CN102229909A - Method for inducing bovine induced pluripotent stem cells - Google Patents

Abstract

The invention discloses a method for inducing bovine induced pluripotent stem cells, which comprises: constructing a fusion protein lentivirus expression vector for inducing bovine induced pluripotent stem cells by using an exogenous defined factor and enhanced green fluorescent protein (EGPF) reporter gene; cultivating and passing bovine fetus fibroblast cells in which the fusion protein of the exogenous defined factor is expressed; gradually separating and cultivating cell colonies with clearly defined colony borders; and obtaining the bovine induced pluripotent stem cells, if the growth states of the cell colonies are stable, the karyotype is normal, the cell colonies are negative in alkaline phosphatase test, the expression of proteins Oct4, Nanog and SSEA-1 is positive in immunocyte chemical tests, teratoma having three embryonic layers can be differentiated in vivo, and the result proves the separated cell conolnies have the characteristics of embryonic stem cells.

Description

A method for inducing bovine induced pluripotent stem cells

technical field

The invention relates to the technical field of cell mutagenesis, in particular to a method for inducing bovine induced pluripotent stem cells.

Background technique

Since 2006 (Takahashi K and Yamanaka S, 2006) it was first reported that mouse skin fibroblasts were reprogrammed into induced pluripotency by exogenously introducing four defined factors (Oct4, Sox2, c-Myc, Klf4) After induced pluripotent stem cells (iPS), so far, induced pluripotent stem cells have been successfully induced from many species and types of somatic cells including human, monkey, rat and pig (Yu J et al ., 2007; Liu H et al., 2008; Liao J et al., 2009; Esteban MA et al., 2009). Also, as research has progressed, approaches have been attempted to address issues of low efficiency and safety in the initial induction of mouse and human iPS cells. And recently, two research groups in my country reported that the mouse induced pluripotent stem cells they induced had obtained living mice completely prepared from induced pluripotent stem cells through tetraploid embryo complementation technology, which strongly proved that induced pluripotent stem cells Stem cells are truly totipotent (Zhao XY et al., 2009; Kang L et al., 2009). Therefore, direct reprogramming of somatic cells into induced pluripotent stem cells using defined factors is a major breakthrough in the field of stem cell biology and has broad application prospects in the fields of genetic engineering and regenerative medicine.

Unlike embryos from animal sources such as mice, rats, and monkeys, embryonic stem cell lines isolated from artiodactyl large animal embryos are unstable in culture and quickly lose their properties. Although scientists from various countries have tried for many years, there is no report on the successful isolation of bovine pluripotent embryonic stem cell lines. Therefore, if bovine induced pluripotent stem cells with totipotency are successfully induced by defined factors, their stem cell characteristics can be effectively used for related scientific research, and their similar characteristics to embryonic stem cells can be used for the production of transgenic livestock animals application. However, there is no literature report on the induction technology of bovine induced pluripotent stem cells at home and abroad.

Contents of the invention

The invention provides a method for inducing bovine induced pluripotent stem cells, using exogenous defined factors and enhanced green fluorescent protein (Enhanced green fluorescent protein, EGFP) to construct a fusion protein lentiviral expression vector for bovine induced pluripotent stem cells Induction, the bovine fetal fibroblasts expressing the fusion protein of the exogenous defined factor were cultured and passaged, and the cell clones with clear borders of the colony were gradually separated and cultured. The cell colony was in a stable growth state, the karyotype was normal, and the alkaline phosphatase test was positive. Immunocytochemical detection showed that the protein expression of Oct4, Nanog, and SSEA-1 were positive, and they could differentiate into teratomas containing three germ layers in vivo. The results confirmed that the isolated and cultured cell clones had embryonic stem cell-like characteristics, and successfully isolated and cultured to obtain bovine inducible pluripotent stem cells.

