A method for efficiently preparing gene edited cloned bovine based on embryonic stem cells

Abstract

The application discloses a method for efficiently preparing gene editing cloned cattle based on embryonic stem cells. The method comprises the following steps: first, an epiblast is separated from an embryo of a high-yield dairy cow at the E7 stage, and a stable cell line bEpiSCs is established; then, an exogenous fragment containing hCSN2 genes and hLF genes is precisely integrated into the third exon region of the bCSN2 gene of the cell line bEpiSCs by using the CRISPR / Cas9 technology; finally, after in-vitro differentiation for 4 generations, the cell line is used as a nuclear donor, and by means of somatic cell nuclear transfer technology, four human milk cloned cattle derived from stem cells are first cultivated. The application breaks through the technical bottleneck of low efficiency of multiple rounds of precise genetic modification in traditional breeding, discovers the feasibility and application potential of the stem cell breeding system in genetic improvement of large animals, provides a new paradigm that can be popularized for the transformation and upgrading of livestock breeding from experience selection to precise design, and has important application prospect.

Description

Technical Field

[0001] This invention belongs to the field of transgenic animals, specifically relating to a method for efficiently preparing gene-edited cloned cattle based on embryonic stem cells. Background Technology

[0002] As a pillar industry of my country's agriculture, animal husbandry urgently requires continuous innovation in breeding technologies for sustainable development. Currently, modern bio-breeding technologies, represented by whole-genome selection, gene editing, somatic cell cloning, and embryo transfer, have significantly improved breeding efficiency. However, common bottlenecks remain, such as difficulties in multi-gene synergistic editing and the challenge of achieving multiple rounds of precise genetic modification, hindering further breakthroughs in breeding effectiveness. Against this backdrop, gene editing breeding technology using embryonic stem cells has emerged. This technology precisely edits the genes of embryonic stem cells, which possess unlimited proliferation and multi-directional differentiation potential, and then combines this with nuclear transfer technology to produce cloned individuals, achieving targeted and efficient improvement of genetic traits and significantly shortening the breeding cycle. It is considered a key pathway to promote the upgrading of modern agricultural breeding.

[0003] In recent years, my country's dairy industry has faced multiple challenges, including rising costs, resource constraints, and the threat of disease, making industrial transformation and upgrading an urgent necessity. Improving dairy cow productivity, enhancing disease resistance, and precisely regulating milk composition to better meet human nutritional needs have become key areas of focus for both scientific research and industry.

[0004] Compared to cow's milk, human milk exhibits significant advantages in both the content and structure of key nutritional proteins. Human milk contains a higher concentration of β-casein (hCSN2, Gene ID: 1447), which is also the more easily digestible A2 type, avoiding absorption and allergy problems that may arise from the predominantly A1 type found in cow's milk. Furthermore, the concentration of lactoferrin (hLF, Gene ID: 4057) in human milk far exceeds that in cow's milk. With its antibacterial, anti-inflammatory, and iron-transporting functions, hLF is crucial for maintaining iron homeostasis and has been proven to effectively reduce the risk of sepsis and necrotizing enterocolitis in premature infants. Therefore, breeding cloned cows carrying human hCSN2 and human hLF genes to secrete milk containing human β-casein and human lactoferrin, thus achieving a nutritional profile closer to human milk, will open up new avenues for modern bio-breeding and has significant application value. Summary of the Invention

[0005] The technical problem to be solved in this application is how to use animal embryonic stem cells to cultivate cloned animals.

[0006] This application first protects the use of animal embryonic stem cells, or animal embryonic stem cells that have undergone one or more rounds (such as one, two, or three rounds) of gene editing, as nuclear transfer donor cells in the cultivation of cloned animals.

[0007] In the above applications, the animal embryonic stem cells that have undergone more than one round of gene editing need to possess pluripotency and / or developmental potential.

[0008] The pluripotency can be manifested in the stable expression of pluripotent core transcription factors. These pluripotent core transcription factors can specifically include OCT4, SOX2, and NANOG, among others.

[0009] The developmental potential can be manifested in excellent blastocyst formation ability, such as a blastocyst formation rate of 0.30 or higher.

[0010] In the above applications, the bovine embryonic stem cells or animal embryonic stem cells that have undergone one or more rounds of gene editing can be differentiated in vitro.

[0011] The number of generations of in vitro differentiation can be one or more.

[0012] The specific generation of the in vitro differentiation can be 3-5 generations (such as 3-4 generations, 4-5 generations, 3 generations, 4 generations, or 5 generations).

