Use of sox17 in improving pluripotency of bovine embryonic stem cells and / or in directed induction of primordial germ cell-like cells

Abstract

This invention provides SOX17 This invention relates to the application of improving the pluripotency of bovine embryonic stem cells and / or the targeted induction of primordial germ cell-like cells, belonging to the field of genetic engineering technology. The invention utilizes overexpression... SOX17 Genes can upregulate core pluripotency factors in bovine embryonic stem cells. NANOG The expression of this substance effectively enhances the pluripotency maintenance capacity of cells, enabling them to maintain typical stem cell morphology, stable proliferative activity, and reduce the proportion of spontaneous differentiation during long-term in vitro culture and passage. It can also directionally induce bovine embryonic stem cells to differentiate into PGC-like cells efficiently in vitro, significantly upregulate the expression level of PGC-specific markers, and improve the positive rate and uniformity of PGC-like cells. At the same time, it can inhibit non-specific differentiation, enhance the specificity of differentiation direction, and improve the stability of the induction system. It has important application value in the rapid breeding of improved livestock breeds, the preservation of germplasm resources, and the study of reproductive and developmental mechanisms.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of SOX17 in improving the pluripotency of bovine embryonic stem cells and / or inducing primordial germ cell-like cells. Background Technology

[0002] Primordial germ cells (PGCs), as precursor cells of germ cells, are the core foundation for gameteogenesis, embryonic development, and the preservation and utilization of animal germplasm resources. The efficient in vitro induction of PGC-like cells not only provides an important research model for elucidating the molecular regulatory mechanisms of mammalian germplasm development but also has significant application value in areas such as rapid breeding of superior livestock, conservation of endangered animal germplasm resources, transgenic animal construction, and cell breeding. As an important economic livestock, cattle benefit from an efficient in vitro induction system for PGC-like cells, which has significant industrial implications for improving the breeding efficiency of beef and dairy cattle and accelerating the genetic propagation of superior traits. Embryonic stem cells possess unlimited in vitro proliferation and multi-lineage differentiation potential, making them ideal seed cells for in vitro induction of PGC-like cells. The pluripotency maintenance state of embryonic stem cells directly determines their directed differentiation efficiency, cell purity, and differentiation stability. Existing research indicates that the homeostatic regulation of the pluripotency transcription factor network is a crucial prerequisite for driving the directed differentiation of stem cells into the PGC lineage. However, bovine embryonic stem cells (BECs) are prone to spontaneous differentiation, unstable expression of pluripotency genes, and decreased pluripotency after long-term passage during in vitro culture. This leads to problems such as low induction efficiency, severe non-specific differentiation, and poor homogeneity of positive cells when inducing differentiation into PGC-like cells, thus restricting the establishment and application of in vitro induction systems for bovine germ cells. Currently, the key factors and molecular mechanisms regulating the maintenance of pluripotency and the directed differentiation of bovine embryonic stem cells into PGCs still require in-depth analysis. Existing induction systems are insufficient to achieve stable, efficient, and highly specific preparation of PGC-like cells. Therefore, identifying key regulatory factors that can effectively maintain the pluripotency of bovine embryonic stem cells and precisely drive their directed differentiation into the PGC lineage, and establishing stable and efficient in vitro induction methods, is an urgent practical need for promoting research on bovine reproductive development and the development of improved breeding technologies. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide... SOX17 Applications in enhancing the pluripotency of bovine embryonic stem cells and / or activating primordial germ cell-like cells, through overexpression in bovine embryonic stem cells. SOX17 Genes can significantly enhance the pluripotency of bovine embryonic stem cells. Simultaneously, overexpression... SOX17 Gene-derived bovine embryonic stem cells can significantly increase the induction rate of PGC-like cells.

[0004] The objective of this invention is achieved through the following technical solution: This invention provides an improvement SOX17 Application of reagents that enhance gene expression levels and / or SOX17 protein levels in improving the pluripotency of bovine embryonic stem cells.

[0005] Preferably, the SOX17 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the amino acid sequence of the SOX17 protein is shown in SEQ ID NO.2.

[0006] Preferably, the enhancement of bovine embryonic stem cell pluripotency includes the following (1) and / or (2): (1) Maintain the typical stem cell morphology and stable proliferation capacity of bovine embryonic stem cells, and prevent spontaneous differentiation after continuous passage; (2) Enhance the ability to maintain cell pluripotency.

[0007] This invention provides a method for improving the pluripotency of bovine embryonic stem cells, comprising: Increasing bovine embryonic stem cells SOX17 Gene expression level.

[0008] Preferred, to improve bovine embryonic stem cells SOX17 Methods for assessing gene expression levels include: SOX17 Gene overexpression vectors were introduced into bovine embryonic stem cells.

[0009] This invention provides an improvement SOX17 Application of reagents that enhance gene expression levels and / or increase SOX17 protein levels in the in vitro directed induction of bovine embryonic stem cells into primordial germ cell-like cells.

[0010] Preferably, the reagent can improve the induction efficiency of bovine embryonic stem cells into primordial germ cell-like cells.

[0011] Preferably, improving induction efficiency includes at least one of the following: (1) Upregulate the mRNA and protein expression levels of PGC-specific marker genes to increase the positive rate and proportion of PGC-like cells; (2) Improve the uniformity and maturity of PGC-like cells after induction.

[0012] This invention provides a method for improving the induction efficiency of bovine embryonic stem cells into primordial germ cell-like cells, comprising: Increasing bovine embryonic stem cells SOX17 After gene expression levels, SOX17 Bovine embryonic stem cells with enhanced gene expression levels were induced to differentiate into primordial germ cell-like cells.

[0013] This invention provides a primer set for identifying whether the pluripotency of bovine embryonic stem cells is enhanced, comprising: bSOX17-F as described in SEQ ID NO.11 and bSOX17-R as described in SEQ ID NO.12.