Technical scheme of the present invention is:

A method for inducing bovine induced pluripotent stem cells, comprising the following steps:

(1), construction of defined factor lentiviral expression vector:

According to the mRNA sequence of pig Sox2, c-Myc, Klf4 gene and the DNA sequence of human Oct4, the upstream and downstream primers of pSox2, pKlf4, pc-Myc, hOct4 gene were designed and synthesized;

Total RNA was extracted from porcine blastocysts, and the pig-specific gene DNA was amplified by RT-PCR using the designed and synthesized upstream and downstream primers of pSox2, pKlf4 and pc-Myc genes. The downstream primers are directly amplified from the human Oct4 gene, and the DNA sequence of the porcine-defined gene and human Oct4 is digested, and then connected to the retroviral vector pLEGFP-N1 to construct a retroviral fusion protein expression vector, from which the retroviral The CMV promoter together with the defined gene and EGFP gene were amplified from the recording virus fusion protein expression vector, and the U6 promoter, CMV promoter and GFP sequence were removed from the pLL3. Gene and EGFP gene were recombined with the digested pLL3.7 to construct a lentiviral-limited gene fusion protein recombinant expression vector;

The upstream and downstream primer sequences of the pSox2, pKlf4, pc-Myc, hOct4 genes are:

Upstream of pSox2: 5'-TTTAAGCTTGCCACCATGTACAACATGATGGAGAC-3';

Downstream: 5'-AAAGGATCCGGCATGTGAGAGAGAGGCAGTGTAC-3';

Upstream of pKlf4: 5'-TATAAGCTTGCCACCATGGCTGTCAGCGACGCAC-3';

Downstream: 5'-GGCGGATCCGGAAAATGCCTCTTCATGTGTAAGGC-3';

Upstream of pc-Myc: 5'-GCCAAGCTTGCCACCCTGGATTTCCTTCGGATAG-3';

Downstream: 5'-AAAGGATCCGCTGGGCAAGAGTTCCGTAGCTG-3';

Upstream of hOct4: 5'-AATGTCGACGCCACCATGGCGGGACACCTGGCTTC-3';

Downstream: 5'-CGCGGATCCGCTCAGTTTGAATGCATGGGAGAGC-3'.

(2), induction of bovine fetal fibroblasts:

The bovine fetal fibroblast cell line was established by the tissue block culture method, and the four-factor plasmid PLL-hOCT4/pSOX2/pMYC/pKLF4, which was the recombinant expression vector of the lentivirus-defined gene fusion protein, was transfected by the calcium phosphate method, and the lentivirus was packaged to produce virus at the same time The required auxiliary plasmids pMDLg, pRSV REV and pVSVg were packaged into 293T cells, and the fresh culture medium of 293T cells was replaced after 12-16 hours. In the plate, after 18-20 days of infection, the initial morphological changes of the cells appeared, and the infected cells were digested by Dispase II, inoculated on the feeder layer of mouse fetal fibroblasts treated with mitomycin C, and cultured for 23-25 days after infection. Days later, bovine embryonic stem cell-like clones were clearly visible.

The specific operation steps for constructing the recombinant expression vector of the lentiviral-limited gene fusion protein include: firstly digest pLL3.7 with Apa I, blunt its end with T4 DNA polymerase, and then single-digest with EcoR I , that is, remove the U6 promoter, CMV promoter and GFP sequence from the pLL3.7 plasmid; design vector modification primers, and amplify the CMV promoter together with the defined Gene and EGFP sequence, and then single-digested with Mun I, after purification, the CMV promoter amplified from the retroviral fusion protein expression vector together with the restricted gene and EGFP gene were digested with pLL3. 7 connection for recombination to construct a recombinant expression vector for a lentiviral-defined gene fusion protein;

The DNA sequence of the designed carrier transformation primer is:

Upstream: 5'-AAAGGGCCCGCGGGACTCTGGGGTTCGTAATA-3';

Downstream: 5'-GCGCAATTGTTACTTGTACAGCTCGTCC-3'.