[0013] The in vitro differentiation method can be as follows: the bovine embryonic stem cells or animal embryonic stem cells that have undergone more than one round of gene editing are passaged in a culture medium (which can be 3i / LAF medium) for 40-60 hours (e.g., 40-48 hours, 48-60 hours, 40 hours, 48 ​​hours or 60 hours). Then, the culture medium is replaced with BM medium containing 8-12 ng / mL BMP4, 3-5 μM SB-431542 and 8-12 ng / mL FGF2. After the cell morphology gradually changes to a fibroblast-like morphology, the cells are passaged and the differentiation culture is continued for 1-2 weeks until the cells exhibit a uniform long spindle-shaped fibrous phenotype.

[0014] In the above applications, the gene editing can be achieved by using the CRISPR / Cas system and homologous recombination technology to integrate exogenous nucleic acid molecules into the genome of animal embryonic stem cells.

[0015] In the above applications, the integration of exogenous nucleic acid molecules into the genome of animal embryonic stem cells using the CRISPR / Cas system and homologous recombination technology can specifically involve integrating the coding gene for human β-casein (hCSN2 gene) and the coding gene for human lactoferrin (hLF gene) into the exon region of the β-casein coding gene in the genome of animal embryonic stem cells using the CRISPR / Cas system and homologous recombination technology.

[0016] In the above applications, the CRISPR / Cas system may include a recombinant expression vector expressing gRNA encoding a gene targeting animal β-casein and a Cas protein.

[0017] In the above applications, the nucleotide sequence of the exogenous nucleic acid molecule is shown as positions 5741-11575 from the 5' end of SEQ ID No. 1.

[0018] In the above applications, the target sequence of the gRNA (i.e., the gRNA encoding the gene that targets animal β-casein) may be as shown in SEQ ID No. 5.

[0019] In any of the applications described above, the animal may be a mammal. Preferably, the mammal may be a cow. Specifically, the cow may be a Chinese Holstein.

[0020] In the above application, the exon region of the β-casein coding gene integrated into the genome of animal embryonic stem cells can specifically be the third exon region of the β-casein coding gene integrated into the genome of animal embryonic stem cells.

[0021] In any of the above-described applications, the step of integrating the coding genes for human β-casein and human lactoferrin into the exon region of the β-casein coding gene of the bovine embryonic stem cell genome using the CRISPR / Cas system and homologous recombination technology can be as follows: (1) Using the CRISPR / Cas system, an exogenous nucleic acid molecule with a nucleotide sequence as shown in SEQ ID No. 1 from position 5741 to 11575 from the 5' end was integrated into the exon region of the gene encoding β-casein in the genome of bovine embryonic stem cells; (2) After completing step (1), bovine embryonic stem cells expressing GFP protein or containing the coding gene of GFP protein are obtained by GFP-positive cell sorting and / or molecular detection. (3) After completing step (2), GFP is removed by the Cre-loxP system, and bovine embryonic stem cells that do not express GFP protein or do not contain the coding gene of GFP protein are obtained by GFP-negative cell sorting and / or molecular detection. Bovine embryonic stem cells that do not express GFP protein or do not contain the gene encoding GFP protein are animal embryonic stem cells that have undergone more than one round of gene editing.

[0022] In the above applications, the target sequence of the gRNA expressed by the CRISPR / Cas system is shown in SEQ ID No. 5.

[0023] In any of the applications described above, the CRISPR / Cas system may be a CRISPR / Cas9 system.

[0024] In any of the above-described applications, the Cas protein may be the Cas9 protein.

[0025] In any of the above applications, the bovine embryonic stem cells are isolated from the epiblast of high-yielding dairy cow embryos and possess stem cell characteristics and genomic stability. Preferably, the high-yielding dairy cow embryo is specifically an E7 stage embryo from a high-yielding dairy cow.