[0014] The beneficial effects of this invention are: This invention provides an improvement SOX17 The application of agents that increase gene expression levels and / or SOX17 protein levels in enhancing bovine embryonic stem cell pluripotency. This invention utilizes overexpression... SOX17 Increasing the level of SOX17 protein in bovine embryonic stem cells can significantly upregulate the core pluripotency factor in bovine embryonic stem cells. NANOG The expression, while maintaining stability OCT4 SOX2 The expression level of this substance effectively enhances the pluripotency maintenance capacity of cells, enabling them to maintain typical stem cell morphology, stable proliferation activity, and reduce the spontaneous differentiation ratio during long-term in vitro culture and passage. This provides seed cells with better condition and stronger stability for subsequent in vitro directed induction to prepare primitive germ cell-like cells, thereby improving induction efficiency and cell purity. It has important application value in livestock breeding, germplasm resource preservation, and reproductive development mechanism research.

[0015] Furthermore, the present invention provides improvements SOX17 The application of reagents that increase gene expression levels and / or enhance SOX17 protein levels in the in vitro directed induction of bovine embryonic stem cell differentiation into primordial germ cell-like cells. This invention utilizes overexpression... SOX17 Gene injections to increase SOX17 protein levels in bovine embryonic stem cells (BECs) can induce efficient differentiation of BECs into primordial germ cell-like cells (PGC-like cells) in vitro. This significantly upregulates the expression levels of PGC-specific markers such as BLIMP1, TFAP2C, NANOS3, and T-Bra, improving the positive rate and homogeneity of PGC-like cells. Simultaneously, rapid silencing of these markers... SOX2 Stable activation NANOG and OCT4 The expression pattern of the gene inhibits non-specific differentiation, enhances the specificity of differentiation direction, and significantly improves the stability and reliability of the induction system, providing a key regulatory means for establishing an efficient and stable in vitro preparation technology system for bovine PGC. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 for SOX17 Overexpression ( SOX17_OEbESCs plasmid transfection flowchart; Figure 2 For 7F- SOX17 Overexpression cell line (7F- SOX17 _OE bESCs) establishment process diagram; top: bright field; bottom: fluorescence; scale bar is 100 μm; Figure 3 WT-bESCs and WT-bESCs in 7F culture medium under low magnification SOX17 _OE bESCs clone morphology diagram; Figure 4 To detect 7F- using RT-qPCR SOX17 Expression levels of pluripotency genes related to _OE bESCs; error bars represent mean ± standard deviation (n = 3); p-values ​​were determined using a two-tailed Student's t-test. p<0.05, p<0.01, p<0.001, p<0.0001); Figure 5 For the detection of WT and protein by Western blot under 7F culture conditions SOX17_ Levels of pluripotent proteins (OCT4, SOX2, NANOG) and SOX17 in OE bESCs; Figure 6 WT and 7F culture SOX17 Immunostaining results of pluripotency proteins (OCT4, SOX2, and NANOG) in _OE; cell nuclei stained with DAPI; scale bar, 50 μm; Figure 7 Flowchart for the induction of differentiation of 7F-bESCs into bPGCLCs; Figure 8 To provide control and induction on days 2, 4, and 6. SOX17 Overexpression of PGC-like cells derived from cells; Top: Bright field; Bottom: Fluorescence; Scale bar, 100 μm; Figure 9 To detect the relative expression levels of core pluripotent genes using RT-qPCR; error bars represent mean ± standard deviation (n = 3); P-values ​​were determined using a two-tailed Student's t-test. p<0.05, p<0.01, p<0.001, p<0.0001; Figure 10 To detect the relative expression levels of marker genes in primordial germ cells using RT-qPCR. Error bars represent mean ± standard deviation (n = 3); P-values ​​were determined using a two-tailed Student's t-test. p<0.05, p<0.01, p<0.001, p<0.0001; Figure 11 Immunostaining results for PGC-like cells (PGC-like cells for short); cell nuclei stained with DAPI, scale bar, 50 μm. Detailed Implementation

[0018] This invention provides an improvement SOX17 The application of reagents that increase gene expression levels and / or SOX17 protein levels in enhancing the pluripotency of bovine embryonic stem cells. This invention primarily involves increasing the expression levels of SOX17 protein in bovine embryonic stem cells in vitro. SOX17 Increased gene expression levels and / or elevated SOX17 protein levels, thereby enhancing bovine embryonic stem cell pluripotency. In this invention, the... SOX17 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the amino acid sequence of the SOX17 protein is shown in SEQ ID NO.2.

[0019] In this invention, improving the pluripotency of bovine embryonic stem cells includes the following (1) and / or (2): (1) maintaining the typical stem cell morphology and stable proliferation capacity of bovine embryonic stem cells, preventing spontaneous differentiation during continuous passage; (2) enhancing the ability to maintain cell pluripotency. This invention does not specifically limit the reagents used; conventional reagents in the art can be used to improve the pluripotency of bovine embryonic stem cells. SOX17 Both increasing gene expression levels and / or increasing SOX17 protein levels are acceptable. As an optional embodiment of the invention, the reagent can be used to induce expression in bovine embryonic stem cells. SOX17 Reagents for gene overexpression; to induce gene overexpression in bovine embryonic stem cells SOX17 Reagents for gene overexpression can be used for overexpression SOX17 Gene vector; the overexpression SOX17 Gene vectors can be TRE- SOX17 -tdtomato overexpression plasmid. This invention relates to TRE- SOX17 There are no particular limitations on the preparation method of the -tdtomato overexpression plasmid; any conventional preparation method in the art can be used. The results of the embodiments of this invention demonstrate that... SOX17 Overexpression significantly upregulated the transcriptional and protein levels of NANOG in bovine embryonic stem cells, enhanced the pluripotency maintenance capacity of bovine embryonic stem cells, and improved the culture stability of the cell line; and SOX17 Overexpression can maintain the typical stem cell morphology and stable proliferation capacity of bovine embryonic stem cells, and spontaneous differentiation does not occur after continuous passage.

