRA gene, the SOX17 gene, the NANOS3 gene, the PRDM1 gene, the DAB2 gene or the CDX2 gene, or any combination thereof;

[0109] In certain embodiments, the marker is used to detect bovine embryonic cells;

[0110] In certain embodiments, the bovine embryo is a bovine embryo E5-E14;

[0111] In certain embodiments, the bovine embryo E5-E14 is selected from E5, E6, E7, E10, E12 or E14, or any combination thereof;

[0112] In certain embodiments, the bovine embryonic E5 is selected from anterior inner mass cells and / or pretrophoblast cells;

[0113] In certain embodiments, the bovine embryonic E6 is selected from inner mass cells and / or trophoblast cells;

[0114] In certain embodiments, the bovine embryonic E7 is selected from inner mass cells and / or trophoblast cells;

[0115] In certain embodiments, the bovine embryo E10 is selected from epiblast cells, hypoblast cells and / or trophoblast cells;

[0116] In certain embodiments, the bovine embryo E12 is selected from epiblast cells, hypoblast cells and / or trophoblast cells;

[0117] In certain embodiments, the bovine embryo E14 is selected from ectoderm cells, mesoderm cells, endoderm cells, primordial germ cells and / or trophoblast cells;

[0118] In certain embodiments, the E5 gene marker, the E6 gene marker and / or the E7 gene marker are used to identify the naive pluripotency stage;

[0119] In certain embodiments, the E10 gene marker and / or the E12 gene marker are used to identify an intermediate pluripotency stage;

[0120] In certain embodiments, the E14 gene marker is used to identify the primed pluripotency stage.

[0121] In another aspect, the present invention provides gene markers, including E10 epiblast gene markers, E10 trophoblast gene markers, E10 hypoblast gene markers,

[0122] The E10 trophoblast expresses or overexpresses one or more of the LGALS3 gene, GPD1L gene, PTGS2 gene, SLC34A2 gene, KRT18 gene, KRT8 gene, PLAC8A gene, DAB2 gene, PLS1 gene, and TKDP1 gene.

[0123] The E10 hypoblast expresses or overexpresses one or more of the ENSBTAG00000026119 gene, APOA2 gene, APOA1 gene, FABP3 gene, FN1 gene, MYL7 gene, RSPO3 gene, CTSV gene, MSMO1 gene, and PPP1R14A gene.

[0124] The E10 epiblast expresses or highly expresses one or more of the STMN1 gene, H3-3A gene, UPP1 gene, MS4A8 gene, STMN2 gene, NANOG gene, ENSBTAG00000051318 gene, BBX gene, SYCP3 gene, and APIP gene.

[0125] In another aspect, the present invention provides a method for identifying bovine embryonic E10 cells, wherein the bovine embryonic cells are tested for the above gene markers.

[0126] If the results are as follows, the bovine embryonic cells to be tested are E10 trophoblasts:

[0127] Express or overexpress one or more of the following genes: LGALS3 gene, GPD1L gene, PTGS2 gene, SLC34A2 gene, KRT18 gene, KRT8 gene, PLAC8A gene, DAB2 gene, PLS1 gene, and TKDP1 gene.

[0128] If the results are as follows, the bovine embryonic cells to be tested are E10 epiblast:

[0129] Express or overexpress one or more of the STMN1 gene, H3-3A gene, UPP1 gene, MS4A8 gene, STMN2 gene, NANOG gene, ENSBTAG00000051318 gene, BBX gene, SYCP3 gene, and APIP gene.

[0130] If the results are as follows, the bovine embryonic cells to be tested are E10 hypoblast:

[0131] Express or overexpress one or more of ENSBTAG00000026119 gene, APOA2 gene, APOA1 gene, FABP3 gene, FN1 gene, MYL7 gene, RSPO3 gene, CTSV gene, MSMO1 gene, and PPP1R14A gene.

[0132] In another aspect, the present invention provides a composition comprising any of the above compositions and / or the above gene markers.

[0133] In another aspect, the present invention provides a method for identifying the development of bovine embryonic cells, the method comprising detecting the bovine embryonic cells to be detected by any one or more methods selected from the following:

[0134] (1) Identification using genetic markers;

[0135] (2) using signaling pathway identification;

[0136] (3) using signaling pathway-related genes for identification;

[0137] The gene marker is the gene marker according to claim 5, the signal pathway is the signal pathway according to any one of claims 1 or 4, and the signal pathway-related gene is the gene according to any one of claims 1 or 4;

[0138] If the results are as follows, the bovine embryonic cell to be tested is an E5 anterior inner cell mass cell:

[0139] Expression or high expression of the PDGFRA gene and / or lack of or low expression of one or more of the NANOG gene, OU5F1 gene, DAB2 gene, or CDX2 gene;

[0140] If the results are as follows, the bovine embryonic cells to be tested are E5 pretrophoblast cells:

[0141] Not expressing or under-expressing the PDGFRA gene and / or expressing or over-expressing one or more of the NANOG gene, OU5F1 gene, DAB2 gene, or CDX2 gene;

[0142] If the results are as follows, the bovine embryonic cell to be tested is an E6 inner cell mass cell:

[0143] expressing or overexpressing one or more of the PDGFRA gene and the NANOG gene and / or not expressing or underexpressing one or more of the POU5F1 gene, the DAB2 gene, or the CDX2 gene;

[0144] If the results are as follows, the bovine embryonic cells to be tested are E6 trophoblast cells:

[0145] Not expressing or under-expressing one or more of the PDGFRA gene and the NANOG gene and / or expressing or over-expressing one or more of the POU5F1 gene, the DAB2 gene, or the CDX2 gene;

[0146] If the results are as follows, the bovine embryonic cell to be tested is an E7 inner cell mass cell:

[0147] expressing or overexpressing one or more of the PDGFRA gene, the NANOG gene, and the POU5F1 gene, and / or not expressing or underexpressing one or more of the DAB2 gene and the CDX2 gene;

[0148] If the results are as follows, the bovine embryonic cells to be tested are E7 trophoblast cells:

[0149] Not expressing or under-expressing one or more of the PDGFRA gene, NANOG gene, and POU5F1 gene, and / or expressing or over-expressing one or more of the DAB2 gene and CDX2 gene;

[0150] If the results are as follows, the bovine embryonic cells to be tested are E10 or E12 epiblast cells:

[0151] expressing or overexpressing one or more of the NANOG gene, POU5F1, and TDGF1 gene, and / or not expressing or underexpressing one or more of the GATA4 gene, GATA6 gene, PDGFRA gene, SOX17 gene, DAB2 gene, and CDX2 gene;

[0152] If the results are as follows, the bovine embryonic cells to be tested are E10 or E12 hypoblast cells:

[0153] expressing or overexpressing one or more of the GATA4 gene, GATA6 gene, PDGFRA gene, and SOX17 gene, and / or not expressing or underexpressing one or more of the NANOG gene, POU5F1 gene, TDGF1 gene, DAB2 gene, or CDX2 gene;

[0154] If the results are as follows, the bovine embryonic cells to be tested are E10 or E12 trophoblasts:

[0155] Not expressing or under-expressing one or more of the NANOG gene, POU5F1 gene, TDGF1 gene, GATA4 gene, GATA6 gene, PDGFRA gene, SOX17 gene and / or expressing or over-expressing one or more of the DAB2 gene or CDX2 gene

[0156] If the results are as follows, the bovine embryonic cells to be tested are E14 ectoderm cells:

[0157] expressing or overexpressing one or more of the NANOG gene, POU5F1 gene, TDGF1 gene, and FGFR1 gene, and / or not expressing or underexpressing one or more of the MESP1 gene, KDR gene, TBX6 gene, FOXF1 gene, PMP22 gene, LAMA1 gene, FN1 gene, IHH gene, FOXA2 gene, PDGFRA gene, SOX17 gene, NANOS3 gene, PRDM1 gene, DAB2 gene, and CDX2 gene;

[0158] If the results are as follows, the bovine embryonic cells to be tested are E14 mesoderm cells:

