Novel non-integrated bovine induced pluripotent stem cell line construction method

Abstract

The invention relates to the technical field of biology, and discloses a novel non-integrated bovine induced pluripotent stem cell line construction method, which comprises the following steps: selecting a proper cell source, constructing an episomal vector system, introducing an episomal vector into bovine primary fibroblasts by using an electrotransfection method, and screening and culturing the transfected bovine primary fibroblasts in a culture medium to obtain the novel non-integrated bovine induced pluripotent stem cell line. According to the method, positive cells successfully transferred into dry factors are obtained, the cell morphology is monitored in the reprogramming process, along with the progress of reprogramming, more and more pluripotent clones appear in a cell population, amplification and identification are performed through clone culture, and the pluripotency and differentiation potential of the successfully established bovine induced pluripotent stem cells are verified. A stable bovine induced pluripotent stem cell line is established, long-term culture is carried out, and pluripotency and stability of the bovine induced pluripotent stem cell line are verified. Through an episomal non-integration method, the cattle non-integration induced pluripotent stem cell line which can be passaged for multiple times in vitro, is relatively good in hereditary stability and can maintain pluripotency and in-vitro differentiation capacity is constructed.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a novel method for constructing a non-integrated bovine induced pluripotent stem cell line. Background Art

[0002] In terms of stem cell construction, scientists transfected mouse fibroblasts with retroviruses to reprogram terminally differentiated primary cells back to stem cells with differentiation ability and pluripotency, and successfully constructed mouse induced pluripotent stem cell lines for the first time. Compared with embryonic stem cells from embryos, induced pluripotent stem cells have similar cell characteristics, such as pluripotency, stemness, and differentiation characteristics. The emergence of this technology has added diversity to different species in the field of stem cells. In recent years, there are more and more ways to reprogram. Scientists have completed the construction of induced pluripotent stem cell lines of animals such as mice, humans, pigs, and bats. There are also many ways to reprogram. At present, induced pluripotent stem cells represented by cattle are used. Induced pluripotent stem cells are obtained by reprogramming somatic cells (such as skin or blood cells). Cattle induced pluripotent stem cells are reversed to a pluripotent state by inducing mature somatic cells. In theory, they can be obtained from any healthy cattle individual, providing more source selection and flexibility, and have good genetic stability. During the reprogramming process, they usually maintain high genetic consistency and integrity, which is suitable for long-term culture and application.

[0003] The current construction of bovine induced pluripotent stem cells has a lower reprogramming efficiency than that of mice and humans, and bovine induced pluripotent stem cells are prone to phenotypic instability in long-term culture, making it difficult to maintain pluripotency. Genome instability may occur during the culture process, especially after long-term passaging, when chromosome mutations may occur in some cells. There are also problems with the long reprogramming process and low efficiency, resulting in long costs and experimental cycles. The pluripotency of bovine induced pluripotent stem cells is still weak, and there are problems in the differentiation process. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and to design a novel method for constructing a non-integrated bovine induced pluripotent stem cell line.

[0005] The present invention provides a novel non-integrated bovine induced pluripotent stem cell line construction method, characterized in that the novel non-integrated bovine induced pluripotent stem cell line construction method comprises the following steps: S1. Select appropriate cell sources: extract skin cells and culture primary fibroblasts as the starting cells for reprogramming; S2, construction of episomal vector system; S3. Cell transfection: The episomal vector was introduced into bovine primary fibroblasts using electrofection method; S4. Screening and culturing: The transfected bovine primary fibroblasts are screened and cultured in a culture medium to obtain positive cells into which the stemness factors have been successfully transferred; S5. Monitoring cell morphology during reprogramming: Induce positive cells on mouse fibroblast feeder cells treated with mitomycin, observe cell morphological changes, and identify whether the induced pluripotent stem cells express specific pluripotency marker genes by immunofluorescence staining or RT-qPCR; S6. Clonal expansion and identification: As reprogramming progresses, more and more pluripotent clones will appear in the cell population, which can be expanded and identified through clonal culture; S7. Induced differentiation and pluripotency verification: The pluripotency and differentiation potential of the successfully established bovine induced pluripotent stem cells were verified; S8. Long-term culture and stability verification: A stable bovine induced pluripotent stem cell line was established and cultured over a long period of time and its stability was verified, including regular testing of the genome for mutations or abnormalities, and continued observation of whether the induced pluripotent stem cells maintained their pluripotency characteristics.