[0026] The inventors of this application first isolated the epiblast from E7 stage embryos of high-yielding dairy cows and established a stable cell line, bEpiSCs. Subsequently, they constructed an HCSN2-HLF-GFP vector (containing the encoding gene for human β-casein (hCSN2 gene) and the encoding gene for human lactoferrin (hLF gene)), and precisely integrated it into the third exon region of the β-casein encoding gene in the bEpiSCs cell line using CRISPR / Cas9 technology. Using PCR technology, they performed cross-homologous arm detection on the stable GFP-expressing cell line (GFP-positive cells sorted by flow cytometry), successfully screening for the HCSN2-HLF-GFP bEpiSCs cell line. Further, they efficiently removed the eGFP marker using the Cre-loxp system, and then screened for GFP-negative clones by flow cytometry, successfully identifying the cell line carrying the HCSN2-HLF gene—the HCSN2-HLF-bEpiSCs cell line. Finally, HCSN2-HLF-bEpiSCs, after four generations of in vitro differentiation, were used as nuclear donors. Using somatic cell nuclear transfer (SCNT) technology, the first humanized milk-like stem cell cloned cattle mediated by embryonic stem cells were successfully prepared. This application marks the first time that two rounds of continuous and precise gene editing have been achieved on embryonic stem cells, resulting in an engineered stem cell line carrying the human milk protein gene. Further combined with somatic cell nuclear transfer technology, four "humanized" cloned cattle were successfully cultivated from stem cells for the first time, validating the feasibility and application potential of stem cell breeding systems in the genetic improvement of large animals.

[0027] Therefore, it is evident that the inventors of this application have established a stable bovine embryonic stem cell line. This system fundamentally overcomes the technical bottlenecks caused by the limited expansion capacity, low editing efficiency, and unstable passage of traditional somatic cells, providing a systematic technical solution for achieving multiple rounds of precise and verifiable genetic modification. The breeding method based on multi-gene editing of bovine embryonic stem cells provided in this application, by establishing a stable bovine embryonic stem cell line and combining multi-gene synergistic editing and nuclear transfer technology, cultivates cloned cattle capable of stably secreting human β-casein and human lactoferrin. This overcomes the technical bottleneck of low efficiency in multiple rounds of precise genetic modification in traditional breeding, achieving targeted and efficient improvement of dairy cow production performance and milk nutritional value. It provides a new, scalable paradigm for the transformation and upgrading of livestock breeding from "experience-based selection" to "precision design," and also demonstrates the feasibility and applicability of embryonic stem cell lines as breeding seed cells in the synergistic improvement of complex traits. This application has significant application value. Attached Figure Description

[0028] Figure 1 To establish a stable cell line bEpiSCs and to detect its pluripotency.

[0029] Figure 2 To construct and identify the HCSN2-HLF-bEpiSCs cell line.

[0030] Figure 3 To detect the pluripotency of the HCSN2-HLF-bEpiSCs cell line and its ability to develop as donor cells.

[0031] Figure 4 To generate and validate cloned cattle using the HCSN2-HLF-bEpiSCs cell line as a nuclear transfer donor cell. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0035] The Escherichia coli DH5α competent cells and Escherichia coli Tran1-T1 competent cells in the following examples are products of TransGen Biotech.

[0036] The primer names and their nucleotide sequences in the following examples are shown in Table 1.

[0037] Table 1

[0038] In the following examples, CD-1® (ICR) IGS mice are products of Spiefer (Beijing) Biotechnology Co., Ltd., used for the preparation of mouse feeder cells. Holstein high-yielding dairy cow E7 embryos and Holstein recipient cows were both provided by Shijiazhuang Tianquan Fine Breed Dairy Co., Ltd. In the following text, Holstein high-yielding dairy cow E7 embryos are referred to as high-yielding dairy cow embryos, and Holstein recipient cows are referred to as recipient cows.

[0039] Implementation examples I. Experimental Methods 1. Preparation of mouse feeder layer cells Fetuses were harvested from the uterus of pregnant CD-1® (ICR) IGS mice (E13.5 genotype). After removing internal organs, head, tail, and limbs, the trunk tissue was collected into 1.5 mL centrifuge tubes. Then, 0.25% Trypsin-EDTA (Gibco, 25300120) was added, and the cells were digested in a 37°C water bath. The digested tissue suspension was transferred to a 10 cm cell culture dish, 8 mL of MEF medium was added, and the mixture was gently mixed and incubated at 37°C, 5% CO2, and saturated humidity. When the primary cells reached the third passage and reached confluence, they were treated with 13.3 ng / mL mitomycin C for 3.5 hours to terminate proliferation. The cells were then washed 2-3 times with DPBS, and digested with 0.1% Trypsin-EDTA at 37°C for 5 minutes to obtain mouse feeder cells.

[0040] MEF medium is DMEM medium (Gibco, 11960-044) containing 10% (v / v) FBS, 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140-122) and 1% GlutaMAX (Thermo Fisher Scientific, 35, 050-061).