[0159] expressing or overexpressing one or more of the MESP1 gene, KDR gene, TBX6 gene, FOXF1 gene, and PDGFRA gene, and / or not expressing or underexpressing one or more of the NANOG gene, POU5F1 gene, TDGF1 gene, FGFR1 gene, PMP22 gene, LAMA1 gene, FN1 gene, IHH gene, FOXA2 gene, SOX17 gene, NANOS3 gene, PRDM1 gene, DAB2 gene, and CDX2 gene;

[0160] If the results are as follows, the bovine embryonic cells to be tested are E14 endoderm cells:

[0161] expressing or overexpressing one or more of the TDGF1 gene, FGFR1 gene, PMP22 gene, LAMA1 gene, FN1 gene, IHH gene, FOXA2 gene, PDGFRA gene, and SOX17 gene, and / or not expressing or underexpressing one or more of the NANOG gene, POU5F1 gene, MESP1 gene, KDR gene, TBX6 gene, FOXF1 gene, NANOS3 gene, PRDM1 gene, DAB2 gene, and CDX2 gene;

[0162] If the results are as follows, the bovine embryonic cells to be tested are E14 primordial germ cell-like cells;

[0163] expressing or overexpressing one or more of the TDGF1 gene, SOX17 gene, NANOS3 gene, and PRDM1 gene, and / or not expressing or underexpressing one or more of the NANOG gene, POU5F1 gene, MESP1 gene, KDR gene, TBX6 gene, FOXF1 gene, FGFR1 gene, PMP22 gene, LAMA1 gene, FN1 gene, IHH gene, FOXA2 gene, PDGFRA gene, DAB2 gene, and CDX2 gene;

[0164] If the results are as follows, the bovine embryonic cells to be tested are E14 trophoblast cells:

[0165] expressing or overexpressing one or more of the FOXF1 gene, DAB2 gene, and CDX2 gene, and / or not expressing or underexpressing one or more of the NANOG gene, POU5F1 gene, TDGF1 gene, MESP1 gene, KDR gene, TBX6 gene, FGFR1 gene, PMP22 gene, LAMA1 gene, FN1 gene, IHH gene, FOXA2 gene, PDGFRA gene, SOX17 gene, NANOS3 gene, and PRDM1 gene;

[0166] If the results are as follows, the bovine embryonic cells to be tested are at the naive pluripotency stage:

[0167] If the test result is positive, the bovine embryonic cell to be tested is an E5 anterior inner cell mass, an E6 inner cell mass, an E7 inner cell mass, or any combination thereof;

[0168] If the results are as follows, the bovine embryonic cells to be tested are at the intermediate pluripotency stage:

[0169] If the test result is positive, the bovine embryonic cell to be tested is E10 epiblast, E12 epiblast, or any combination thereof;

[0170] If the results are as follows, the bovine embryonic cells to be tested are at the primed pluripotent stage:

[0171] If the test result is yes, then the bovine embryonic cell to be tested is E14 ectoderm;

[0172] In certain embodiments, the detection is expression level detection;

[0173] In certain embodiments, the detection is single-cell or multi-cell transcriptome analysis;

[0174] In certain embodiments, the bovine embryonic cell to be tested is selected from bovine embryos E5-E14;

[0175] In certain embodiments, the bovine embryo E5-E14 is selected from E5, E6, E7, E10, E12 or E14, or any combination thereof.

[0176] In certain embodiments, the method further comprises comparing the test results of the bovine embryonic cell to be tested with the embryonic lineage and pluripotency stage characteristics established for pigs to determine the bovine embryonic cell developmental characteristics of the bovine embryonic cell to be tested;

[0177] In certain embodiments, the bovine embryonic cell is selected from the group consisting of anterior inner cell mass, inner cell mass, epiblast, and ectoderm.

[0178] In certain embodiments, the bovine embryo is a bovine embryo E1-E14;

[0179] In certain embodiments, the bovine embryos E1-E14 are selected from E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, E12, E13 or E14, or any combination thereof.

[0180] In another aspect, the present invention provides use of any of the above compositions or markers in identifying the pluripotency stage of bovine embryonic cells.

[0181] In another aspect, the present invention provides use of any of the above compositions or markers in identifying the developmental stage of bovine embryonic cells.

[0182] Beneficial effects

[0183] The present invention discloses a method for identifying the pluripotency state of bovine epiblast, achieving the technical effect of identifying the pluripotency state of bovine embryos, particularly the pluripotency state of bovine epiblast. Specifically disclosed are gene markers for different pluripotency stages and developmental stages. Using these gene markers for different pluripotency stages and developmental stages of bovine embryos, the pluripotency and developmental stage of bovine embryonic cells or cell clusters to be tested can be rapidly identified. BRIEF DESCRIPTION OF THE DRAWINGS

[0184] Figure 1 Deciphering lineage differentiation in bovine embryos by single-cell transcriptome sequencing.

[0185] A: Morphology of bovine embryos collected for scRNA-seq analysis, covering six different developmental stages: E5 (late morula), E6 (early blastocyst), E7 (late blastocyst), E10 (early double embryo), E12 (late double embryo), and E14 (primitive streak embryo). The scale bar for E5-E7 and E10 embryos is 100 μm, while the scale bar for E12 and E14 embryos is 1 mm. The scale bar for the E14 embryonic disc is 200 μm. The dotted box indicates the location of the embryonic disc at the E10, E12, and E14 stages.

[0186] B; UMAP plot showing transcriptional similarity across all bovine embryonic cells. Colored dots represent the indicated embryonic days, and arrows indicate known developmental trajectories.

[0187] C: UMAP plots provide insights into cell typing at each embryonic stage, with different cell types represented by dots of different colors.

[0188] DF: Dot plot visualization of expression patterns of signature genes for different cell types in E5-E7 embryos (D), E10 and E12 embryos (E), and E14 embryos (F). Dot size encodes the percentage of cells within a cell type, and color encodes the average expression level. See also Figure 4 and Figure 5 .

[0189] Figure 2 Dynamic tracking of bovine epiblast pluripotency.

[0190] A: UMAP plots depicting scRNA-seq data obtained from bovine embryonic lineages, including E5-E7 ICMs, E10 and E12 epiblast, and E14 ectoderm.

[0191] B: Spearman correlation coefficient based on the mean expression levels of uniquely expressed genes in cells of each embryonic lineage.

[0192] C: GO term map of pre-ICMs / ICMs, EPIs, and ECTOs-specific genes enriched.

[0193] D: Representative gene clusters with similar expression trends are shown in pre-ICM, ICM, epiblast, and epiblast cells from E5 to E14. Changes in formative and primed pluripotency genes.

[0194] E: Heat map showing expression changes of genes related to the JAK / STAT3, TGFβ / SMADs, FGF / ERK, and Wnt / β-catenin signaling pathways in selected bovine embryonic lineages.

[0195] Figure 3 Comparative analysis of lineage development and pluripotency changes in bovine and porcine embryos.

[0196] A: Principal component analysis of single-cell data from bovine and porcine embryos. Circles and triangles represent bovine and porcine, respectively. Clusters are color-coded according to species and embryonic lineage.

[0197] B: Spearman correlation coefficients were calculated for bovine and porcine embryonic lineages.

[0198] C; Pseudotemporal analysis of bovine and porcine embryonic lineages.

[0199] D: Schematic representation of the different pluripotent states in the bovine and porcine embryonic lineages.

[0200] E: Heat map showing the lineages of bovine and porcine embryos Representative marker genes for formative and primed pluripotent states.

[0201] F: Heatmaps of representative gene expression related to signaling pathways in bovine and porcine embryonic lineages.

[0202] G: GO / KEGG enrichment terms of DEGs during the pluripotency changes of selected bovine and porcine embryonic lineages.

[0203] Figure 4 ForFigure 1 Segregation and identification of related bovine embryonic developmental lineages.

[0204] A and B: Developmental trajectories inferred by cell type or lineage (A) and visualized (B) using pseudotime analysis of Mono3.

[0205] C: Representative GO biological process enrichment terms are proposed based on the genes associated with the identified cell types.