[0020] This invention provides a method for improving the pluripotency of bovine embryonic stem cells, comprising: increasing the pluripotency of bovine embryonic stem cells... SOX17 Gene expression levels. As an optional embodiment of the present invention, increasing gene expression levels in bovine embryonic stem cells... SOX17 Methods for assessing gene expression levels include: SOX17 Gene overexpression vectors are introduced into bovine embryonic stem cells. As an optional embodiment of the present invention, the... SOX17 Gene overexpression vectors include TRE- SOX17 -tdtomato overexpression plasmid.

[0021] This invention provides an improvement SOX17 The application of reagents that increase gene expression levels and / or SOX17 protein levels in the in vitro directed induction of bovine embryonic stem cells into primordial germ cell-like cells. This invention improves induction efficiency by at least one of the following: (1) upregulating the mRNA and protein expression levels of PGC-specific marker genes, thereby increasing the positive rate and proportion of PGC-like cells; (2) enhancing the uniformity and maturity of induced PGC-like cells. In this invention, the PGC-specific marker genes include any one or more of BLIMP1, TFAP2C, NANOS3, and T-Bra. This invention preferably utilizes rapid silencing... SOX2 ,activation NANOG ,Stablize OCT4 This expression enables the inhibition of non-specific differentiation of cells into non-reproductive lineages and enhances the specificity of differentiation direction.

[0022] As an optional embodiment of the present invention, the reagent can improve the induction efficiency of bovine embryonic stem cells into primordial germ cell-like cells. The present invention does not specifically limit the reagent; conventional reagents in the art can be used to improve the differentiation efficiency of bovine embryonic stem cells. SOX17 Both increasing gene expression levels and / or increasing SOX17 protein levels in bovine embryonic stem cells are acceptable. As an optional embodiment of the invention, the reagent can be used to increase the expression level of SOX17 protein in bovine embryonic stem cells. SOX17 Reagents for gene overexpression; to induce gene overexpression in bovine embryonic stem cells SOX17 Reagents for gene overexpression can be used for overexpression SOX17 Gene vector; the overexpression SOX17 Gene vectors can be TRE- SOX17 -tdtomato overexpression plasmid. This invention relates to TRE- SOX17 There are no particular limitations on the preparation method of the -tdtomato overexpression plasmid; any conventional preparation method in this field may be used.

[0023] This invention provides a method for improving the induction efficiency of bovine embryonic stem cells into primordial germ cell-like cells, comprising: Increasing bovine embryonic stem cells SOX17 After gene expression levels, SOX17 Bovine embryonic stem cells with enhanced gene expression levels were induced to differentiate into primordial germ cell-like cells.

[0024] This invention improves the quality of bovine embryonic stem cells. SOX17 There are no particular limitations on the methods used to improve gene expression levels; any conventional method in the field may be used. As an optional embodiment of this invention, this method aims to increase the expression level of bovine embryonic stem cells. SOX17 Methods for assessing gene expression levels include: SOX17 Gene overexpression vectors are introduced into bovine embryonic stem cells. As an optional embodiment of the present invention, the... SOX17 Gene overexpression vectors include TRE- SOX17 -tdtomato overexpression plasmid.

[0025] The present invention does not specifically limit the method of inducing differentiation, and any conventional method of inducing differentiation in the art can be used.

[0026] This invention provides a primer set for identifying whether the pluripotency of bovine embryonic stem cells is enhanced, comprising: bSOX17-F as described in SEQ ID NO. 11 and bSOX17-R as described in SEQ ID NO. 12. Using this primer set, real-time quantitative PCR can be performed on the cDNA of bovine embryonic stem cells to determine... SOX17 Gene expression levels. If SOX17 A significant increase in gene expression levels indicates that the pluripotency of bovine embryonic stem cells has been enhanced.

[0027] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] The primer information for RT-qPCR is detailed in Table 1.

[0029] Table 1 RT-qPCR primers

[0030] The cell culture media involved in the following technical solutions are shown in Tables 2-3.

[0031] Table 2. mTeSR 1. Components of the 7-factor bESCs culture system based on basal culture medium (50 mL)

[0032] Table 3. Induction culture medium for bPGCLCs (10 mL)

[0033] The cell line used in this invention is from Inner Mongolia Saikexing Breeding Biotechnology Co., Ltd.

[0034] Example 1 SOX17 Effect of overexpression on the pluripotency of bESCs 1. SOX17 The Gene ID (Entrez ID) in NCBI is 534010. SOX17 For detailed mRNA information, please refer to NCBI Reference Sequence: NM_001206251.1, as shown in SEQ ID NO.1. For detailed amino acid sequence information of SOX17, please refer to NCBI Reference Sequence: NP_001193180.1, as shown in SEQ ID NO.2.

[0035] SEQ ID NO.1:

[0036] SEQ ID NO.2: MSSPDAGYASDEQSQPRSALPAVMAGLGPCPWAESLSPLGDMKMKGEAAASAGAPAGPAGRAKGESRIRRPMNAFMVWAKDERKRLAQQNPDLHNAELSKMLGKSWKALTLAEKRPFVEEAERLRVQHMQDHPNYKYRPRRRKQVKRLKRVEGGFLHGLAEPPAAALGPEGGRVAMDGLGLPFPEQGFPAGPPLLPPHLGGHYRD CPGLGAPQLDGYPLPTPDTSPLDGVEPDPAFFAAPLPADCPAPGPYSYAPAADYAGPPEPPGAGPLHPRLGPEPAGPAMPGLLAPPSALHMYYGPVGSQAAAAGGGGRGFQMPPQPPPHGPGQPSPPPEALHCRDSAEPGAPAELLGEVERTEFEQYLHFVCKPEMGLPYPGHDAGVTLPDGHGALSSVVSDASSAVYYCNYPDV.