[0006] Furthermore, in the above-mentioned novel non-integrated bovine induced pluripotent stem cell line construction method, it is characterized in that in step S2, an episomal vector containing key transcription factors is constructed, and the transcription factors include Oct4, Sox2, Klf4 and c-Myc.

[0007] Furthermore, in the above-mentioned novel non-integrated bovine induced pluripotent stem cell line construction method, it is characterized in that step S3 specifically includes the following process: the constructed episomal vector DNA and bovine primary fibroblasts in the logarithmic growth phase are placed together in an electroporation cup, and the cell membrane of the bovine primary fibroblasts is temporarily porous by electric shock, so that the episomal vector DNA enters the bovine primary fibroblasts, and after electroporation, the bovine primary fibroblasts continue to be cultured.

[0008] Furthermore, in the above-mentioned novel non-integrated bovine induced pluripotent stem cell line construction method, it is characterized in that step S4 specifically includes the following process: screening and culturing the bovine primary fibroblasts transfected in step S3, and selecting the successfully reprogrammed cell population based on whether cell clones appear, wherein the culture medium contains LIF, CHIR99021, MinocyclineHCl, Dimethindenemale growth factor, and N2B27basal culture medium with epigenetic modifier Vc2p added to maintain the undifferentiated state of the cells, and regularly replacing the culture medium to keep the cell growth active.

[0009] Furthermore, in the above-mentioned novel non-integrated bovine induced pluripotent stem cell line construction method, it is characterized in that step S5 specifically includes the following process: when the immunofluorescence staining method is selected, the cells screened in step S4 are fixed, contacted with an antibody solution against a protein related to a pluripotency marker gene, the antibody is bound to the target protein in the cell, a fluorescently labeled secondary antibody is used to bind to the primary antibody, and the fluorescent signal of the cell is observed under a fluorescence microscope; When the RT-qPCR method is used, the total RNA from the cells screened in step S4 is extracted, and after the genome is removed, a reverse transcription reaction is performed to convert the RNA into cDNA, and a qPCR reaction is performed using primers for pluripotency marker genes, and the relative expression of the gene is calculated based on the Ct value of the qPCR reaction.

[0010] Furthermore, in the above-mentioned novel non-integrated bovine induced pluripotent stem cell line construction method, it is characterized in that in step S5, the cells are observed to be closely arranged, with small cell volume and round shape.

[0011] Furthermore, in the above-mentioned novel non-integrated bovine induced pluripotent stem cell line construction method, it is characterized in that step S6 specifically includes the following processes: collecting cloned bovine induced pluripotent stem cell line samples, obtaining nucleic acid samples for detection by cell lysis or extraction of genomic DNA, configuring a PCR reaction system, setting PCR reaction conditions, placing the reaction system in a PCR instrument for cyclic amplification, taking out the PCR product after the reaction is completed, and using gel electrophoresis analysis to identify whether the bovine induced pluripotent stem cell cell line has exogenous genes transferred into it.

[0012] Furthermore, in the above-mentioned novel non-integrated bovine induced pluripotent stem cell line construction method, it is characterized in that step S7 specifically includes the following processes: selecting mice in good health, collecting fertilized eggs of mice, culturing embryos in culture medium, injecting bovine induced pluripotent stem cells into mouse embryos using microinjection technology, transplanting the embryos injected with bovine induced pluripotent stem cells into the uterus of a surrogate mother mouse, regularly observing the pregnancy status of the surrogate mother mouse and the development process of the embryo, and collecting embryonic tissues for analysis at different stages of embryonic development.