[0206] D: Heatmap of lineage-specific gene expression. In each cell type, genes were ranked by unsupervised hierarchical clustering (UHC).

[0207] Figure 5 are predicted marker genes for EPI, HYPO and TE lineages in bovine E10 embryos. Figure 1 Related.

[0208] AC: Prediction of EPI (A), HYPO (B), and TE (C) lineage marker genes in bovine E10 embryos. DETAILED DESCRIPTION

[0209] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0210] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0211] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.

[0212] The gene names involved in this application are all human gene names. The RNA sequences of the genes involved in this application have 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60% and / or lower similarities with the RNA sequences of the corresponding human genes, and have the same functions as the corresponding human genes.

[0213] Experimental model and study participant details

[0214] Material availability

[0215] All bEpiSCs generated in this study are available from the primary contact with a complete material transfer agreement.

[0216] Data and code availability

[0217] The scRNA-seq and RNA-seq datasets generated during this study are available on Gene Expression Omnibus (GEO) with the following accession code: GSE256201.

[0218] Animal treatment and ethics statement

[0219] All experimental procedures for mice and dairy cows were approved in advance by the Laboratory Animal Welfare and Animal Experimentation Ethics Review Committee of China Agricultural University, approval number AW10204202-3-1.

[0220] mice

[0221] (ICR) IGS and BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and used for the isolation of mouse embryonic fibroblasts (MEFs) and the bEpiSCs teratoma formation assay. MEFs were treated with mitomycin C (Selleckchem, S8146) to prepare feeder cells for bEpiSCs.

[0222] ox

[0223] Bovine single-cell collection and single-cell transcriptome analysis were performed using Holstein-Friesian embryos at stages E5, E6, E7, E10, E12, and E14. Embryonic day n (E(n)) embryos were obtained at day n post-coitum. For the derivation of bEpiSCs, embryos at E7, E10, E12, and E14 were used.

[0224] Bovine embryo collection and embryonic single cell isolation

[0225] The embryonic cells used in this study were derived entirely from Holstein cow embryos. E5-E7 embryos were obtained by thawing and culturing frozen bovine embryos until the desired developmental stage was reached. The zona pellucida was treated with Pronase (Protease Pronase, Sigma, 10165921001) for 15-30 seconds and then washed and removed using a solution consisting of DPBS+0.1% BSA. Subsequently, the embryonic cells were mechanically separated and transferred to a lysis buffer. E10, E12, and E14 embryos were obtained by in vivo transplantation and subsequent rinsing, and single embryonic cells were separated and collected by a combination of enzyme treatment and mechanical manipulation.

[0226] Derivation of bovine ICMs, epiblast, and epiblast bEpiSCs

[0227] Bovine ICMs, epiblast, and ectoderm were separated by mechanical dissociation and stained with TrypLE TM The cells were treated with 5% FcGen Express (an animal-derived recombinant enzyme primarily used to dissociate various adherent mammalian cells, Gibco, 12605010) for 3 minutes and then plated onto feeder cells supplemented with 3i / LAF medium. The cultures were incubated at 37°C in 5% O₂ and 5% CO₂. Spherical outgrowths were harvested and digested using Accutase Cell Dissociation Reagent (Gibco, A11105-01) and passaged every 3 days at a 1:4 ratio.

[0228] Experimental methods

[0229] Single-cell RNA library preparation and sequencing

[0230] As previously studied 7,8Single-cell RNA-seq libraries were prepared using a modified Smart-seq2 protocol as described in . Briefly, single embryonic cells were transferred to lysis buffer containing an 8 bp barcode. Subsequently, first-strand cDNA was reverse synthesized and amplified in a reverse transcription (RT) mix consisting of 4 U RNase inhibitor, 100 U SuperScript II reverse transcriptase (Invitrogen, 18064071), 1 mM dNTPs (TAKARA, 4019), 60 mM MgCl2, and 3 μM RT primer with 10 μM TSO primer. After PCR amplification, the product was purified using 0.8× Beckman's AMPure XP beads (A63882). Biotin PCR enrichment was then performed to further improve library quality. Finally, single-cell RNA-seq libraries were constructed according to the instructions provided by the KAPA PCR Library Amplification / Illumina Series (KAPA KK8054). High-quality libraries were sequenced using the Illumina HiSeq Xten platform (Novogene), with paired-end reads of 150 bp in length. The primers used in the experiments are listed in the Key Resources table.

[0231] RT–qPCR

[0232] Total RNA was extracted from bEpiSCs using the RNA prep Pure Cell / Bacteria kit (TIANGEN, DP430) and then reverse transcribed into cDNA using 5× All-In-One RT Master Mix (Abm, G490). PCR amplification was then performed on the Archimed Real Time System (ROCGENE) using 2× RealStar Green Power Mixture (GenStar, A311-05). CT (2 -ΔΔCT ) method. ΔC values ​​were calculated using GAPDH as an internal control. All experiments were performed in triplicate. Primer sequences used for real-time PCR can be found in the Key Resources table.

[0233] RNA-seq for rRNA depletion

[0234] Total RNA was extracted from four bEpiSCs samples using the RNeasy Mini kit (Qiagen, 74106). To construct strand-specific RNA-seq libraries, we used an rRNA removal protocol (Globin-Zero Gold rRNARemoval Kit, Illumina, GZG1224) combined with Ultra TM Directional RNA Library PrepKit for All libraries were quantified using the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen, Life Technologies, Q32851) and sequenced on the Illumina HiSeq4000 platform.

[0235] Quantitative statistical analysis

[0236] Single-cell RNA-seq low-level processing and filtering

[0237] For the STRT-seq dataset, raw reads were segmented by the 8-bp cell barcode located on Read 2, allowing for two mismatches. In addition, the 8-bp unique molecular identifiers (UMIs) located on Read 2 were switched to the identifier line of the paired Read 1. Read 1 was then processed to remove template switching oligonucleotide (TSO) primers, low-quality bases, and polyA sequences. 7 The trimmed reads were aligned to their respective reference genomes (bovine: Bos_taurus.ARS_UCD.12; porcine: Sscrofa1.1). Unique molecular identifiers (UMIs) were generated using kallisto (v-0.46.0). 5 count.

[0238] Identification of differentially expressed genes at embryonic stages across different lineages

[0239] Based on the differentiation process during bovine embryonic development, we divided bovine embryonic cells into three major lineages: the embryonic lineage, including the pre-ICM (inner cell mass) at E5, the ICM (inner cell mass) at E6 and E7, the epiblast at E10 and E12, and the epiblast at E14; the TE (trophectoderm) lineage, including the pre-TE at E5 (pre-trophectoderm at E5 (late morula)), the TE at E6, E7, E10, E12, and E14; and the hypoblast lineage, including the hypoblast at E10 and E12, and the definitive endoderm ( Figure 1 Middle C).

[0240] Construction of expression tendency

[0241] To track the dynamic changes of DEGs during embryonic development (differentially expressed genes (DEGs) in the trophectoderm (TE) lineage exhibit distinct expression trends, and the expression patterns of these genes at different developmental stages reflect the differentiation and functional changes of the trophectoderm), we constructed the expression trends of DEGs in the ectoderm lineage. We first calculated the average expression level of each gene in each lineage at a specific embryonic developmental time point. The average expression levels of the embryonic lineages were rescaled and analyzed using the k-means clustering method with parameters k = 10 and iter.max = 100. DEGs with similar trends during embryonic development were grouped into separate clusters.