The 293T cells were confluent one day before the virus packaging, and the 293T cell culture medium was: high-sugar DMEM containing 8-12% fetal bovine serum, 95-105 U/ml penicillin, and 0.08-0.12 mg/ml streptomycin, and the cells were confluent 50-60% of the virus packaging is carried out; the calcium phosphate method is used to transfect the four-factor plasmid PLL-hOCT4/pSOX2/pMYC/pKLF4 of the recombinant expression vector of the lentivirus-defined gene fusion protein, and the lentivirus packaging is produced at the same time The helper plasmids pMDLg, pRSV REV and pVSVg required by the virus were packaged into 293T cells, and the fresh culture medium of 293T cells was replaced after 12-16 hours. After 48 hours, the virus-containing culture medium was collected and filtered through a 0.45μm filter membrane, and then transferred to Millipore’s 100KD ultrafiltration tube Centrifuge at 4000rpm for 20-40min at 3-5°C to obtain concentrated virus liquid.

The day before the virus begins to infect the bovine fetal fibroblasts, inject the bovine fetal fibroblasts into the bovine fetal fibroblast culture plate, the cell density is 1×10 ⁵ /well, and the bovine fetal fibroblast culture medium is: High-glucose DMEM containing 8-12% fetal bovine serum, 95-105 U/ml penicillin, 0.08-0.12 mg/ml streptomycin, and after 24 hours, replace it with bovine milk supplemented with concentrated virus solution and Polybrene at a concentration of 10 μg/mL. Fetal fibroblast culture medium, 12 hours after the beginning of virus infection, change to bovine fetal fibroblast culture medium without virus liquid, and replace it with bovine fetal fibroblast culture medium added with concentrated virus liquid and Polybrene at a concentration of 10 μg/mL on the second day after the virus begins to infect. The fibroblast culture solution was re-infected once, and the bovine fetal fibroblast culture medium without virus solution was changed after 12 hours. The culture medium was replaced with bovine induced pluripotent stem cell induction and maintenance medium. After 18-20 days, it was digested with Dispase II and inoculated on the feeder layer of 12.5-day mouse fetal fibroblasts treated with mitomycin C, and new bovine induced pluripotent stem cells were added. Induced pluripotent stem cell induction and maintenance culture medium continued to be cultured at 37.5°C and 5% CO ₂ saturated humidity.

The specific steps for culturing the bovine fetal fibroblast cell line include: taking 2.5-month-old Holstein cow fetal skin tissue, adopting the tissue block culture method to establish the fibroblast cell line, the culture medium is containing 8-12% fetal bovine serum, 95 -105U/ml penicillin, 0.08-0.12mg/ml streptomycin in high-sugar DMEM; the preparation method of the mouse fetal fibroblast feeder layer is to select the 2-4 generations of mouse embryos that cover the bottom of the dish. Fibroblasts were treated with 9-11ug/ml mitomycin C for 2-3 hours, routinely digested with trypsin and inoculated into a culture dish with a concentration of 0.008-0.012g/L gelatin, and the seeding cell density was 2× 10 ⁵ pieces/ml.

The bovine induced pluripotent stem cell induction and maintenance medium is: containing 1.5-2.5mM glutamine, 1.5-2.5mM sodium pyruvate, 0.8-1.2% non-essential amino acids, 0.08-0.12mM β-mercaptoethanol , 990-1100U/ml LIF, 3.5-4.5ng/ml bFGF, 13-17% FBS and 0.8-1.2% penicillin and streptomycin high sugar DMEM.

The % average value described in the present invention accounts for the volume percentage of the total volume.

The mRNA sequence information of the porcine Sox2, c-Myc, and Klf4 genes comes from the database of the National Center for Biotechnology Information of the United States.

The upstream and downstream primers of the pSox2, pKlf4 and pc-Myc genes refer to the specific DNA sequences that can amplify the corresponding genes from the DNA fragments of the species pig. The upstream and downstream primers of the hOct4 gene refer to the specific DNA sequence of the hOct4 gene amplified from the direct human Oct4 gene.