[0041] The mouse feeder cells were counted and then divided into tubes of 2.4 × 10⁻⁶ cells. 6 Each cell was aliquoted into cryovials and preserved long-term using a programmed gradient cryopreservation method.

[0042] 2. Establish a stable cell line bEpiSCs The detailed flowchart for isolating epiblasts from high-yielding dairy cow embryos and establishing stable cell lines (bEpiSCs) can be found in [link to flowchart]. Figure 1 In (A) (Jiang S, Li H, Zhang L, et al. Generic Diagramming Platform (GDP): a comprehensive database of high-quality biomedical graphics. Nucleic AcidsRes. 2025;53(D1):D1670-D1676. doi:10.1093 / nar / gkae973.).

[0043] Specifically, high-yielding dairy cow embryos were used to establish a stable cell line, bEpiSCs, using the method described in the reference (Elucidation of the pluripotent potential of bovine embryonic lineages facilitates the establishment of formal stem cell lines. Cellular & Molecular Life Sciences. 2024). In brief, the epiblast cells of high-yielding dairy cow embryos were mechanically isolated and processed using TrypLE... TM Digested with Express (Gibco, 12,605,010) for 3 minutes, then inoculated onto mouse feeder cells containing 3i / LAF medium (described in the following literature: Generation and characterization of stable pig pregastrulation epiblast stem cell lines. Cell Research. 2022), and cultured in a 5% O2, 5% CO2 saturated humidity incubator. After clonal growth for 2-3 days, digested with Accutase (Gibco, A11105-01), adding an equal volume of 3i / LAF medium to stop digestion when most clones detached, then passaged at a 1:4 ratio, repeated every 3 days.

[0044] 3. AP staining AP staining was performed using the Alkaline Phosphatase Detection Kit (Millipore, SCR004). For AP staining methods, reagent preparation, and precautions, please refer to the kit described above.

[0045] 4. Genomic DNA extraction Genomic DNA was extracted from cell and tissue samples using the TIANamp Genomic DNA Kit (TIANGEN, DP304).

[0046] 5. Plasmid construction (1) The plasmid PX330-Puro was constructed using the method described in the reference (Multiplex Genome Engineering Using CRISPR / CasSystems.Science. 2013).

[0047] (2) Construction of plasmids PX330-bCSN2 sgRNA-1-Puro, PX330-bCSN2 sgRNA-2-Puro, PX330-bCSN2 sgRNA-3-Puro and PX330-bCSN2 sgRNA-4-Puro ① Four sgRNA target sequences were synthesized by BGI Genomics: sgRNA-1: 5'-aggctttccacaatctataa-3' (SEQ ID No. 2), sgRNA-2: 5'-ctggaagaactcaatgtacc-3' (SEQ ID No. 3), sgRNA-3: 5'-agtaacagtctctaatgatc-3' (SEQ ID No. 4), and sgRNA-4: 5'-agaactcaatgtacctggtg-3' (SEQ ID No. 5). These sequences contain a 20 bp target region immediately adjacent to PAM. The target gene corresponding to all four sgRNA targets is the BCSN2 gene, which encodes the bovine bCSN2 protein (Gene ID: 281099).

[0048] ② The plasmid PX330-Puro was digested with the restriction endonuclease BbsI (37℃, 1 h) to obtain the linearized plasmid PX330-Puro. Then, the annealing products of single-stranded sgRNA-1, sgRNA-2, sgRNA-3, and sgRNA-4 were ligated to the linearized plasmid PX330-Puro using T4 ligase, resulting in plasmids PX330-bCSN2 sgRNA-1-Puro, PX330-bCSN2 sgRNA-2-Puro, PX330-bCSN2 sgRNA-3-Puro, and PX330-bCSN2 sgRNA-4-Puro.

[0049] (3) Construction of plasmid gbCSN2-Donor-hCSN2-hLF-loxp-eGFP-Puro-loxp (hereinafter referred to as HCSN2-HLF-GFP vector) The nucleotide sequence of the HCSN2-HLF-GFP vector (circular) is shown in SEQ ID No. 1.

[0050] The construction method of the HCSN2-HLF-GFP vector is briefly described below: ① The donor vector used Donor-OCT4-A5-3×flag-PNP-tdtomato as its backbone. The left homologous arm was designed 5.5 kb upstream of the sgRNA target site, and the right homologous arm was designed 2.0 kb downstream of the sgRNA target site. The plasmid and the amplified 5'HA fragment were double-digested with MluI and AgeI enzymes, and the target fragment was recovered and ligated with T4 ligase. The 3'HA fragment was ligated with ClaI and PacI restriction enzymes in the same way as the 5'HA fragment.