[0242] Cross-species comparative analysis

[0243] We downloaded the orthologous gene lists of the four species using the BioMart tool in the genome browser 105 (http: / / dec2021.archive.ensembl.org / index.html) and retained 16,841 genes, all of which were 1:1 orthologous genes. We then retained 1:1 orthologous genes in the bovine and porcine embryonic lineage datasets. For the integrated analysis between the porcine and bovine embryonic datasets, we used the Seurat CCA method for anchoring and dataset alignment. The top 2,000 features with repeated variables in the dataset were selected, and the “FindIntegrationAnchors” function with the following parameters was used to identify anchors: “reduction = 'cca, k, anchor = 5, normalization. Method = 'SCT'”. Then, based on the identified anchors, the dataset was integrated using the “IntegrateData” function with the following parameters: “dims = 1:30, k.weight = 50, normalization. Method = 'SCT'”. The “AverageExpression” function was used to obtain the average expression value of genes in the comprehensive determination, and the “cor” function was used to calculate the Spearman correlation coefficient between different cell types of the two species. The Wilcoxon rank sum test was used to determine the expression of genes in the two species using the “FindMarkers” function. Fold changes in gene expression levels between the formative state and the primed state, and between the formative state and the primed state. Only those with |'avg_logFC'|>0.25 and 'p_val'<0.05 were considered DEGs.

[0244] Pseudo-time analysis

[0245] Using the R package Monocle3 (v-1.3.1) 45The developmental trajectory of embryonic cells was reconstructed. The UMI matrix was used as input, and the cells were pseudo-timed using the variable genes obtained from Seurat. Destiny (v-2.14.0) R package was used to further analyze the cells. 46 The developmental order of individual cells from the two species was determined according to the differentially expressed genes (DEGs) between all embryonic day cells of pigs calculated by the “FindAllMarkers” function.

[0246] RNA-seq data processing and analysis

[0247] The expression levels of protein-coding genes (from the gene annotation file [GTF] of Ensembl-Bos_taurus.ARS_UCD.12) were quantified as transcripts per million (TPM) using kallisto (v-0.46.0). 6 To identify DEGs between different cell types, we used a Benjamini-Hochberg-adjusted false discovery rate (FDR) < 0.05 and an absolute log2 (fold change) > 2 as cutoffs for statistical significance.

[0248] Correlation analysis of transcriptomes between bEpiSCs and embryonic cells

[0249] For the integrated analysis between the bEpiSC and bovine embryo datasets, we used the Seurat RPCA method for anchoring and dataset alignment. We selected the top 2000 features with reproducible variability across the datasets and identified anchors using the "FindIntegrationAnchors" function with the following parameters: "reduction = 'rpca', k, anchor = 15." The datasets were then integrated based on the identified anchors using the "IntegrateData" function with the following parameters:

[0250] Scaling and PCA were applied on the combined dataset and the integrated PCA coordinates were used as input to the clustering and t-SNE visualization workflow with the following parameters: ‘dims=1:5’. Key markers were visualized using the ‘FeaturePlot’ function.

[0251] Functional enrichment analysis

[0252] Using Metascape http: / / metascape.org) Functional enrichment analysis was performed on the selected genes. Bovine genes were mapped to their human orthologs, with humans (Homo sapiens) being the target species for the analysis. Enrichment analysis was performed using all genes in the genome as a background set, with Gene Ontology (GO)-Biological Process (GO-BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways as ontology sources. Terms with a minimum count ≥ 3, adjusted P < 0.01, and enrichment factor ≥ 1.5 were considered significant, and similar terms were grouped into clusters. Using -log 10 (P value) Histogram depicts key pathways.

[0253] Table 3 Key resources

[0254]

[0255]

[0256]

[0257]

[0258] Example 1: Elucidating lineage separation during bovine embryonic development by single-cell transcriptome sequencing

[0259] To elucidate the molecular basis of early bovine embryonic development and pluripotency changes, combined with our insights into livestock embryonic development, we optimized single-cell collection at key time points (E5, E6, E7, E10, E12, and E14) in bovine embryos. Figure 1 The transcriptomes of 616 single cells were sequenced using a modified single-cell tag reverse transcription sequencing (STRT-seq) protocol (method details), and 456 single cells were ultimately retained for subsequent analysis after strict quality control (Table 2).

[0260] By identifying key markers of different embryonic lineages during embryonic development, we defined the inner cell mass (ICM) and trophectoderm (TE) in E5-E7 embryos, and the epiblast (EPI), hypoblast (HYPO), and TE in E10 and E12 embryos. In addition, we characterized various cell types in embryos at the early primitive streak stage at E14, including the ectoderm (ECTO), mesoderm, definitive endoderm, TE, and primordial germ cells (PGCs) ( Figure 1B, C, and Table 1), and further described the lineage differentiation process during bovine embryonic development at the single-cell level using pseudotime analysis ( Figure 4 A in 4 and B in 4).

[0261] Based on this comprehensive characterization, we performed gene ontology analysis to identify differential expression patterns across these different embryonic lineages ( Figure 4 C and D). Gene expression analysis showed:

[0262] The pre-ICM lineage highly expresses PDGFRA, and the pre-TE lineage highly expresses CDX2, DAB2, POU5F1, and NANOG genes, which is similar to what we observed in the first lineage differentiation of pig embryos. 1 With the formation of ICM and TE lineages, POU5F1, NANOG and other pluripotency genes gradually began to be upregulated in ICM (PDGFRA and NANOG genes were highly expressed in E6-ICM, PDGFRA, NANOG and POU5F1 genes were highly expressed in E7-ICM), but were downregulated in TE (CDX2, DAB2 and POU5F1 genes were highly expressed in E6-TE, CDX2 and DAB2 genes were highly expressed in E7-TE) ( Figure 1 (D in the middle). Embryos at E10-E12 already have clear epi, hyper, and endothelial cells (EPIs), which are highly expressed in E10 and E12 epithelial cells (EPIs), including TDGF1, POU5F1, and NANOG. Hypothelial cells at E10 and E12 highly express SOX17, PDGFRA, GATA6, and GATA4. TEs at E10 and E12 highly express CDX2 and DAB2. Figure 1 Middle E):

[0263] E5 anterior inner cell mass: associated with the expression of PDGFRA gene, NANOG gene, OU5F1 gene, DAB2 gene or CDX2 gene; E5 anterior trophoblast: associated with the expression of PDGFRA gene, NANOG gene, OU5F1 gene, DAB2 gene or CDX2 gene; E6 inner cell mass: associated with the expression of PDGFRA gene, NANOG gene, POU5F1 gene, DAB2 gene or CDX2 gene; E6 trophoblast: associated with the expression of PDGFRA gene, NANOG gene, POU5F1 gene, DAB2 gene or CDX2 gene; E7 inner cell mass: associated with the expression of PDGFRA gene, NANOG gene, POU5F1 gene, DAB2 gene or CDX2 gene OG gene, POU5F1 gene, DAB2 gene, CDX2 gene expression; E7 trophoblast: related to the expression of PDGFRA gene, NANOG gene, POU5F1, DAB2 gene, CDX2 gene; E10 or E12 epiblast: express or highly express one or more of NANOG gene, POU5F1, TDGF1 gene and / or do not express or low express one or more of GATA4 gene, GATA6 gene, PDGFRA gene, SOX17 gene, DAB2 gene, CDX2 gene; E10 or E12 hypoblast: express or highly express one or more of NANOG gene, POU5F1, TDGF1 gene and / or do not express or low express one or more of GATA4 gene, GATA6 gene, PDGFRA gene, SOX17 gene, DAB2 gene, CDX2 gene; The trophoblast of E10 or E12 is related to the expression of NANOG gene, POU5F1 gene, TDGF1 gene, DAB2 gene or CDX2 gene; the ectoderm of E14 is related to the expression of NANOG gene, POU5F1 gene, TDGF1 gene, FGFR1 gene, MESP1 gene, KDR gene, TBX6 gene, FOXF1 gene, PMP22 gene, LAMA1 gene, F E14 mesoderm: correlated with the expression of MESP1, KDR, TBX6, FOXF1, PDGFRA, NANOG, POU5F1, TDGF1, FGFR1, PMP22, LAMA1, FN1, IHH, FOXA2, SOX17, NANOS3, PRDM1, DAB2, and CDX2 genes;E14 endoderm: associated with the expression of TDGF1 gene, FGFR1 gene, PMP22 gene, LAMA1 gene, FN1 gene, IHH gene, FOXA2 gene, PDGFRA gene, SOX1, NANOG gene, POU5F1 gene, MESP1 gene, KDR gene, TBX6 gene, FOXF1 gene, NANOS3 gene, PRDM1 gene, DAB2 gene, and CDX2 gene; E14 primordial germ cell-like cells; associated with the expression of TDGF1 gene, SOX17 gene, NANOS3 gene, PRDM1 gene, NANOG gene, POU5F1 gene, MESP1 gene, KDR gene, and TBX6 gene , FOXF1 gene, FGFR1 gene, PMP22 gene, LAMA1 gene, FN1 gene, IHH gene, FOXA2 gene, PDGFRA gene, DAB2 gene, and CDX2 gene expression; E14 trophoblast: correlated with the expression of FOXF1 gene, DAB2 gene, CDX2 gene, NANOG gene, POU5F1 gene, TDGF1 gene, MESP1 gene, KDR gene, TBX6 gene, FGFR1 gene, PMP22 gene, LAMA1 gene, FN1 gene, IHH gene, FOXA2 gene, PDGFRA gene, SOX17 gene, NANOS3 gene, and PRDM1 gene (; Figure 1 Middle DF);