[0013] The beneficial effect lies in that the present invention selects a suitable cell source, constructs an episomal vector system, uses an electrofection method to introduce the episomal vector into bovine primary fibroblasts, screens and cultures the transfected bovine primary fibroblasts in a culture medium, obtains positive cells into which stemness factors are successfully transferred, monitors cell morphology during the reprogramming process, and as the reprogramming progresses, more and more pluripotent clones will appear in the cell population, which are amplified and identified through clonal culture, and the pluripotency and differentiation potential of the successfully established bovine induced pluripotent stem cells are verified, and finally a stable bovine induced pluripotent stem cell line is established, which is cultured for a long time and its stability is verified, including regular detection of whether the genome has mutations or abnormalities, and continued observation of whether the induced pluripotent stem cells maintain pluripotency characteristics. The bovine induced pluripotent stem cell line constructed in the present invention is a non-integrated cell line constructed by an episomal vector, which reduces the risk of genomic instability and is safer; the effect on gene editing and improvement is more prominent; the bovine induced pluripotent stem cell line has a faster proliferation rate and can maintain a good cell morphology in vitro; through the episomal non-integration method, a bovine non-integrated induced pluripotent stem cell line is successfully constructed, which can be passaged multiple times in vitro, has good genetic stability, and can maintain pluripotency and in vitro differentiation ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the following detailed description of the preferred embodiment.The drawings are only for the purpose of illustrating the preferred embodiments and are not to be construed as limiting the invention.

[0015] Figure 1 This is a flow chart of a method for constructing a novel non-integrated bovine induced pluripotent stem cell line provided in an embodiment of the present invention.

[0016] Figure 2 This is a diagram of the expression of pluripotency factors of bovine induced pluripotent stem cells identified by immunofluorescence staining in the novel non-integrated bovine induced pluripotent stem cell line construction method provided in an embodiment of the present invention.

[0017] Figure 3 This is a diagram of the expression of pluripotency factors of bovine induced pluripotent stem cells identified by RT-qPCR in the novel non-integrated bovine induced pluripotent stem cell line construction method provided in an embodiment of the present invention.

[0018] Figure 4 A diagram for detecting whether exogenous genes have been introduced into the genome of the novel non-integrated bovine induced pluripotent stem cell line construction method provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0019] The embodiments of the present invention are described in detail below. Unless otherwise specified, all scientific and technological terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. All patents and publications to which the present invention relates are incorporated herein by reference in their entirety. The term "comprising" or "including" is an open expression, i.e., including the contents specified in the present invention, but does not exclude the contents of other aspects.

[0020] For ease of understanding, the specific process of the embodiment of the present invention is described below. Figure 1 As shown, a novel method for constructing a non-integrated bovine induced pluripotent stem cell line comprises the following steps: S1. Select appropriate cell sources: extract skin cells and culture primary fibroblasts as the starting cells for reprogramming; S2, construction of episomal vector system; S3. Cell transfection: The episomal vector was introduced into bovine primary fibroblasts using electrofection method; S4. Screening and culturing: The transfected bovine primary fibroblasts are screened and cultured in a culture medium to obtain positive cells into which the stemness factors have been successfully transferred; S5. Monitoring cell morphology during reprogramming: Inducing positive cells by inoculating them on mouse fibroblast feeder cells treated with mitomycin, observing cell morphological changes, and identifying whether the induced pluripotent stem cells express specific pluripotency marker genes by immunofluorescence staining and RT-qPCR; S6. Clonal expansion and identification: As reprogramming progresses, more and more pluripotent clones will appear in the cell population, which will be expanded and identified through clonal culture; S7. Induced differentiation and pluripotency verification: The pluripotency and differentiation potential of the successfully established bovine induced pluripotent stem cells were verified; S8. Long-term culture and stability verification: A stable bovine induced pluripotent stem cell line was established and cultured over a long period of time and its stability was verified, including regular testing of the genome for mutations or abnormalities, and continued observation of whether the induced pluripotent stem cells maintained their pluripotency characteristics.

[0021] In this embodiment, in step S2, an episomal vector containing key transcription factors is constructed, and the transcription factors include Oct4, Sox2, Klf4 and c-Myc.