[0051] ② The key functional element hCSN2-hLF-LOXP-EF-1α-EGFP-P2A-PuroR-SV40 poly(A)-LOXP was also synthesized by BGI and integrated into the donor vector through homologous recombination.

[0052] (4) Construction of pCAG-Cre-IRES-Puro plasmid The DNA fragment between EcoRI and AgeRI in the pCAG-IRES-Puro vector was replaced with the CDS of the gene encoding the Cre protein (GeneID: 2777477), while all other sequences remained unchanged, to obtain the pCAG-Cre-IRES-Puro plasmid.

[0053] The nucleotide sequence of the pCAG-IRES-Puro vector (circular) is shown in SEQ ID No. 6.

[0054] Plasmids PX330-Puro, PX330-bCSN2 sgRNA-1-Puro, PX330-bCSN2 sgRNA-2-Puro, PX330-bCSN2 sgRNA-3-Puro, PX330-bCSN2 sgRNA-4-Puro, and HCSN2-HLF-GFP all carry the ampicillin resistance gene and exhibit ampicillin (Amp) resistance.

[0055] 6. Detection of SgRNA cleavage efficiency (1) Four sgRNAs (sgRNA-1: 5'-aggctttccacaatctataa-3', sgRNA-2: 5'-aggctttccacaatctataa-3', sgRNA-3: 5'-agtaacagtctctaatgatc-3' and sgRNA-4: 5'-agaactcaatgtacctggtg-3') were designed and synthesized using the CRISPOR online tool (http: / / crispor.tefor.net / ) to systematically evaluate their targeted cleavage efficiency. Based on the backbone of plasmid PX330-Puro, plasmids PX330-bCSN2 sgRNA-1-Puro, PX330-bCSN2 sgRNA-2-Puro, PX330-bCSN2 sgRNA-3-Puro and PX330-bCSN2 sgRNA-4-Puro were constructed (see step 5 (2)).

[0056] (2) To evaluate the cleavage activity of sgRNA, bEpiSCs cultured to 70-80% confluence were first seeded into 24-well plates and transfected using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015): Each well transfection system contained 2 μg of plasmid (plasmid PX330-bCSN2 sgRNA-1-Puro, plasmid PX330-bCSN2 sgRNA-2-Puro, plasmid PX330-bCSN2 sgRNA-3-Puro or plasmid PX330-bCSN2 sgRNA-4-Puro) and 1 μL of P3000 reagent (transfection aid to improve transfection efficiency), which were diluted with 1.5 μL of Lipofectamine 3000 in 50 μL of Opti-MEM and then incubated at room temperature for 15 minutes to form a transfection complex.

[0057] (3) Cells were collected and genomic DNA was extracted 48 hours after transfection. A fragment of about 500 bp was amplified using target-specific primers. After purification, denaturation-renaturation treatment was performed (95℃, 5 min; then reduced to 85℃ at 0.1℃ / s, and then reduced to 25℃ at 0.3℃ / s) to form heteroduplexes. T7E1 endonuclease (NEB, M0302L) was added and reacted at 37℃ for 30 min. The enzyme digestion products were analyzed by 2% agarose gel electrophoresis. The mutagenicity of each sgRNA was evaluated by calculating the proportion of cleavage bands.

[0058] The results showed that sgRNA-4 had the highest cleavage efficiency and was used in subsequent experiments.

[0059] 7. bEpiSCs Transfection and Screening (1) Before electroporation, bEpiSCs were digested using Accutase (Gibco, A11105-01).

[0060] (2) Each time the electrical circuit is operated, the Neon NxT system is used, with 1×10 of bEpiSCs. 6 Using individual cells as units, under conditions of 1200V, 20ms, and 2 pulses, 2 μg of plasmid PX330-bCSN2 sgRNA-4-Puro (see step 5 (2)) and 2 μg of HCSN2-HLF-GFP vector (see step 5 (3)) were co-transfected to achieve site-specific integration of the hCSN2-hLF-loxp-eGFP-Puro-loxp sequence (nucleotide sequence as shown in SEQ ID No. 1 from position 5741 to 11575 from the 5' end) into the third exon of the BCSN2 gene. After transfection, GFP-positive cells were sorted using a flow cytometer (MoFlo XDP, Beckman) at 488 nm excitation with a 710 / 50 bandpass filter (the first round of cell line screening was performed using a GFP reporter system), and then seeded into 48-well plates for single-clone amplification. To verify the gene editing, genomic DNA was directly extracted from the cell line using cell lysis buffer (Invitrogen, AM8723) and used as a template for PCR amplification. Sequencing of the product confirmed correct integration, and the HCSN2-HLF-GFP bEpiSCs cell line was screened by cross-homologous arm detection.