[0264] We also defined genes that are highly expressed within the EPI / HYPO / TE lineages in E10 embryos. These genes could serve as potential markers for lineage identification during bovine embryonic development ( Figure 5 (AC):

[0265] E10 TE alone or compared with E10 HYPO and E10 EPi, expressed or overexpressed LGALS3 gene, GPD1L gene, PTGS2 gene, SLC34A2 gene, KRT18 gene, KRT8 gene, PLAC8A gene, DAB2 gene, PLS1 gene, and TKDP1 gene.

[0266] E10 HYPO alone or compared with E10 TE and E10 EPi, expressed or overexpressed ENSBTAG00000026119 gene, APOA2 gene, APOA1 gene, FABP3 gene, FN1 gene, MYL7 gene, RSPO3 gene, CTSV gene, MSMO1 gene, and PPP1R14A gene.

[0267] E10 EPi alone or compared with E10 TE and E10 HYPO, expressed or overexpressed STMN1 gene, H3-3A gene, UPP1 gene, MS4A8 gene, STMN2 gene, NANOG gene, ENSBTAG00000051318 gene, BBX gene, SYCP3 gene, and APIP gene.

[0268] We further identified genes specifically expressed in the pre-inner cell mass (pre-ICM), pre-trophoblast (pre-TE), inner cell mass (ICM), trophoblast (TE), epiblast, hypoblast, ectoderm, mesoderm, endoderm, or primordial germ cell-like cells (PSCs). After data analysis, the top 10 most significantly expressed genes were selected as specific markers, as shown in Tables 4-13.

[0269] The anterior inner cell mass-specifically expressed genes are shown in Table 4, specifically as follows:

[0270] Table 4

[0271] p_val avg_log2FC pct.1 pct.2 p_val_adj cluster gene humangenename 1.90044E-37 2.419605993 1 0.444 3.27179E-33 Pre-ICM SPIC SPIC 7.37727E-35 1.034388633 0.976 0.466 1.27007E-30 Pre-ICM MYOM1 MYOM1 7.72316E-35 1.312843231 1 0.995 1.32962E-30 Pre-ICM PARP1 PARP1 1.43452E-31 1.782432224 1 0.744 2.46967E-27 Pre-ICM GPCPD1 GPCPD1 1.09351E-29 1.445692589 1 0.867 1.88258E-25 Pre-ICM RPIA RPIA 3.17351E-29 0.944336713 0.976 0.49 5.46351E-25 Pre-ICM RAB7B RAB7B 5.09014E-28 0.76619255 0.976 0.44 8.76319E-24 Pre-ICM PPM1K PPM1K 9.85096E-28 2.067490615 1 0.998 1.69594E-23 Pre-ICM CKB CKB 8.09644E-26 1.217605742 1 1 1.39388E-21 Pre-ICM PRDX... ​ 2.53492E-25 1.263317735 1 0.814 4.36412E-21 ​ ​ ​

[0272] The anterior trophoblast-specific expression genes are shown in Table 5, specifically as follows:

[0273] Table 5

[0274] ​ ​ ​ ​ ​ ​ ​ ​ 2.62163E-30 2.3780502 1 0.991 4.51339E-26 ​ ​ ​ 1.11682E-28 1.557172541 1 0.984 1.92271E-24 ​ ​ ​ 3.73159E-28 0.860124702 0.967 0.197 6.4243E-24 ​ ​ ​ 5.84551E-28 1.363970637 1 0.754 1.00636E-23 ​ ​ ​ 6.66135E-28 0.541466986 0.933 0.124 1.14682E-23 ​ ​ ​ 1.20131E-27 1.753168587 1 0.662 2.06818E-23 ​ ​ ​ 1.20795E-27 1.572720418 1 0.995 2.07961E-23 ​ ​ ​ 1.35031E-27 1.895420744 1 0.845 2.32469E-23 ​ ​ ​ 1.71531E-27 1.229724515 1 0.998 2.95308E-23 ​ ​ ​ 6.4747E-27 1.508933453 1 0.995 1.11468E-22 ​ ​ ​

[0275] The inner cell mass-specifically expressed genes are shown in Table 6, specifically as follows:

[0276] Table 6

[0277]

[0278]

[0279] The trophoblast-specific expression genes are shown in Table 7, specifically as follows:

[0280] Table 7

[0281] ​ ​ ​ ​ ​ ​ ​ ​ 1.31205E-83 2.808084206 1 0.993 2.25882E-79 ​ ​ ​ 4.93624E-82 2.508683301 1 1 8.49824E-78 ​ ​ ​ 1.17543E-79 1.797629693 0.994 0.985 2.02362E-75 ​ ​ ​ 3.30758E-79 0.972310216 0.95 0.84 5.69432E-75 ​ ​ ​ 5.81398E-79 1.314622004 0.983 0.924 1.00093E-74 ​ ​ ​ 2.33163E-78 1.524149994 1 0.996 4.01413E-74 ​ ​ ​ 9.32546E-78 2.402358508 0.994 0.902 1.60547E-73 ​ ​ ​ 1.46721E-76 2.269401505 1 1 2.52595E-72 ​ ​ ​ 1.79987E-76 1.333846454 0.95 0.818 3.09865E-72 ​ ​ ​ 1.02192E-75 1.272208464 0.917 0.669 1.75935E-71 ​ ​ ​

[0282] The epiblast-specific expression genes are shown in Table 8, specifically as follows:

[0283] Table 8

[0284] ​ ​ ​ ​ ​ ​ ​ ​ 1.95861E-65 1.681851002 1 0.602 3.37194E-61 ​ ​ ​ 1.66336E-60 1.220865874 1 0.77 2.86363E-56 ​ ​ ​ 8.44314E-59 0.986264012 0.977 0.629 1.45357E-54 ​ ​ ​ 5.73944E-58 1.904835483 1 0.954 9.88101E-54 ​ ​ ​ 1.18309E-57 1.644438779 1 0.984 2.0368E-53 ​ ​ ​ 7.2469E-56 1.431036991 1 1 1.24763E-51 ​ ​ ​ 1.64531E-54 1.580356586 1 0.965 2.83256E-50 ​ ​ ​ 5.82176E-51 1.534677558 1 0.813 1.00227E-46 ​ ​ ​ 1.51171E-49 0.878442723 0.989 0.64 2.60256E-45 ​ ​ ​ 3.63487E-48 0.831050015 0.989 0.553 6.25779E-44 ​ ​ ​

[0285] The hypoblast-specific expression genes are shown in Table 9, specifically as follows:

[0286] Table 9

[0287]

[0288]

[0289] Ectoderm-specific expression genes are shown in Table 10, specifically as follows:

[0290] Table 10

[0291] ​ ​ ​ ​ ​ ​ ​ ​ 1.73623E-30 2.950222435 1 0.895 2.98909E-26 ​ ​ ​ 1.30242E-24 0.757479357 1 0.584 2.24225E-20 Ectoderm IDH2 IDH2 2.63496E-24 0.723016113 0.964 0.421 4.53634E-20 Ectoderm TAPBP TAPBP 3.86205E-24 1.370859417 1 0.738 6.64891E-20 Ectoderm SLC7A3 SLC7A3 6.30732E-24 0.861270288 1 0.523 1.08587E-19 Ectoderm LCK LCK 3.76971E-22 0.766050635 1 0.862 6.48993E-18 Ectoderm CIAPIN1 CIAPIN1 4.40726E-22 1.542101271 1 0.734 7.58754E-18 Ectoderm ERAS ERAS 5.5239E-22 1.751173192 1 0.942 9.50995E-18 Ectoderm UCHL1 UCHL1 1.87086E-21 1.080798398 1 0.787 3.22088E-17 Ectoderm SCCPDH SCCPDH 3.46771E-21 1.229138871 1 0.998 5.97002E-17 Ectoderm ENO1 ENO1

[0292] Mesoderm-specific expression genes are shown in Table 11, specifically as follows:

[0293] Table 11

[0294] p_val avg_log2FC pct.1 pct.2 p_val_adj cluster gene humangenename 3.03204E-12 0.308500421 0.857 0.045 5.21996E-08 Mesoderm GPM6A GPM6A 4.01872E-12 0.268572372 1 0.071 6.91864E-08 Mesoderm MESP1 MESP1 5.20024E-12 1.916123786 1 0.459 8.95273E-08 Mesoderm MYL4 MYL4 1.74011E-11 1.259497301 1 0.659 2.99578E-07 Mesoderm TUBA4A TUBA4A 2.02738E-11 0.39909473 0.857 0.111 3.49035E-07 Mesoderm TBX6 TBX6 1.06023E-10 1.550622891 1 0.768 1.8253E-06 Mesoderm CRABP2 CRABP2 1.0671E-10 1.50252446 1 1 1.83712E-06 Mesoderm ALDOA ALDOA 6.51571E-10 1.0042629 1 0.494 1.12175E-05 Mesoderm IGFBP4 IGFBP4 1.43035E-09 0.795709652 1 0.205 2.46248E-05 Mesoderm FGF17 FGF17 2.78982E-09 1.373059873 1 0.993 4.80296E-05 Mesoderm PGK1 PGK1

[0295] Endoderm-specific expression genes are shown in Table 12, specifically as follows:

[0296] Table 12

[0297]

[0298]

[0299] The primordial germ cell-like cell-specifically expressed genes are shown in Table 13, specifically as follows:

[0300] Table 13

[0301] p_val avg_log2FC pct.1 pct.2 p_val_adj cluster gene humangenename 1.852E-09 1.71549255 1 0.117 3.1884E-05 PGCs NANOS3 NANOS3 1.852E-09 2.671807172 1 0.618 3.1884E-05 PGCs FABP7 FABP7 1.852E-09 2.685802392 1 0.947 3.1884E-05 PGCs PLIN2 PLIN2 1.852E-09 2.92967847 1 1 3.1884E-05 PGCs SLC25A5 SLC25A5 1.852E-09 1.115794283 1 0.274 3.1884E-05 PGCs MFSD2A MFSD2A 8.0267E-09 1.298095314 1 0.585 0.000138188 PGCs ACSF2 ACSF2 1.79874E-08 2.8228623 1 0.854 0.000309671 PGCs IFI30 IFI30 3.01954E-08 1.059648471 1 0.351 0.000519843 PGCs DDIT4L DDIT4L 5.3397E-08 0.840703438 1 0.007 0.000919282 PGCs PTPRC PTPRC 5.96832E-08 1.457974096 1 0.605 0.001027506 PGCs LGALS3BP LGALS3BP

[0302] In E14 embryos at the early gastrula stage, we found a cell population with high expression of NANOS3 and PRDM1 genes ( Figure 1 Middle F), which marks the beginning of PGC (primordial germ cell) formation 2、3、9 However, POU5F1 and CDX2 genes showed no differential expression in early bovine embryos during the initial lineage separation period, as observed in pig and human embryos, but different from mouse embryos. 4 .

[0303] In conclusion, single-cell transcriptome sequencing of bovine embryos comprehensively depicts the embryonic lineage and accurately delineates the differentiation trajectory of each germ layer lineage.

[0304] Table 1

[0305]

[0306] Table 2

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321] Example 2 Tracking the dynamic changes of pluripotency during bovine ectoderm development

[0322] To further understand the existence of naive, formative, or primed pluripotency stages during bovine embryonic ectoderm development and to elucidate the potential core regulatory signaling pathways involved, we performed pairwise comparisons and classified 2,267 differentially expressed genes from cells at the six stages into 16 clusters.

[0323] First, we identified 594 upregulated genes in pre-ICMs / ICMs, 552 upregulated genes in EPIs, and 375 upregulated genes in ECTOs. Gene Ontology (GO) term enrichment analysis showed that the biological processes of "respiratory electron transport chain" and "oxidative phosphorylation" were significantly enriched in pre-ICMs / ICMs compared with "glycolysis process" and "hexose metabolism process" observed in ECTOs ( Figure 2 This metabolic shift from oxidative phosphorylation to glycolysis is thought to be the primary mechanism by which embryonic stem cells Pluripotency to Primed Pluripotency10 Interestingly, E10 and E12 EPIs exhibited an intermediate metabolic and developmental state, enriched with graphene oxide associated with “oxidative phosphorylation,” “typical glycolysis,” and “germ cell development.” Figure 2 Middle C). These findings suggest that E10 and E12 EPIs may possess features that establish pluripotency.

[0324] We observed that at the E5-E7 ICM stage, the expression of representative genes associated with naive pluripotency (such as ESRRB, SOX15, SUZ12, KLF4, IL6R, TBX3, and ZFP42) decreased significantly.

[0325] In contrast, representative genes associated with formative pluripotency, including DNMT3B, NANOG, TDGF1, and ZIC2, showed high expression levels in the E10 and E12 ectoderm. In addition, FETUB, GPX4, ID1, ID3, and other genes associated with primed pluripotency and embryonic morphogenesis were upregulated from E7 ICM to E14 ectoderm ( Figure 2 Middle D).

[0326] For ease of understanding and subsequent analysis, we defined E5-E7 ICM as the naive stage, E10-E12 ectoderm as the formative stage, and E14 ectoderm as the primed stage.

[0327] We studied the AK / STAT3 signaling pathway, FGF / ERK signaling pathway, TGFβ / SMAD signaling pathway and WNT / β-catenin signaling pathway, and marked the main related genes, including JAK / STAT3 signaling pathway related genes: IL6ST gene, STAT3 gene, PIK3R2 gene, PIK3R1 gene, LIFR gene, KLF4 gene, TBX3 gene, IL6R gene, PIK3CA gene and JAK2 gene; TGFβ / SMADs signaling pathway related genes: ID1 gene, BMP1 gene, ACVR1 gene, TGFBR2 gene, SMAD5 gene, ID3 gene, BMP7 gene, BMPR2 gene, TGFB1 gene, BMPR1B gene, ACVR1B gene, INGBA gene, NODAL gene , ACVR2A gene, ACVR2B gene, SMAD9 gene, TDGF1 gene, BMP4 gene, SMAD4 gene and BMPR1A gene; FGF / ERK signaling pathway-related genes: MAPK14 gene, MAPK1 gene, ETS1 gene, FGF2 gene, MAPK3 gene, FGFR2 gene, JUN gene, KDR gene, MAP2K2 gene, PDGFA gene, FGFR1 gene, ITGA6 gene, ETS2 gene and ITGA3 gene; WNT / β-catenin signaling pathway-related genes: GSK3B gene, TCF7 gene, CDH12 gene, APC gene, WNT11 gene, FZD1 gene, FZD10 gene, CTNNB1 gene, FZD2 gene, WNT3A gene, WNT5B gene and WNT7B gene.