[0037] 2. SOX17 Construction process of overexpression cell lines (1) Constructing PiggyBac-TET on / off- SOX17 -tdtomato inducible overexpression plasmid, which has inducible regulation and fluorescent tracking properties, provides a basis for subsequent stable transfection in bovine embryonic stem cells and SOX17 Functional studies laid a solid experimental foundation. This plasmid is referenced in [Zhao, Lixia, et al., Establishment of bovine expanded potential stem cells. Proceedings of the National Academy of Sciences, 118.15 (2021): e2018505118.].

[0038] The specific method is as follows: using cDNA derived from differentiated bovine embryonic stem cells as a template, according to bovine... SOX17Specific primers were designed for the coding region of the gene, and amplification was performed using polymerase chain reaction (PCR). The upstream primer sequence is shown in SEQ ID NO. 19: ATCGGCCGGATATCGAATTCATGAGCAGCCCGGATGCGGG; the downstream primer sequence is shown in SEQ ID NO. 20: GTTTGAGTAAATCAAAGTTAAGAGTTTGTTTGACAGGAGCGACAATTTTACTAGACACGTCGGGATAGTTGCAGTA. Cattle were successfully obtained through amplification. SOX17 The complete coding sequence (CDS) of the gene was obtained. The PCR products were separated and identified by agarose gel electrophoresis, and the results showed that the specific amplified fragment was approximately 1230 bp in size, consistent with the theoretically expected length, indicating that the amplified product was correct. Simultaneously, the PiggyBac-TET on / off-tdtomato basic expression vector was linearized by double digestion with EcoRI and BamHI restriction endonucleases. After separation by agarose gel electrophoresis, a linearized vector backbone fragment of 7641 bp was recovered. This vector system can achieve tetracycline-induced regulation, providing a basis for subsequent... SOX17 The induced expression provides a controllable expression platform. The purified and recovered [material / product]... SOX17 Gene fragments and linearized vector backbones were directionally ligated under the catalysis of T4 DNA ligase to construct bovine vectors. SOX17 Overexpression of recombinant plasmids.

[0039] To further verify the accuracy of the recombinant plasmid, double enzyme digestion was performed for identification. The digestion products were analyzed by agarose gel electrophoresis, and the results showed that the size of the digested fragments was consistent with expectations, proving that the target gene had been correctly inserted into the vector. Subsequently, the positive recombinant plasmid was sent for sequencing, and Sanger sequencing results further confirmed that the inserted sequence was bovine. SOX17 The gene coding regions are completely identical, with no mutations or frameshifts. Therefore, the PiggyBac-TET on / off-Genome has been successfully constructed. SOX17 -tdtomato inducible overexpression plasmid, which has inducible regulation and fluorescent tracking properties, provides a basis for subsequent stable transfection in bovine embryonic stem cells and SOX17 Functional studies have laid a reliable experimental foundation.

[0040] (2) In the experimental preparation stage, the culture dish was first coated with 0.1% gelatin and left to stand at 37℃ for 30 min, after which the excess liquid was discarded. Bovine embryonic stem cells cultured on the feeder layer were digested by ACC, centrifuged and resuspended, and then seeded into gelatin-coated dishes and incubated at 38.5℃ for 30-60 min. Taking advantage of the difference in adhesion rate between feeder layer cells and embryonic stem cells, feeder layer cells were preferentially adhered to the culture dish. The suspended high-purity bovine embryonic stem cells were collected to obtain feeder-free bovine embryonic stem cells (7F-bESCs), which were used for feeder-free culture.

[0041] (3) The successfully constructed TRE- SOX17 -tdtomato overexpression plasmid (or Tet-on-TRE- SOX17 The plasmid was introduced into 7F-bESCs cultured without a feeder layer via liposome-mediated transfection to establish... SOX17 Inducible overexpression stable cell lines, methods as follows Figure 1 As shown, the specific steps are as follows: Cell preparation and transfection system preparation: Bovine embryonic stem cells in the logarithmic growth phase were collected. After routine digestion and centrifugation, the cell pellet was aliquoted at a 1:3 ratio to prepare for subsequent transfection. The liposome transfection system was prepared strictly according to the two-component method: First, solution A was prepared by gently mixing 4 μL of Lipofectamine 2000 transfection reagent with 50 μL of OPTI-MEM serum-free medium; then solution B was prepared by mixing Tet-on-TRE- SOX17 The plasmid (2 μg), PB-EF1α-transposase plasmid (2 μg), and PB-EF1α-rTTA plasmid (2 μg) were thoroughly mixed with 50 μL of OPTI-MEM medium. Equal volumes of solution A and solution B were then mixed in a new 0.6 mL centrifuge tube and incubated at room temperature for 10 minutes to form a stable liposome-DNA complex.

[0042] Transfection procedure and stable cell line selection: Take an appropriate amount of the prepared cell pellet and gently resuspend it in the above-mentioned mixture of A and B to ensure full contact between the cells and the transfection complex. Incubate the mixture in a 37°C, 5% CO2 incubator for 30 min, gently tapping the tube wall every 5 min to maintain cell suspension. After incubation, discard the original coating medium in the culture plate and resuspend the cells in 900 μL of doxycycline-free complete culture medium, and evenly seed them into the culture wells. After the cells adhere and their growth is stable, change the culture medium to induction medium containing 1 μg / mL Dox to initiate exogenous gene expression. 12 h after transfection, observe cell morphology and red fluorescent protein expression under an inverted fluorescence microscope. Subsequently, add 3 μg / mL puromycin for continuous selection for 3-5 days. Based on the red fluorescent label carried by the vector, use a pipette to accurately pick single clonal cell clusters or single cells with strong fluorescence intensity and typical morphology, and transfer them to 96-well plates for amplification culture.

[0043] Monoclonal expansion and cell line cryopreservation: After the selected monoclonal cells reached a passageable state in 96-well plates, they were progressively expanded to 48-well and 24-well plates for large-scale culture. Once sufficient cells were obtained, another round of puromycin drug screening was performed until no dead cells or only a very small number of dead cells remained in the culture system for purification. SOX17 Overexpressing cell lines. Finally, the overexpressing cell lines were expanded, aliquoted and cryopreserved using standard cell cryopreservation buffer for subsequent experiments.