The vector modification primers refer to the specific DNA sequences that amplify the CMV promoter together with the defined gene and EGFP sequence from the constructed retroviral vector plasmid pLEGFP--hOCT4/pSOX2/pMYC/pKLF4, and the primers are generally designed by ourselves. , synthesized by a specialized biological company.

The purpose of replacing the fresh culture medium of 293T cells is to remove excess calcium phosphate precipitation and cell metabolites that have not entered the cells, and replace them with the culture medium required for normal 293T cell culture, which is conducive to the growth of 293T cells and the subsequent time. The virus-containing culture medium produced by packaging of 293T cells was collected.

The advantages of the present invention are:

(1), the present invention utilizes exogenous defined factors and EGFP to construct fusion protein lentiviral expression vectors for the induction of bovine induced pluripotent stem cells, and cultures and passages bovine fetal fibroblasts expressing exogenous defined factor fusion proteins, which can Cell clones with clear borders of colonies were gradually isolated and cultured. The growth state of the cell colonies was stable and the karyotype was normal. A teratoma containing three germ layers was formed, and the results confirmed that the isolated and cultured cell clones were pluripotent stem cells, which can be called bovine induced pluripotent stem cells; for the first time, bovine induced pluripotent stem cells were successfully induced;

(2) Compared with the usual induced pluripotent stem cell induction technology, the present invention uses a fusion protein composed of defined factors and EGFP to induce bovine induced pluripotent stem cells. At the gene level, the coding regions of two proteins or protein domains are connected end-to-end according to the reading frame, and the gene expression product is controlled by the same regulatory sequence; the fusion protein technology is due to the expression of the target protein and the construction of a bifunctional target protein It has an irreplaceable role in research, and has important uses in the production and research of medicine, agriculture, environment, etc.; EGFP is a commonly used one in the fluorescent protein family, and its fluorescence is highly stable. The mechanism and characteristics of the role in the process of cell reprogramming. The fusion protein composed of exogenous defined factors and EGFP can effectively detect, track and grasp the characteristics of exogenous defined factors, which will help to study the mechanism of bovine induced pluripotent stem cells in the future. Research;

(3) The formula of the culture medium used in the present invention to induce and maintain the stem cell characteristics of bovine induced pluripotent stem cells has a certain reference value for the future isolation and cultivation of bovine embryonic stem cell lines.

Utilizing exogenous defined factors and EGFP to form a fusion protein applied to the induction of bovine induced pluripotent stem cells can track and grasp the mechanism and characteristics of exogenous defined factors in the reprogramming process. It can be seen from the present invention that exogenous defined factors can Most of the induced pluripotent stem cell colonies are not expressing or expressing at a low level, while a few cells are in a state of high expression, which shows that after the complete reprogramming of somatic cells to form induced pluripotent stem cells, foreign genes do not express and the incomplete reprogramming Somatic exogenous genes are still highly expressed. Zhao et al. (Zhao Y et al., 2008) used EGFP as a fluorescent marker to induce human induced pluripotent stem cells and found that the silence of EGFP expression indicated the complete reprogramming of induced pluripotent stem cells, and the expression of EGFP showed the induction of pluripotent stem cells. Competent stem cells are still incompletely reprogrammed. Yu et al. (Yu J et al., 2009) found that the expression level of Oct4 in fully reconstituted human induced pluripotent stem cells was consistent with that of human ES cells through gene chip detection. Maherali et al. (Maherali N et al., 2007) confirmed that the expression of exogenous defined factors after the formation of mouse induced pluripotent stem cells was not necessary for the growth of induced pluripotent stem cells when using the induced expression of exogenous defined factors. Numerous research results have shown that the endogenous pluripotency genes activated after the formation of induced pluripotent stem cells are sufficient to maintain the growth and development of stem cells, and the exogenous limiting factors are in a silent state in induced pluripotent stem cells. Using defined factors and EGFP to form a fusion protein This invention is used for the reprogramming of bovine somatic cells, and can successfully induce bovine induced pluripotent stem cells. However, there are no literature reports on bovine induced pluripotent stem cell technology at home and abroad. Therefore, the present invention is the first at home and abroad, and it shortens the distance from induced pluripotency to animal husbandry application, which has far-reaching significance. Secondly, by using the characteristic of EGFP with stable green fluorescence, the mechanism and characteristics of exogenous defined factors in the process of cell reprogramming can be tracked and mastered, laying the foundation for further research on the use of bovine induced pluripotent stem cells in the future. Thirdly, because bovine embryonic stem cell lines have not been established at home and abroad, the formula of culture medium for inducing and maintaining bovine induced pluripotent stem cells used in the technology of the present invention can provide a reference for future research on establishing bovine embryonic stem cell lines.