[0061] PX330-bCSN2 sgRNA-4-Puro expresses Cas9 protein and sgRNA. After binding, the genome is cleaved. Using the principle of homologous recombination, the homologous arm in the HCSN2-HLF-GFP vector binds to the chromosome, and then the middle sequence of the homologous arm, namely the hCSN2-hLF-loxp-eGFP-Puro-loxp sequence, is inserted into the genome.

[0062] (3) To remove the eGFP marker, after completing step (2), electroporate 2 μg of pCAG-Cre-IRES-Puro plasmid into the HCSN2-HLF-GFP bEpiSCs cell line. GFP-negative clones are screened by flow cytometry and further amplified to obtain the HCSN2-HLF-bEpiSCs cell line. That is, the cell line carrying the HCSN2-HLF gene is obtained by efficiently removing the eGFP marker using the Cre-loxp system.

[0063] 8. Differentiation of HCSN2-HLF-bEpiSCs cell lines To improve the success rate of cloned embryos, the HCSN2-HLF-bEpiSCs cell line was differentiated. The specific steps are as follows: After passage of the HCSN2-HLF-bEpiSCs cell line obtained in step 7 in 3i / LAF medium for 48 hours, the medium was replaced with BM medium containing BMP4 (10 ng / mL), SB-431542 (5 μM), and FGF2 (10 ng / mL) (described in the following literature: Generation and characterization of stable pig pregastrulation epiblaststem cell lines. Cell Research. 2022). After the cell morphology gradually changed to a fibroblast-like morphology, it was passaged and cultured for another 1 to 2 weeks until the cells showed a uniform long spindle-shaped fibrous phenotype. The cells obtained at this time (i.e., cells differentiated from the HCSN2-HLF-bEpiSCs cell line for 4 generations in vitro) will be used as donor cells for nuclear transfer.

[0064] 9. Immunofluorescence staining After fixation with 4% paraformaldehyde for 30 minutes, the samples were permeabilized with 0.5% Triton X-100 at room temperature for 15 minutes. After removing the permeabilization solution, the samples were washed three times with washing buffer (DPBS containing 0.1% Tween 20 and 0.1% Triton X-100) at 70 rpm on a shaker, followed by blocking with 3% BSA for 1 hour. Primary antibodies (OCT4 antibody (Santa Cruz, sc-5279) for OCT4 detection; SOX2 antibody (Santa Cruz, sc-365823) for SOX2 detection; and NANOG antibody (PeproTech, 500-P236)) were diluted according to the recommended ratio and incubated overnight with the samples at 4°C on a shaker. After incubation, the samples were washed three times with washing buffer for 10 minutes each time. Then, the corresponding species-derived fluorescent secondary antibody was added, and the samples were incubated at room temperature in the dark for 1 hour. The washing steps were repeated. Finally, DAPI (Beyotime, P0131) was used to stain the cell nuclei to mark their locations.

[0065] 10. Preparation and transfer of cloned embryos Bovine ovaries were collected from the slaughterhouse, preserved in physiological saline at 30°C, and transported to the laboratory within 2 hours. Upon arrival, the ovaries were first washed three times with preheated physiological saline at 38°C. Then, follicles with a diameter of 2-8 mm were aspirated using a 10mL syringe equipped with an 18-gauge needle, and cumulus-oocyte complexes (COCs) were collected. COCs matured in vitro for 17-18 hours in an incubator at 38.5°C, 5% CO2, and saturated humidity. Afterward, they were treated with 0.1% hyaluronidase to remove excess cumulus cells, selecting only mature oocytes that had extruded the first polar body for subsequent experiments. Oocyte enucleation was performed in M199 processing solution (Gibco, 11150-059) containing 10% FBS, followed by injection of donor cells into M199 solution containing 2% FBS. Cell fusion was performed using an electrofusion instrument (CFB16-HB) with parameters set at 22V and 10μs. The reconstructed embryos were then treated with 5 μM iomycin (Sigma, D2629) for 5 minutes, and then transferred to 2 mM 6-DMAP (Sigma, I3909) for 4 hours. The activated embryos were then cultured in IVC medium (Bioscience, BOIVC2501) for 7 days, and finally, morphologically sound blastocysts were selected for transfer into recipient cattle that had undergone estrus synchronization.