[0044] 3. SOX17 Verification of overexpression effect and clonal morphology analysis Following continuous screening with puromycin, a single clonal cell line with typical morphology, clear boundaries, and a dense, dome-shaped growth pattern was successfully isolated and expanded using limiting dilution combined with single-cell picking technology. This cell line maintained stable clonal morphology and proliferative capacity throughout passage up to the 20th generation.

[0045] After screening with a continuous gradient of puromycin concentrations, surviving positive cells were collected and prepared into a homogeneous single-cell suspension. Using a limiting dilution method, the cells were serially diluted to an appropriate density and seeded into 96-well culture plates, ensuring that theoretically no more than one cell was seeded per well. Physical separation of single cells was achieved through statistical distribution. After seeding, the cells were cultured under standard conditions, with periodic half-volume medium replacements to maintain a stable cell growth environment.

[0046] After the cell clusters are cultured to the point where they are visible to the naked eye / microscope, single-cell picking is performed under an inverted microscope: only single-clonal cell clusters that are formed by the proliferation of a single cell, have typical morphology, clear boundaries, and grow in a dense dome shape are selected. They are precisely picked out using the cloning loop method or micro-trypsin digestion method and then seeded sequentially into 24-well plates, 6-well plates, and culture flasks for expansion culture.

[0047] In subsequent continuous passage culture, conventional adherent cell culture methods were used, maintaining consistent culture medium, serum concentration, and culture conditions, and cell morphology and proliferation status were continuously observed. The results showed that... SOX17 The overexpression stable cell line maintained a stable clonal morphology and good proliferation capacity throughout the 20th generation, without significant changes in differentiation or morphological heterogeneity or loss of fluorescence signal, demonstrating that the cell line has good genetic stability.

[0048] Fluorescence microscopy showed that the selected monoclonal antibodies gradually increased in size after culture, and the addition of doxycycline (DOX) activated the exogenous... SOX17 Gene overexpression, synchronous expression of reporter genes tdtomato The red fluorescent signal, such as Figure 2 . Figure 2 WT cells in the figure represent control cells; SOX17-OE for SOX17 Overexpression cell lines; p10 and P8 refer to the number of passages; Day 1 and Day 5 refer to the number of days of induction; the scale bar in the figure is 100 μm. Figure 3 At the 20th passage, WT-bESCs and WT-bESCs in 7F culture medium under low magnification SOX17_OE bESCs clone morphology; Top: Bright field; Bottom: Fluorescence; Scale bar, 250 μm.

[0049] 4. To SOX17 RT-qPCR was performed on overexpressed bESCs and WT bESCs to detect the expression levels of related genes. Primer sequences are detailed in Table 1 at the end.

[0050] At the mRNA level, WT and SOX17_OE in bESCs SOX17 Expression levels and levels of pluripotency-related genes were compared.

[0051] (1) The bESCs used in the experiment were cultured routinely in a 38.5℃, 5% CO2 incubator. Before passage, the bESC culture medium, DPBS, ACC digestion solution and cell termination solution were preheated thoroughly using a dry bath. After removing the cells to be passaged from the incubator, the original culture medium was discarded, and 1 mL of DPBS was added for gentle washing and then discarded. 200 μL of ACC digestion solution was added to the adherent cells and incubated in the incubator for about 2 min. The digestion status of the cells was then observed under a stereomicroscope. Immediately after digestion, 800 μL of termination solution was added, and the cells were gently pipetted and then transferred to a 1.5 mL centrifuge tube for centrifugation. The supernatant was discarded. An appropriate amount of fresh culture medium was added to resuspend the cell pellet, and the cells were seeded onto the prepared feeder layer cells. The cells were gently shaken to mix and then placed in a 38.5℃ incubator for further culture. After seeding, the growth status of the cells should be continuously observed, and the culture medium should be changed every 48 h to avoid excessive medium changes affecting cell growth. When the cell density reaches more than 80%, the cells can be passaged again at a ratio of 1:3. After completing the passage expansion of bESCs following the steps described above, the cells can be cryopreserved once the desired density is reached. Before cryopreservation, remove the cryopreservation box from -80°C and add an appropriate amount of isopropanol to bring it to room temperature. Then, perform routine digestion and centrifugation to collect the cells. Discard the supernatant and resuspend the cells in pre-chilled (4°C) cell cryopreservation buffer (500 μL per well is recommended). Aliquot the cell suspension into cryovials, clearly labeling them with cell name, culture system, passage number, and date. Place them in the cryopreservation box and incubate at -80°C using a gradient cooling process. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.

[0052] (2) Collect cell pellets Based on cell growth status and density, when the sample collection criteria are met (70%–80% confluence), cell samples should be collected according to the following standardized procedure: First, gently wash the culture wells with 1 mL of DPBS buffer to remove dead cells and metabolic products, then discard the washing solution. Next, following the standard procedures for cell passage, add appropriate amounts of digestion and stop solution sequentially, and transfer the cell suspension to centrifuge tubes for centrifugation.

[0053] After centrifugation, carefully discard the supernatant and resuspend the cell pellet in 1 mL of DPBS buffer. Transfer the resuspended solution to a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 5 min. This step is to thoroughly remove residual liquid and any potential impurities. After centrifugation, discard the supernatant again and immediately freeze the centrifuge tube containing the cell pellet in liquid nitrogen. Then, transfer it to a -80°C freezer for stable storage, providing a high-quality sample base for subsequent RNA extraction experiments.

[0054] (3) Extraction of RNA from the sample Because RNA molecules are unstable and highly susceptible to degradation, the entire extraction process must be strictly controlled in a low-temperature and RNase-free environment. Before operation, the high-speed centrifuge was pre-cooled to 4°C, and ice packs were used to maintain the low temperature throughout the experiment. All pipetting steps required the use of certified RNase-free pipette tips. First, the biosafety cabinet was sterilized by 15 minutes of UV irradiation. Then, the work surface, pipette surfaces, and operating areas were thoroughly wiped with a dedicated cleaning agent containing RNase inhibitors to ensure the experimental environment met sterile and RNase-free standards. Total RNA extraction from cells was performed strictly following the operating procedures of the Qiagen RNA Extraction Kit (Qiagen, 74106).