[0022] Specifically, episomal vectors are special DNA vectors that can remain non-integrated in host cells and can be passed to daughter cells during cell division. This type of vector has important application value in gene therapy, protein production, and the generation of induced pluripotent stem cells (iPSCs).

[0023] Non-integrative: Episomal vectors do not insert into the host cell’s genome, thus reducing the risk of insertional mutagenesis, which is critical for long-term safety.

[0024] Replication-competent: They contain one or more origins of replication derived from a virus or plasmid (e.g. OriP from EBV), which enables the vector to replicate itself within the cell and maintain sufficient copy numbers to ensure expression levels.

[0025] Stability: Some episomal vectors contain elements from sources such as EBV (Epstein-Barr Virus) that help stabilize free-standing DNA molecules in the cell nucleus.

[0026] In this protocol, the episomal method was used to successfully establish a non-integrated bovine induced pluripotent stem cell (iPS) cell line. The definition of non-integrated iPS cells is that reprogramming factors (such as Oct4, Sox2, Klf4, c-Myc, etc.) are not integrated into the genome in the cell. The specific advantages of this type of iPS cells are as follows: 1. Advantages of fast growth rate 1) Improved yield and efficiency: Rapidly proliferating induced pluripotent stem cells can reach the required cell number more quickly, thereby greatly improving the efficiency of experiments, especially when a large number of cells are required for subsequent research (such as gene editing, drug screening or stem cell therapy, etc.).

[0027] 2) Accelerate the research process: Rapidly proliferating induced pluripotent stem cells can shorten the time from reprogramming to application, promoting the rapid development of basic research and clinical applications.

[0028] 3) Reduce costs: The rapid proliferation of cells means that less culture time and resources are required during the experiment, thereby reducing the cost of the experiment.

[0029] 4) Increased application flexibility: Rapidly proliferating cells can be more easily used in different applications, such as large-scale cell production, gene repair, tissue engineering, etc.

[0030] Genome safety and stability 1) Avoid genomic mutations: Since the reprogramming factors will not be integrated into the genome, the risk of insertion mutations and genomic instability is reduced. Integrative reprogramming methods may cause genomic instability and may even activate oncogenes or cause chromosomal rearrangements.

[0031] 2) Reduce the risk of tumor formation: Non-integrated induced pluripotent stem cells do not involve genomic integration of exogenous DNA, which reduces the risk of tumor occurrence, especially the potential carcinogenicity of transcription factors such as c-Myc and Klf4 during the integrative reprogramming process.

[0032] Less immune rejection 1) Reduce immune system response: Non-integrated induced pluripotent stem cells do not carry foreign genes (such as viral vectors, etc.), so they may cause lower immune responses when transplanted into animals or humans. This is very important for transplantation and regenerative medicine in clinical applications.

[0033] 2) Improved safety: During clinical treatment or cell transplantation, non-integrated cell lines are safer than integrated cell lines, reducing the risk of immune rejection.

[0034] Simplify cell line preparation 1) No permanent changes in genetic material: Non-integrative reprogramming avoids the need to integrate exogenous genes into the cell genome. Therefore, no additional gene elimination or screening steps are required in the later stages after cell reprogramming, thereby simplifying the cell line preparation process.

[0035] 2) Easier to culture for a long time: Since there is no need to eliminate integration factors, non-integrated induced pluripotent stem cells can maintain pluripotency for a longer period of time, and their proliferation rate and plasticity remain stable.

[0036] Genetically modified organisms have no side effects Eliminate transgenic side effects: Integrated induced pluripotent stem cells usually leave genetic traces of exogenous genes, while non-integrated induced pluripotent stem cells avoid this problem. In the study of transgenic applications, non-integrated induced pluripotent stem cells can be safer in subsequent applications, and there is no long-term effect of exogenous genes on recipient cells.

[0037] Controllability Exogenous genes can be removed at any time: Non-integrated induced pluripotent stem cells allow the removal of exogenous transcription factors when needed, avoiding side effects that may be caused by long-term expression of exogenous genes. By reducing the expression of exogenous factors or eliminating plasmids, induced pluripotent stem cells can restore a more "natural" phenotype, close to the state of pluripotent stem cells in the body.