[0328] Corresponding to the above-mentioned pluripotent state, we observed downregulation of JAK / STAT3 signaling pathway-related genes, such as IL6ST gene, STAT3 gene, LIFR gene, IL6R gene, PIK3CA gene, and JAK2 gene, at the initial stage of E5-E7. However, TGFβ / SMADs signaling pathway-related genes, such as INHBA gene, NODAL gene, ACVR2A / 2B gene, BMP4, and BMPR1A / 1B, and FGF / ERK signaling pathway-related genes, such as MAPK1 gene, MAPK14 gene, FGF2 gene, PDGFA gene, FGFR1 gene, and FGFR2 gene, showed sustained expression during the formation and priming stages. In addition, the expression of WNT / β-catenin signaling-related genes (including TCF7 gene, APC gene, WNT11 gene, CTNNB1 gene, FZD2 gene, and WNT3A gene) gradually increased in the primed stage ( Figure 2These results characterize the molecular basis of bovine embryonic pluripotency and elucidate the unique signaling pathway dependencies of the EPI (epidermal pluripotency) at various stages of pluripotency. They indicate that the maintenance of bovine intermediate pluripotency depends on the activation of the FGF / ERK and TGFβ / SMADs signaling pathways, while the inhibition of the WNT / β-catenin signaling pathway is consistent with our observations in early porcine embryos. 1 .

[0329] Example 3 Comparative Analysis of Development and Pluripotency Changes in Cattle and Pigs

[0330] Next, we investigated the similarities and differences in the changes in pluripotency from ICM to EPI to ECTO (ectoderm) during bovine and porcine embryonic development. To mitigate potential bias from stage mismatches between species, we initially performed principal component analysis (PCA) and Spearman correlation analysis. PCA of bovine and porcine embryonic lineages showed that samples were tightly clustered according to developmental stage and time ( Figure 3 Middle A). Spearman correlation analysis showed that the pedigree development of bovine and porcine embryos was highly similar, representing two distinct groups.

[0331] Specifically, the E5 pre-ICM and E6 ICM of cattle were very similar to the E5 pre-ICM to E7 EPI of pigs. In addition, similar results were observed between the E10-E12 EPI of cattle and the E10 EPI of pigs, and similar results were also observed between the E14 ECTO of cattle and the E11-E13 ECTO of pigs ( Figure 3 We then plotted the individual transcriptomes of bovine and porcine samples over time. Embryonic lineage- and pluripotency-specific groups were largely distinct ( Figure 3 Figure C) summarizes the relative duration of bovine and porcine embryonic lineage development and pluripotent lineage progression. We selected representative stages of the bovine and porcine embryonic lineages, namely the naive, formative, and primed states, to analyze the differences ( Figure 3 The differences in gene expression observed at each stage clearly reflect distinct pluripotent characteristics ( Figure 3 Middle E).

[0332] During the transition from naive to formative and then to primed pluripotency, we observed consistent expression patterns among genes associated with signaling pathways, including the JAK / STAT3, FGF / ERK, TGFβ / SMAD, and WNT / β-catenin pathways in both bovine and porcine embryonic lineages ( Figure 3F). GO / KEGG enrichment analysis showed that during the transition from the naive state to the formative state, the expression of genes related to the "signaling pathway regulating stem cell pluripotency" and the "TGFβ signaling pathway" in the bovine and porcine embryonic lineages was upregulated, while genes related to "oxidative phosphorylation" were downregulated. During the transition from the intermediate state to the primed pluripotent state, the expression of genes related to the "TGFβ signaling pathway" and the "Hippo signaling pathway" in the bovine and porcine embryonic lineages was upregulated, while the expression of genes related to the "cellular response to leukemia inhibitory factor" was downregulated, which further reflects the consistency between the development and pluripotency changes of the bovine and porcine embryonic lineages ( Figure 3 These findings demonstrate a high degree of similarity in embryonic development and pluripotency dynamics between bovine and porcine embryos, suggesting the existence of similar regulatory networks controlling the pluripotency of PSCs in bovine and porcine embryos.

[0333] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

[0334] References:

[0335] 1.Zhi,ML,Zhang,JY,Tang,QZ,Yu,DW,Gao,S.,Gao,DF,Liu,PL,Guo,JX,Hai,T.,Gao,J.,et al.(2022).Generation and characterization of stable pigpregastrulation epiblast stem cell lines.Cell Res.32,383-400.10.1038 / s41422-021-00592-9.

[0336] 2.Saitou,M.,and Hayashi,K.(2021).Mammalian in vitrogametogenesis.Science 374,ARTN eaaz6830.10.1126 / science.aaz6830.

[0337] 3.Oikawa,M.,Kobayashi,H.,Sanbo,M.,Mizuno,N.,Iwatsuki,K.,Takashima,T.,Yamauchi,K.,Yoshida,F.,Yamamoto,T.,Shinohara,T.,et al.(2022).Functionalprimordial germ cell-like cells from pluripotent stem cells in rats.Science376,176-179.10.1126 / science.abl4412.

[0338] 4.Yao,C.,Zhang,W.,and Shuai,L.(2019).The first cell fate decisioninpre-implantation mouse embryos.Cell Regeneration 8,51-57.10.1016 / j.cr.2019.10.001.

[0339] 5.Bray,N.L.,Pimentel,H.,Melsted,P.,and Pachter,L.(2016).Near-optimalprobabilistic RNA-seq quantification.Nat.Biotechnol.34,525-527.10.1038 / nbt.3519

[0340] 6.Love,M.I.,Huber,W.,and Anders,S.(2014).Moderated estimation offoldchange and dispersion for RNA-seq data with DESeq2.Genome Biol.15,550.ARTN 550.10.1186 / s13059-014-0550-8.

[0341] 7.Gao,S.,Yan,L.,Wang,R.,Li,J.,Yong,J.,Zhou,X.,Wei,Y.,Wu,X.,Wang,X.,Fan,X.,et al.(2018).Tracing the temporal-spatial transcriptomelandscapes ofthe human fetal digestive tract using single-cell RNA-sequencing.Nat.CellBiol.20,721-734.10.1038 / s41556-018-0105-4.

[0342] 8.Wang,M.,Liu,X.X.,Chang,G.,Chen,Y.D.,An,G.,Yan,L.Y.,Gao,S.,Xu,Y.W.,Cui,Y.L.,Dong,J.,et al.(2018).Single-Cell RNA Sequencing AnalysisRevealsSequential Cell Fate Transition during Human Spermatogenesis.Cell StemCell23,599-614.e4.10.1016 / j.stem.2018.08.007.

[0343] 9.Zhu,Q.F.,Sang,F.,Withey,S.,Tang,W.,Dietmann,S.,Klisch,D.,Ramos-Ibeas,P.,Zhang,H.X.,Requena,C.E.,Hajkova,P.,et al.(2021).Specification andepigenomic resetting of the pig germline exhibit conservationwith the humanlineage.Cell Rep.34,108735.ARTN 108735.10.1016 / j.celrep.2021.108735.

[0344] 10.van Oosten,A.L.,Costa,Y.,Smith,A.,and Silva,J.C.R.(2012).JAK / STAT3signalling is sufficient and dominant over antagonistic cues for theestablishment ofnaive pluripotency.Nat.Commun.3,817.10.1038 / ncomms1822.

Claims

1. Use of a substance for identifying gene markers in the preparation of a product for identifying the developmental stage or pluripotency stage of bovine embryonic cells, wherein the gene markers are selected from one or more genes shown in Tables 4 to 13, or any combination thereof.

2. The use according to claim 1, characterized in that The bovine embryonic cell developmental stages include pre-inner cell mass, pre-trophoblast, inner cell mass, trophoblast, epiblast, hypoblast, ectoderm, mesoderm, endoderm or primordial germ cell-like cells.

3. The use according to claim 2, characterized in that The anterior inner cell mass and / or anterior trophoblast are selected from bovine embryo E5; or, the inner cell mass is selected from bovine embryo E6 and / or E7; or, the trophoblast is selected from bovine embryo E6-E14; or, the epiblast is selected from bovine embryo E10-E12; or, the hypoblast is selected from bovine embryo E10-E12; or, the ectoderm, mesoderm, endoderm and / or primordial germ cell-like cells are selected from bovine embryo E14; Preferably, the bovine embryos E10-E12 are selected from E10 or E12, or any combination thereof; Preferably, the bovine embryos E6-E14 are selected from E6, E7, E10, E12 or E14, or any combination thereof.