[0055] (4) RNA to cDNA experimental procedure Based on the measured RNA concentration, the required volume for each sample was calculated using 1 μg of RNA as a template, and a 14 μL reverse transcription reaction system was prepared. After preparation, the system was gently mixed and briefly centrifuged, then placed in a PCR instrument and run according to the preset program: the first stage reaction conditions were 42℃ for 2 min, and immediately after the reaction, the reaction tubes were briefly centrifuged and placed on ice for 3 min; then the second stage reaction was performed, with the program set to 42℃ for 30 min, followed by heating at 95℃ for 3 min to terminate the reaction and inactivate the reverse transcriptase. After the reaction was completed, the desired cDNA product was obtained and stored at -20℃ for later use to ensure the stability and reproducibility of subsequent experiments.

[0056] (5) Real-time quantitative PCR This experiment employed real-time quantitative PCR (qPCR) technology for highly sensitive and specific gene expression quantification analysis. Using the KAPA SYBRFAST qPCR Kit, cDNA obtained through reverse transcription was used as a template. Following strict adherence to the reaction system preparation standards, specific upstream and downstream primers were used to prepare a 10 μL reaction volume for real-time quantitative PCR amplification. GAPDH was used as an internal control gene, and its expression level was used as a relative quantification benchmark to eliminate potential differences between samples. The obtained qPCR data were analyzed using relative quantification methods, with the most commonly used being 2-1... -ΔΔCt The core of this method lies in normalizing the expression level of the target gene to a stably expressed internal reference gene (GAPDH was used in this experiment) to eliminate systematic errors caused by differences in the initial cDNA amount and reaction efficiency between samples.

[0057] The specific calculation steps are as follows: Calculate the ΔCt value: ΔCt = Ct (target gene) - Ct (internal reference gene, GAPDH). This step calibrates the expression level of the target gene to the same internal reference standard.

[0058] Calculate the ΔΔCt value: Select the control group sample as the calibration sample, ΔΔCt = ΔCt(experimental group) - ΔCt(control group). This value reflects the degree of change in target gene expression in the experimental group relative to the control group.

[0059] Calculate relative expression levels: according to Formula 2 -ΔΔCt The results showed the fold change in the expression of the target gene in the experimental group relative to the control group.

[0060] Finally, the obtained relative expression levels were visualized using GraphPad Prism professional statistical software, and the significance of differences between groups was analyzed and labeled using appropriate statistical test methods (such as t-test).

[0061] RT-qPCR was performed on cells that had been passaged 25 times. Figure 4 The results showed that, compared with the control group, SOX17 Overexpression of bovine embryonic stem cells SOX17 The mRNA levels of the gene were significantly upregulated, confirming the effectiveness of the inducible expression system. Among the core pluripotency genes, OCT4 and SOX2 The expression level did not change significantly, while NANOG The expression level of [something] was significantly increased. Overexpression SOX17 No reduction OCT4 , SOX2 It can increase the expression of core pluripotency genes and significantly upregulate core pluripotency factors. NANOG The transcriptional level suggests SOX17 Overexpression does not disrupt the pluripotency of bovine embryonic stem cells; on the contrary, it can positively regulate the pluripotency network and enhance the cell's ability to maintain pluripotency.

[0062] 5. The overexpression effect was assessed at the protein level, comparing WT and... SOX17_OE Western blot analysis was performed on the cells, with β-ACTIN used as an internal reference protein.

[0063] The Western Blot experimental method is as follows: (1) Extraction and concentration determination of total cell protein Sufficient cell volume is required for cell protein extraction. First, transfer cells cultured in 24-well plates to 6-well plates and culture for several days until the cell density reaches the target level (approximately two wells of a 6-well plate, with confluence exceeding 90%). Wash cells three times with DPBS to remove dead cells, digest and centrifuge to obtain cell pellet. Mix the pellets from both wells and add pre-prepared cell lysis buffer, lysing on ice for 20 min. Then, centrifuge at 12000 rpm for 10 min in a pre-chilled centrifuge at 4°C. Carefully aspirate the supernatant (avoiding aspiration of the pellet) and transfer it to a new centrifuge tube. Take 15 μL of the supernatant and dilute it 1:5 with Lysis as the protein concentration assay sample; repeat the assay three times for each sample. Add 6× Loading Buffer to the remaining supernatant proportionally, dilute the 6× Loading Buffer to 1×, boil in water for 5 min to denature the proteins, and then store at -20°C for later use.

[0064] For protein concentration determination, solution A+B was prepared at a volume ratio of 1:50 between solution B (copper sulfate solution) and solution A (BCA reagent). The test area was marked with a marker in a 96-well plate. 200 μL of the prepared BCA working solution and 25 μL of the test sample were added to each well. The plate was placed in a constant temperature incubator and incubated at 37°C in the dark for 30 min. Finally, the protein concentration was determined using a microplate reader.

[0065] Cell lysis buffer preparation: Lysis: phosphatase inhibitor; protease inhibitor = 100:10:1. Generally, 250 μL of lysis buffer is needed for one six-well plate.

[0066] (2) Western Blot detection The experimental steps are as follows: Calculate the average value based on the protein concentration measured in the previous step, and then calculate the loading amount based on a concentration of 20 μg.

[0067] Gel preparation: Prepare 10% gels according to the instructions. The separating gel preparation method is: 4 mL lower gel solution, 4 mL upper gel buffer, and 80 μL modified electrophoresis reagent; the stacking gel preparation method is: 1 mL upper gel solution, 1 mL upper gel buffer, and 20 μL modified electrophoresis reagent.

[0068] Remove the gel plate from the gel casting rack, wipe the liquid off the outer surface of the glass plate with paper, and then attach it to the gel holder with electrodes. Next, pour 1×Running Buffer into the electrophoresis tank.