[0038] Potential for clinical translation Feasibility of clinical application: Non-integrated induced pluripotent stem cells have a higher potential for clinical translation because they are closer to the natural state in the body and do not contain potential exogenous genetic material. This makes them safer in clinical treatment, especially in the field of cell therapy and regenerative medicine.

[0039] In this embodiment, step S3 specifically includes the following process: the constructed episomal vector DNA and bovine primary fibroblasts in the logarithmic growth phase are placed together in an electroporation cup, and the cell membrane of the bovine primary fibroblasts is temporarily pore-formed by electric shock, so that the episomal vector DNA enters the bovine primary fibroblasts. After electroporation, the bovine primary fibroblasts continue to be cultured.

[0040] In this embodiment, step S4 specifically includes the following process: screening and culturing the bovine primary fibroblasts transfected in step S3, and selecting the cell population that has been successfully reprogrammed based on whether cell clones appear, wherein the culture medium contains LIF, CHIR99021, MinocyclineHCl, Dimethindenemale growth factor, and N2B27basal culture medium with epigenetic modifier Vc2p is added to maintain the undifferentiated state of the cells, and the culture medium is replaced regularly to keep the cell growth active.

[0041] like Figure 2 , Figure 3 As shown, in this embodiment, step S5 specifically includes the following process: when the immunofluorescence staining method is selected, the cells screened in step S4 are fixed on a glass slide, the cells on the glass slide are contacted with an antibody solution against a protein related to a pluripotency marker gene, the antibody is bound to the target protein in the cell, a fluorescently labeled secondary antibody is used to bind to the primary antibody, and the fluorescence signal of the cell is observed under a fluorescence microscope; When the RT-qPCR method is used, the total RNA from the cells screened in step S4 is extracted, and after the genome is removed, a reverse transcription reaction is performed to convert the RNA into cDNA, and a qPCR reaction is performed using primers for pluripotency marker genes, and the relative expression of the gene is calculated based on the Ct value of the qPCR reaction.

[0042] Specifically, RT-qPCR (Real-Time Quantitative Polymerase Chain Reaction) is a method for quantitative detection of specific RNA sequences. It combines the two technologies of reverse transcription (RT) and quantitative PCR (qPCR), allowing accurate measurement of gene expression at the transcriptional level. The operation steps are: extract total RNA; perform quality control (such as checking purity by gel electrophoresis or spectrophotometer); remove genome; reverse transcribe and synthesize cDNA; set up qPCR reaction system, including cDNA template, specific primers, fluorescent dye or probe; run the program on a real-time fluorescence quantitative PCR instrument; analyze data and interpret results.

[0043] In this embodiment, in step S5, it is observed that the cells are closely arranged, small in size and round in shape.

[0044] like Figure 4 As shown, in this embodiment, step S6 specifically includes the following processes: collecting cloned bovine induced pluripotent stem cell cell line samples, obtaining nucleic acid samples for detection by cell lysis or extraction of genomic DNA, configuring a PCR reaction system, setting PCR reaction conditions, placing the reaction system in a PCR instrument for cyclic amplification, taking out the PCR product after the reaction is completed, and using gel electrophoresis analysis to identify whether the bovine induced pluripotent stem cell cell line has exogenous genes transferred into it.

[0045] In this embodiment, step S7 specifically includes the following processes: selecting mice in good health, collecting fertilized eggs of the mice, culturing the embryos in a culture medium, injecting bovine induced pluripotent stem cells into the embryos of the mice using microinjection technology, implanting the embryos injected with bovine induced pluripotent stem cells into the uterus of a surrogate mother mouse, regularly observing the pregnancy status of the surrogate mother mouse and the development process of the embryo, and collecting embryonic tissues for analysis at different stages of embryonic development.