4. The use according to claim 3, characterized in that The gene markers for identifying the anterior inner cell mass are the gene markers shown in Table 4, or any combination thereof; or the gene markers for identifying the anterior trophoblast are the gene markers shown in Table 5, or any combination thereof; or the gene markers for identifying the inner cell mass are the gene markers shown in Table 6, or any combination thereof; or the gene markers for identifying the trophoblast are the gene markers shown in Table 7, or any combination thereof; or the gene markers for identifying the epiblast are the gene markers shown in Table 8, or any combination thereof; or the gene markers for identifying the hypoblast are the gene markers shown in Table 9, or any combination thereof; or the gene markers for identifying the ectoderm are the gene markers shown in Table 10, or any combination thereof; or the gene markers for identifying the mesoderm are the gene markers shown in Table 11, or any combination thereof; or the gene markers for identifying the endoderm are the gene markers shown in Table 12, or any combination thereof; or the gene markers for identifying primordial germ cell-like cells are the gene markers shown in Table 13, or any combination thereof.

5. The use according to claim 1, characterized in that The bovine embryonic cell developmental stages include the primitive pluripotency stage, the intermediate pluripotency stage, and the initial pluripotency stage.

6. The use according to claim 5, characterized in that The gene markers for identifying the primitive pluripotency stage are gene markers for identifying the pre-inner cell mass, gene markers for identifying the pre-trophoblast, gene markers for identifying the inner cell mass and / or gene markers for identifying the trophoblast; or the gene markers for identifying the intermediate pluripotency stage are gene markers for identifying the epiblast and / or gene markers for identifying the hypoblast; or the gene markers for identifying the initial pluripotency stage are gene markers for identifying the ectoderm, gene markers for identifying the mesoderm, gene markers for identifying the endoderm and / or gene markers for identifying primordial germ cell-like cells.

7. The use according to claim 5, characterized in that The substance used to identify the primitive state stage of bovine embryonic cells is a substance for detecting the gene marker for identifying the pluripotency stage of bovine embryonic cells is a substance for detecting the JAK / STAT3 signaling pathway, or the substance used to identify the intermediate pluripotency stage of bovine embryonic cells is a substance for detecting the FGF / ERK signaling pathway and / or a substance for detecting the TGFβ / SMAD signaling pathway, or the substance used to identify the initial state stage of bovine embryonic cells is a substance for detecting the JWNT / β-catenin signaling pathway.

8. The use according to claim 7, characterized in that The substances for detecting the JAK / STAT3 signaling pathway include substances for detecting the IL6ST gene, STAT3 gene, PIK3R2 gene, PIK3R1 gene, LIFR gene, KLF4 gene, TBX3 gene, IL6R gene, PIK3CA gene or JAK2 gene, or any combination thereof; Or, the substance for detecting the TGFβ / SMADs signaling pathway includes a substance for detecting ID1 gene, BMP1 gene, ACVR1 gene, TGFBR2 gene, SMAD5 gene, ID3 gene, BMP7 gene, BMPR2 gene, TGFB1 gene, BMPR1B gene, ACVR1B gene, INGBA gene, NODAL gene, ACVR2A gene, ACVR2B gene, SMAD9 gene, TDGF1 gene, BMP4 gene, SMAD4 gene or BMPR1A gene, or any combination thereof; Or, the substance for detecting the FGF / ERK signaling pathway includes a substance for detecting the MAPK14 gene, the MAPK1 gene, the ETS1 gene, the FGF2 gene, the MAPK3 gene, the FGFR2 gene, the JUN gene, the KDR gene, the MAP2K2 gene, the PDGFA gene, the FGFR1 gene, the ITGA6 gene, the ETS2 gene or the ITGA3 gene, or any combination thereof; Or, the substance for detecting the WNT / β-catenin signaling pathway includes a substance for detecting the GSK3B gene, TCF7 gene, CDH12 gene, APC gene, WNT11 gene, FZD1 gene, FZD10 gene, CTNNB1 gene, FZD2 gene, WNT3A gene, WNT5B gene or WNT7B gene, or any combination thereof; Preferably, the bovine embryonic cells are selected from the group consisting of anterior inner cell mass, inner cell mass, epiblast and ectoderm; Preferably, the bovine embryo is a bovine embryo E5-E14; More preferably, the bovine embryos E5-E14 are selected from E5, E6, E7, E10, E12 or E14, or any combination thereof.

9. A method for identifying the developmental stage of bovine embryonic cells, characterized in that: The method comprises detecting the bovine embryonic cells to be detected by any one or more methods selected from the following: (1) Identification using genetic markers; (2) using signaling pathway identification; (3) using signaling pathway-related genes for identification; The gene marker is the gene marker in any one of claims 1 to 6, the signal pathway is the signal pathway in the application of claim 7 or 8, and the signal pathway-related gene is the gene in claim 8; If the results are as follows, the bovine embryonic cell to be tested is an anterior inner cell mass cell: expressing one or more of the genes described in Table 4; If the results are as follows, the bovine embryonic cells to be tested are pretrophoblast cells: expressing one or more of the genes described in Table 5; If the results are as follows, the bovine embryonic cell to be tested is an inner cell mass cell: expressing one or more of the genes described in Table 6; If the results are as follows, the bovine embryonic cells to be tested are trophoblast cells: expressing one or more of the genes described in Table 7; If the results are as follows, the bovine embryonic cells to be tested are epiblast cells: express one or more of the genes described in Table 8; If the results are as follows, the bovine embryonic cells to be tested are hypoblast cells: express one or more of the genes described in Table 9; If the results are as follows, the bovine embryonic cells to be tested are ectoderm cells: expressing one or more of the genes described in Table 10; If the results are as follows, the bovine embryonic cells to be tested are mesoderm cells: expressing one or more of the genes described in Table 11; If the results are as follows, the bovine embryonic cells to be tested are endoderm cells: express one or more of the genes described in Table 12; If the results are as follows, the bovine embryonic cells to be tested are primordial germ cell-like cells: Express one or more of the genes described in Table 13.

10. A method for identifying the pluripotency stage of bovine embryonic cells, characterized in that: The method comprises detecting the bovine embryonic cells to be detected by any one or more methods selected from the following: (1) Identification using genetic markers; (2) using signaling pathway identification; (3) using signaling pathway-related genes for identification; The gene marker is the gene marker in any one of claims 1 to 6, the signal pathway is the signal pathway in the application of claim 7 or 8, and the signal pathway-related gene is the gene in claim 8; If the results are as follows, the bovine embryonic cells to be tested are at the naive pluripotency stage: The test result is that the bovine embryonic cell to be tested is a pre-inner cell mass, a pre-trophoblast, an inner cell mass, a trophoblast, or any combination thereof; If the results are as follows, the bovine embryonic cells to be tested are at the intermediate pluripotency stage: If the test result is positive, the bovine embryonic cell to be tested is epiblast, hypoblast, or any combination thereof; If the results are as follows, the bovine embryonic cells to be tested are at the primed pluripotent stage: If the test result is yes, the bovine embryonic cells to be tested are ectoderm, mesoderm, endoderm, or primordial germ cell-like cells; Preferably, the detection is expression level detection; Preferably, the detection is single-cell or multi-cell transcriptome analysis; Preferably, the bovine embryonic cells to be tested are selected from bovine embryos E5-E14; Preferably, the bovine embryos E5-E14 are selected from E5, E6, E7, E10, E12 or E14, or any combination thereof.

11. The method according to claim 10 or 11, characterized in that The method further includes comparing the test results of the bovine embryonic cell to be tested with the embryonic lineage and pluripotency stage characteristics established for pigs to determine the bovine embryonic cell developmental characteristics of the bovine embryonic cell to be tested; Preferably, the bovine embryonic cells are selected from the group consisting of anterior inner cell mass, inner cell mass, epiblast and / or ectoderm; More preferably, the bovine embryo is a bovine embryo E1-E14; Further preferably, the bovine embryos E1-E14 are selected from E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, E12, E13 or E14, or any combination thereof.

Images