[0069] Sample loading: Pull the comb vertically upwards, then add the protein sample to the SDS-PAGE gel wells according to the calculated protein concentration. Perform electrophoresis using the following program: 60 V, 30 min; 90 V, 1.5 h. After electrophoresis, remove the glass plate from the electrode holder and mark the front and back sides immediately. Cut a PVDF film to the same size as the gel, activate it in methanol for 15 s, and then place it in distilled water for 10 min. After activation, immerse it, the gel, and a sponge in 1× transfer buffer and shake for 20 min.

[0070] Place the sponge-gel-PVDF membrane-sponge in the center of the transfer clamp and clamp tightly. Transfer program: 110 V, 300 mA, 50 min, start transfer.

[0071] Blocking: After the transfer is complete, remove the transfer clamp and place the PVDF membrane in the pre-prepared blocking solution on a shaker for 1 hour. The blocking solution consists of 0.2 g of skim milk powder and 40 mL of 1×TBST.

[0072] After sealing is complete, discard the sealing solution, add an appropriate amount of 1×TBST to wash the membrane, repeat 3 times, 10 min each time.

[0073] Cut the desired protein band from the corresponding position according to the size of the marker band, put it into the diluted primary antibody, and incubate overnight at 4°C.

[0074] Wash three times with 1×TBST, 10 min each time. After washing, place in the prepared secondary antibody and incubate at room temperature for 60 min. After secondary antibody incubation, wash three more times with 1×TBST, 10 min each time.

[0075] Development solution preparation: The development solution was prepared using Pierce ECL Western blot substrate at a 1:1 ratio to visualize the protein signal, and the protein was detected using the e-BLOT contact non-destructive quantitative WB imaging system.

[0076] Western blot analysis was performed on cells passaged 25 times, and the results are as follows: Figure 5 As shown. The effects of overexpression were detected at the protein level. SOX17 Effects of overexpression on the levels of key pluripotency proteins in bovine embryonic stem cells, on WT and SOX17_OE Western blot analysis was performed on the cells, with β-ACTIN as an internal control protein. The results showed... SOX17 The level of SOX17 protein was significantly increased in bESCs of _OE ( Figure 5 The results were consistent with those of RT-qPCR, indicating that we successfully obtained... SOX17 Overexpression cell lines.

[0077] 6. Immunofluorescence staining was used to detect wild-type (WT) and... SOX17 Overexpression ( SOX17_OE The expression and protein localization of pluripotent genes in cells.

[0078] Cell preparation and fixation: The day before the experiment, Matrigel was pre-coated in 8-well plates and feeder cells were seeded. Cells to be tested were seeded into the wells and fixed when the cell density reached 60%-70% confluence. The culture medium was removed, and an appropriate amount of 4% cell fixative was added to each well. Fixation was performed at room temperature for 30 min. If subsequent staining was not planned immediately after fixation, the fixative was discarded, DPBS buffer was added, and the plate was stored at 4°C.

[0079] Cell permeability and non-specific site blocking: Discard the fixative or preservation solution, add 500 μL of DPBS buffer to each well and wash 3 times for 5 min each time to thoroughly remove residual fixative. After washing, add 200 μL of immunofluorescence buffer (IF Buffer: DPBS containing 1% BSA and 0.1% Triton X-100) to each well and permeabilize at room temperature for 30 min.

[0080] Antibody Incubation and Washing: Primary Antibody Incubation: Dilute the primary antibody with IF Buffer according to the manufacturer's recommended ratio. After discarding the permeabilization buffer, add an appropriate amount of diluted primary antibody working solution to each well. Wrap the plate with aluminum foil to avoid light exposure and incubate overnight at 4°C. Primary Antibody Washing: Discard the primary antibody working solution and add 200 μL of IF Buffer to each well for 3 washes, 5 min each time. Secondary Antibody Incubation: Select the appropriate secondary antibody according to the species and fluorescent labeling requirements of the primary antibody and dilute it appropriately with IF Buffer. After discarding the washing buffer, add the secondary antibody working solution and incubate at room temperature in the dark for 1 h. Secondary Antibody Washing: Discard the secondary antibody working solution and add 200 μL of IF Buffer to each well for 3 washes, 5 min each time. DAPI Staining and Mounting: Add 200 μL of DAPI staining solution (recommended concentration 1 μg / mL) to each well and incubate at room temperature in the dark for 5 min. Final Washing: Discard the DAPI staining solution and perform the following washes in sequence: wash 3 times with IF Buffer for 5 min each time, and wash once with DPBS for 5 min. Mounting and Preservation: After preliminary observation of the staining effect under a fluorescence microscope, add 5 μL of anti-fluorescence quenching mounting medium to each well, gently cover with a coverslip, and seal the edges with clear nail polish. After complete drying, the slides can be stored at -20℃ in the dark for subsequent image acquisition using a laser confocal microscope.

[0081] Immunofluorescence staining was used to localize the expression of key pluripotency factors such as OCT4, SOX2, and NANOG. The immunofluorescence staining results showed that WT and... SOX17_OE In the bovine embryonic stem cells group, NANOG, SOX2, and OCT4 were all specifically localized in the cell nucleus, consistent with the typical characteristics of pluripotent stem cells. Compared with the WT control group, SOX17_OE The protein expression levels of SOX2 and OCT4 in the group showed no significant changes, while the fluorescence signal intensity of NANOG increased significantly, confirming... SOX17 Overexpression can upregulate NANOG expression at the protein level without disrupting the core pluripotency network, suggesting... SOX17 Overexpression can enhance the pluripotency maintenance capacity of bovine embryonic stem cells and improve the culture stability of cell lines. Figure 6 ).