[0046] Specifically, the induced pluripotent stem cell line in the present invention is a non-integrated cell line constructed by episomal vectors. Most of the currently established bovine induced pluripotent stem cell lines, especially the first few generations of bovine induced pluripotent stem cell lines, rely on the introduction of exogenous genes, which are usually introduced into cells through viral vectors or other transfection techniques (such as mRNA or small molecule compounds) and integrated into the genome. The role of these exogenous genes is to start the reprogramming process of bovine somatic cells, returning them to a pluripotent state, thereby obtaining the characteristics of induced pluripotent stem cells. The introduction of exogenous factors means that these cell lines may carry transgenic sequences in their genomes. Although most exogenous genes are helpful for maintaining the pluripotency of cells, they may also bring about problems of genetic stability, especially when transgenic insertion occurs in important genes or regulatory regions. During long-term culture, the proliferation ability and differentiation potential of cells may be affected, and even genomic instability may be caused. Although bovine induced pluripotent stem cells maintain high pluripotency in many cases, how to ensure the genetic stability of the cell line and the stability of multi-generation transmission is still a difficulty in research. Therefore, constructing a bovine non-integrated induced pluripotent stem cell line not only reduces the risk of genomic instability and is safer, but also plays a very important role in agricultural biological breeding, such as: (1) Gene editing and improvement: Bovine induced pluripotent stem cells can be used for gene editing to help develop more productive, disease-resistant or adaptable cattle breeds. For example, specific genes in induced pluripotent stem cells can be modified through technologies such as CRISPR, and then the desired cattle population can be generated through in vitro differentiation. This provides a new means for precision breeding and improvement. (2) Disease models and drug screening: Bovine induced pluripotent stem cells can be used to establish different disease models to help study and screen anti-disease drugs or vaccines. For example, induced pluripotent stem cell technology can be used to simulate genetic diseases or infectious diseases (such as foot-and-mouth disease) in cattle, providing support for vaccine development and disease prevention. (3) Cloning technology: Induced pluripotent stem cells can serve as the basis for cloning technology. In agricultural production, induced pluripotent stem cells can provide unlimited cell resources for target varieties, thereby improving the reproductive efficiency of excellent varieties. Bovine induced pluripotent stem cells provide a more flexible tool, allowing cattle with high yields or special traits to be more widely replicated through somatic cell cloning technology. (4) Regenerative medicine and biotherapy: Bovine induced pluripotent stem cells can also be used in animal regenerative medicine research. For example, induced pluripotent stem cells can be used to differentiate into specific cell types to treat injured or sick cattle and improve health management. (5) Bioreactors: Bovine induced pluripotent stem cells can be used to produce valuable biological products, such as vaccines, antibodies and other biopharmaceutical products. By culturing and differentiating into specific cell types (such as bovine mammary epithelial cells) in vitro, biological drugs can be produced on a large scale.

[0047] The induced pluripotent stem cell line in the present invention is a new type of stem cell culture condition. The bovine induced pluripotent stem cell line under this culture condition has a faster proliferation rate, can be passaged for more than 100 generations, and can maintain a good cell morphology in vitro. At present, there are various culture conditions for bovine induced pluripotent stem cell lines. We optimize the concentration of small molecule addition based on the original conditions, and add epigenetic small molecule compounds at the same time to promote the proliferation rate of bovine iPSC cell lines, maintain clone morphology, etc.

[0048] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A novel method for constructing a non-integrated bovine induced pluripotent stem cell line, characterized in that: The novel non-integrated bovine induced pluripotent stem cell line construction method comprises the following steps: S1. Select appropriate cell sources: extract skin cells and culture primary fibroblasts as the starting cells for reprogramming; S2, construction of episomal vector system; S3. Cell transfection: The episomal vector was introduced into bovine primary fibroblasts using electrofection method; S4. Screening and culturing: The transfected bovine primary fibroblasts are screened and cultured in a culture medium to obtain positive cells into which the stemness factors have been successfully transferred; S5. Monitoring cell morphology during reprogramming: Induce positive cells on mouse fibroblast feeder cells treated with mitomycin, observe cell morphological changes, and identify whether the induced pluripotent stem cells express specific pluripotency marker genes by immunofluorescence staining or RT-qPCR; S6. Clonal expansion and identification: As reprogramming progresses, more and more pluripotent clones will appear in the cell population, which can be expanded and identified through clonal culture; S7. Induced differentiation and pluripotency verification: The pluripotency and differentiation potential of the successfully established bovine induced pluripotent stem cells were verified; S8. Long-term culture and stability verification: A stable bovine induced pluripotent stem cell line was established and cultured over a long period of time and its stability was verified, including regular testing of the genome for mutations or abnormalities, and continued observation of whether the induced pluripotent stem cells maintained their pluripotency characteristics.