[0066] 11. Preparation of bovine ear fibroblasts Bovine ear margin tissue was collected from the cattle farm and then washed once with 75% alcohol and three times with DPBS containing antibiotics to disinfect the surface. Subsequently, hair and cartilage were thoroughly removed with a blade to obtain clean tissue blocks. The processed tissue was transferred to 1.5 mL centrifuge tubes, minced, and mixed with 200 μL of FBS (Gibco, 16000-044) to form a tissue paste. This paste was then evenly applied to the bottom of a T25 culture flask containing MEF medium and finally placed in a 37°C, 5% CO2, and saturated humidity incubator for primary culture to obtain bovine ear margin fibroblasts.

[0067] 12. Karyotype analysis Before karyotype analysis, 1% KaryoMAX® Colcemid solution (Gibco, 15212012) was added to the culture medium for bEpiSCs, and the cells were incubated for 2-3 hours to arrest cell division at metaphase. Cells were then digested using TrypLE™ Express (Gibco, 12605010), and after centrifugation, approximately 50 μL of culture medium was retained to resuspend the cell pellet. 5 mL of pre-warmed 0.075 M KCl solution (Sigma, P5405) was slowly added, and the cells were hypotonic at 37°C for 30 minutes. Then, 500 μL of freshly prepared fixative (a mixture of 3 parts methanol and 1 part acetic acid) was added, and the mixture was gently inverted to mix. The mixture was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The fixation process was repeated twice, using 5 mL of fixative each time, with the final cell pellet resuspended in 1 mL of fixative. The resuspended cell pellet was dropped from a height of 1 m onto a pre-cooled glass slide tilted at 45° and allowed to air dry at room temperature. After drying, the slides are stained with 10% Giemsa staining solution (Sangon, E6073140001) in the dark for 10-15 minutes, and then dried in an oven at 37°C. The slides are then examined and photographed under an oil immersion microscope.

[0068] 13. Statistical Analysis Experimental data were analyzed using SAS statistical software. One-way ANOVA and Duncan's test were used to determine the significance of differences between different treatments. A p-value < 0.05 was considered significant.

[0069] II. Experimental Results 1. Establishment of stable cell line bEpiSCs Three stable cell lines bEpiSCs were successfully established from the epiblast cells of six high-yielding dairy cow embryos. Figure 1 (C) Based on pluripotency (including AP staining) and karyotype analysis, all three stable cell lines bEpiSCs were positive for alkaline phosphatase staining and had normal chromosome numbers (2n=60). Figure 1 (B) Immunofluorescence staining results further confirmed that the three stable cell lines bEpiSCs stably expressed pluripotent core transcription factors such as OCT4, SOX2, and NANOG. Figure 1 (D) Therefore, the three stable cell lines bEpiSCs established in this application possess typical stemness characteristics and genomic stability, and can be used for subsequent gene editing.

[0070] 2. Obtaining the HCSN2-HLF-bEpiSCs cell line The plasmids PX330-bCSN2 sgRNA-4-Puro and HCSN2-HLF-GFP vector were constructed, and the hCSN2-hLF-loxp-eGFP-Puro-loxp sequence was precisely integrated into the third exon of the BCSN2 gene using the CRISPR / Cas9 system. The integration target site was sgRNA-4: 5'-agaactcaatgtacctggtg-3' ( Figure 2 (A) Subsequently, genotyping of eight stable GFP-expressing cell lines was performed using cross-homologous arm PCR. The correct size of the 3' cross-homologous arm was 4.1 kb, and the correct size of the 5' cross-homologous arm was 6.1 kb. The results showed that six of them were positive clones with correct integration (i.e., HCSN2-HLF-GFP bEpiSCs cell lines), with an integration efficiency of 75%. Figure 2 (B)

[0071] 3. Assessment of pluripotency and developmental capacity of HCSN2-HLF-bEpiSCs cell lines Alkaline phosphatase (AP) staining and immunofluorescence assays showed that the HCSN2-HLF-bEpiSCs cell line remained AP-positive and stably expressed pluripotency core transcription factors such as OCT4, SOX2, and NANOG, indicating that the pluripotency of the cells was not affected after gene editing. Figure 3 (A)

[0072] Further developmental potential was assessed using HCSN2-HLF-bEpiSCs cell lines or bovine ear marginal fibroblasts (as controls) as nuclear transfer donor cells. Results showed ( Figure 3 In the middle (B) section, the blastocyst rate of the HCSN2-HLF-bEpiSCs cell line (0.36±0.032) was significantly higher than that of bovine ear fibroblasts (0.28±0.035) (p<0.05).