[0082] Example 2 SOX17 Effect of overexpression on the directed differentiation of bovine embryonic stem cells into PGC-like cells 1. Obtaining 7F-bESCs cultured under feeder-free conditions SOX17 Overexpression cell lines, resistant to WT and SOX17 Overexpression ( SOX17_OE Bovine embryonic stem cells (BECs) were used to induce PGCLCs. To this end, based on a fibronectin-coated culture system, an induction system consisting of Activin A, IWR-1, bFGF, and BMP4 was used to induce 7F-bESCs and... SOX17_OE bESCs cells were induced to adhere to the culture medium, such as Figure 7 .

[0083] 7F-bESCs and SOX17 Overexpression of bovine embryonic stem cells ( SOX17_OE After digestion with ACC, bESCs were gently resuspended in bovine primordial germ cell-like cell induction medium (bPGCLCs induction medium) to form a single-cell suspension, which was then seeded into pre-coated 12-well plates. During induction, the induction medium was changed every 24 hours, and the proliferation status and morphological changes of the cells were continuously observed and recorded.

[0084] During the induction process, cell morphology was observed and images were recorded on days 2, 4, and 6. Figure 8 Following induction treatment, the initially typical bovine embryonic stem cell clonal colonies gradually dispersed, and on day 4, smaller, highly refractive cell colonies appeared in bright-field microscopy, characterized by large, round nuclei. Their numbers gradually increased with prolonged induction. Notably, compared to the control group, SOX17The number of these bright, round cells was greater in the overexpressing cells, and the cells showed reporter gene fluorescence signals under a fluorescence microscope.

[0085] 2. Real-time quantitative PCR analysis Cell samples were collected on days 2, 4, and 6 of induction using real-time quantitative PCR. The method was the same as in Example 1, and the results are as follows. Figure 9 The results showed that, compared with the 7F-WT control group, 7F- SOX17_OE In the group SOX2 The expression of [a specific substance] was completely suppressed throughout the differentiation cycle and remained at an extremely low level, significantly lower than that of the WT group during the same period. NANOG The expression of the group peaked on day 4 of differentiation, which was 2.5 times that of the WT group, and remained high on day 6. OCT4 The expression of [specific component] remained stable during the differentiation process, reversing the downward trend over time in the WT group, and there were no significant differences between the two groups at any time point. These results indicate that... SOX17 Overexpression can be mitigated by rapid silencing. SOX2 Continuous activation NANOG ,Stablize OCT4 The regulatory model reshapes the pluripotent gene network, guides bovine embryonic stem cells to differentiate into the primordial germ cell lineage, and achieves efficient in vitro induction of PGC-like cells.

[0086] Real-time quantitative PCR was performed to detect the expression level of PGC-specific genes, and the results are as follows: Figure 10 As shown. SOX17 Overexpression can significantly enhance PGC-specific genes BLIMP1 , NANOS3 , TFAP2C The level of expression. Moreover, SOX17 Overexpression of key genes in primordial germ cells increased with the number of induction days, from day 2 to day 6. BLIMP1 , NANOS3 , TFAP2C The expression level gradually increased. This result indicates that... SOX17 Overexpression can activate the expression of reproduction-related genes, thereby promoting the induction efficiency of PGCLCs.

[0087] 3. Immunofluorescence staining Based on the gene expression detection results above, we further selected PGC-like cell samples with the highest expression levels of PGC-related genes on day 6 of induction for immunofluorescence staining, using the same method as in Example 1. The results are as follows... Figure 11 As shown, compared with the WT control group, SOX17In the _OE group, the fluorescence signal intensity and the proportion of positive cells of PGC lineage-specific markers such as BLIMP1, T-Bra, and TFAP2C were significantly increased, and all markers were specifically localized to the cell nucleus and co-localized with DAPI staining. These results are highly consistent with transcriptional level data from real-time quantitative PCR, confirming the protein level. SOX17 Overexpression can significantly improve the efficiency and purity of bovine embryonic stem cells differentiating into PGC-like cells, providing key support for the establishment of an efficient PGC in vitro induction system.

[0088] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Improve SOX17 Application of reagents that enhance gene expression levels and / or SOX17 protein levels in improving the pluripotency of bovine embryonic stem cells.

2. The application according to claim 1, characterized in that, The SOX17 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the amino acid sequence of the SOX17 protein is shown in SEQ ID NO.

2.

3. The application according to claim 1 or 2, characterized in that, The improvement of bovine embryonic stem cell pluripotency includes the following (1) and / or (2): (1) Maintain the typical stem cell morphology and stable proliferation capacity of bovine embryonic stem cells, and prevent spontaneous differentiation after continuous passage; (2) Enhance the ability to maintain cell pluripotency.

4. A method for improving the pluripotency of bovine embryonic stem cells, characterized in that, include: Increasing bovine embryonic stem cells SOX17 Gene expression level.

5. The method according to claim 4, characterized in that, Increasing bovine embryonic stem cells SOX17 Methods for assessing gene expression levels include: SOX17 Gene overexpression vectors were introduced into bovine embryonic stem cells.

6. Improve SOX17 Application of reagents that enhance gene expression levels and / or increase SOX17 protein levels in the in vitro directed induction of bovine embryonic stem cells into primordial germ cell-like cells.

7. According to claim 6, the reagent can improve the induction efficiency of bovine embryonic stem cells into primordial germ cell-like cells.

8. The application according to claim 7, characterized in that, Improving induction efficiency includes at least one of the following: (1) Upregulate the mRNA and protein expression levels of PGC-specific marker genes to increase the positive rate and proportion of PGC-like cells; (2) Improve the uniformity and maturity of PGC-like cells after induction.

9. A method for improving the induction efficiency of bovine embryonic stem cells into primordial germ cell-like cells, characterized in that, include: Increasing bovine embryonic stem cells SOX17 After gene expression levels, SOX17 Bovine embryonic stem cells with enhanced gene expression levels were induced to differentiate into primordial germ cell-like cells.

10. A primer set for identifying whether the pluripotency of bovine embryonic stem cells is enhanced, characterized in that, include: bSOX17-F as described in SEQ ID NO. 11 and bSOX17-R as described in SEQ ID NO. 12.

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