2. A method for constructing a novel non-integrated bovine induced pluripotent stem cell line according to claim 1, characterized in that: In step S2, an episomal vector containing key transcription factors is constructed, and the transcription factors include Oct4, Sox2, Klf4 and c-Myc.

3. The method for constructing a novel non-integrated bovine induced pluripotent stem cell line according to claim 1, characterized in that: Step S3 specifically The method includes the following steps: placing the constructed episomal vector DNA and bovine primary fibroblasts in the logarithmic growth phase into an electroporation cup, temporarily pore-forming the cell membrane of the bovine primary fibroblasts by electric shock, thereby allowing the episomal vector DNA to enter the bovine primary fibroblasts; after electric transformation, the bovine primary fibroblasts continue to be cultured.

4. The method for constructing a novel non-integrated bovine induced pluripotent stem cell line according to claim 1, characterized in that: Step S4 specifically includes the following process: The bovine primary fibroblasts transfected in step S3 were screened and cultured, and the cell population successfully reprogrammed was selected based on whether cell clones appeared. The culture medium contained LIF, CHIR99021, Minocycline HCl, Dimethindenemale growth factor, and N2B27 basal culture medium with epigenetic modifier Vc2p added to maintain the undifferentiated state of the cells. The culture medium was replaced regularly to keep the cells growing actively.

5. The method for constructing a novel non-integrated bovine induced pluripotent stem cell line according to claim 1, characterized in that: Step S5 specifically The process includes the following steps: when the immunofluorescence staining method is used, the cells screened in step S4 are fixed, contacted with an antibody solution targeting a protein related to a pluripotency marker gene, the antibody is bound to a target protein in the cell, a fluorescently labeled secondary antibody is used to bind to the primary antibody, and the fluorescent signal of the cell is observed under a fluorescent microscope; When the RT-qPCR method is used, the total RNA from the cells screened in step S4 is extracted, and after the genome is removed, a reverse transcription reaction is performed to convert the RNA into cDNA, and a qPCR reaction is performed using primers for pluripotency marker genes, and the relative expression of the gene is calculated based on the Ct value of the PCR reaction.

6. The method for constructing a novel non-integrated bovine induced pluripotent stem cell line according to claim 1, characterized in that: In step S5, it was observed that the cells were closely packed, small in size and round in shape.

7. The method for constructing a novel non-integrated bovine induced pluripotent stem cell line according to claim 1, characterized in that: Specifically in step S6 The process includes: collecting cloned bovine induced pluripotent stem cell cell line samples, obtaining nucleic acid samples for detection by cell lysis or extraction of genomic DNA, configuring a PCR reaction system, setting PCR reaction conditions, placing the reaction system in a PCR instrument for cyclic amplification, taking out the PCR product after the reaction is completed, and using gel electrophoresis analysis to identify whether exogenous genes have been transferred into the bovine induced pluripotent stem cell cell line.

8. The method for constructing a novel non-integrated bovine induced pluripotent stem cell line according to claim 1, characterized in that: Step S7 specifically includes the following processes: selecting mice in good health, collecting fertilized eggs of the mice, culturing the embryos in a culture medium, injecting bovine induced pluripotent stem cells into mouse embryos using microinjection technology, transplanting the embryos injected with bovine induced pluripotent stem cells into the uterus of a surrogate mother mouse, regularly observing the pregnancy status of the surrogate mother mouse and the development process of the embryo, and collecting embryonic tissues for analysis at different stages of embryonic development.

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