[0073] The above results indicate that the HCSN2-HLF-bEpiSCs cell line, while maintaining pluripotency, possesses the in vivo developmental potential to serve as a nuclear transfer donor, providing a reliable cell source for the subsequent production of gene-edited cloned cattle.

[0074] 4. Verification of cloned cattle Using the HCSN2-HLF-bEpiSCs cell line as nuclear transfer donor cells, a total of 45 cloned embryos were constructed and transferred into 31 recipient cows. The average pregnancy rates at 30 days, 60 days, and 120 days were 38.7%, 35.4%, and 35.4%, respectively, with a birth rate of 19.3%. Ultimately, four healthy, surviving cloned calves were obtained (see...). Figure 4 (C) and (A)).

[0075] Gene insertion identification was performed on cloned calves. The correct size of the 3' homologous arm was 2.1 kb, and the correct size of the 5' homologous arm was 6.1 kb. The length of the exogenous inserted gene was 360 bp in both cases. PCR identification results showed ( Figure 4 In the study (B), none of the four recipient cows expressed the HCSN2 and HLF genes, and all newborn calves stably integrated the HCSN2 and HLF genes, confirming them as offspring obtained through stem cell gene editing.

[0076] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of animal embryonic stem cells or animal embryonic stem cells that have undergone one or more rounds of gene editing as nuclear transfer donor cells in the cultivation of cloned animals.

2. The application according to claim 1, characterized in that: The animal embryonic stem cells that have undergone more than one round of gene editing possess pluripotency and / or developmental potential.

3. The application according to claim 1, characterized in that: The bovine embryonic stem cells or animal embryonic stem cells that have undergone one or more rounds of gene editing can be differentiated in vitro.

4. The application according to claim 1, characterized in that: The gene editing involves integrating exogenous nucleic acid molecules into the genome of animal embryonic stem cells using the CRISPR / Cas system and homologous recombination technology.

5. The application according to claim 4, characterized in that: The integration of exogenous nucleic acid molecules into the genome of animal embryonic stem cells using the CRISPR / Cas system and homologous recombination technology involves integrating the coding genes for human β-casein and human lactoferrin into the exon region of the β-casein coding gene in the genome of animal embryonic stem cells using the CRISPR / Cas system and homologous recombination technology.

6. The application according to claim 5, characterized in that: The CRISPR / Cas system includes a recombinant expression vector that expresses gRNA encoding a gene targeting animal β-casein and a Cas protein; The nucleotide sequence of the exogenous nucleic acid molecule is shown in SEQ ID No. 1, positions 5741-11575 from the 5' end.

7. The application according to claim 6, characterized in that: The target sequence of the gRNA is shown in SEQ ID No.

5.

8. The application according to any one of claims 1 to 7, characterized in that: The animal in question is a mammal; Preferably, the mammal is a cow.

9. The application according to any one of claims 4 to 8, characterized in that: The steps for integrating the coding genes for human β-casein and human lactoferrin into the exon region of the β-casein coding gene of bovine embryonic stem cells using the CRISPR / Cas system and homologous recombination technology are as follows: (1) Using the CRISPR / Cas system, an exogenous nucleic acid molecule with the nucleotide sequence shown in SEQ ID No. 1 from position 5741 to 11575 from the 5' end is integrated into the exon region of the gene encoding β-casein in the genome of bovine embryonic stem cells; the target sequence of the gRNA expressed by the CRISPR / Cas system is shown in SEQ ID No. 5; (2) After completing step (1), bovine embryonic stem cells expressing GFP protein or containing the coding gene of GFP protein are obtained by GFP-positive cell sorting and / or molecular detection. (3) After completing step (2), GFP is removed by the Cre-loxP system, and bovine embryonic stem cells that do not express GFP protein or do not contain the coding gene of GFP protein are obtained by GFP-negative cell sorting and / or molecular detection. Bovine embryonic stem cells that do not express GFP protein or do not contain the gene encoding GFP protein are animal embryonic stem cells that have undergone more than one round of gene editing.

10. The application according to claim 1, characterized in that: The bovine embryonic stem cells were isolated from the epiblast of high-producing dairy cow embryos and possess stem cell characteristics and genomic stability.

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