Description of drawings

Figure 1 is a schematic diagram of the plasmid map of pLL3.7.

Fig. 2 is a schematic diagram of construction map of retroviral vector of defined factor fusion protein.

Fig. 3 is a schematic diagram of the construction map of the lentiviral vector plasmid pLL-hOCT4/pSOX2/pMYC/pKLF4-GFP of the defined factor fusion protein.

Fig. 4 is a schematic diagram of the induction process of bovine induced pluripotent stem cells.

Fig. 5 is a morphological diagram of bovine fetal fibroblasts before virus infection.

Fig. 6 is a morphological diagram of bovine fetal fibroblasts on day 19 under stem cell culture medium culture conditions after virus infection twice.

Figure 7 is a morphological diagram of bovine induced pluripotent stem cell clones.

Fig. 8 is a phase diagram of metaphase of bovine induced pluripotent stem cells at passage 13.

Fig. 9 is a map of pluripotency genes detected by RT-PCR, in which 1 represents bovine fetal fibroblasts, 2-3 represent bovine induced pluripotent stem cells, and 4 represents mouse ES cells.

Fig. 10 is a micrograph of AP staining of bovine induced pluripotent stem cells expressing embryonic stem cells.

Fig. 11 is a micrograph showing the expression of embryonic stem cell Oct4 protein expressed by bovine induced pluripotent stem cells.

Fig. 12 is a micrograph showing the expression of embryonic stem cell Nanog protein expressed by bovine induced pluripotent stem cells.

Fig. 13 is a micrograph showing the expression of embryonic stem cell SSEA-1 protein expressed by bovine induced pluripotent stem cells.

Fig. 14 is a micrograph of the neural-like tissue (ectoderm) of bovine induced pluripotent stem cells in nude mice.

Fig. 15 is a micrograph of muscle-like tissue (mesoderm) of bovine induced pluripotent stem cells in nude mice.

Fig. 16 is a micrograph of gland-like tissue (endoderm) of bovine induced pluripotent stem cells in nude mice.

Detailed ways

Specific example: induction of Holstein cow fetal fibroblasts into induced pluripotent stem cells method:

(1), construction of defined factor lentiviral expression vector:

According to the mRNA sequences of porcine Sox2 (NM 001123197), c-Myc (NM 001005154), Klf4 (NM001031782) genes on NCBI, the upstream and downstream primers were designed and synthesized. The primer sequences are as follows:

Upstream of pSox2: 5'-TTTAAGCTTGCCACCATGTACAACATGATGGAGAC-3'

Downstream: 5'-AAAGGATCCGGCATGTGAGAGAGAGGCAGTGTAC-3'

Upstream of pKlf4: 5'-TATAAGCTTGCCACCATGGCTGTCAGCGACGCAC-3'

Downstream: 5'-GGCGGATCCGGAAAATGCCTCTTCATGTGTAAGGC-3'

Upstream of pc-Myc: 5'-GCCAAGCTTGCCACCCTGGATTTCCTTCGGATAG-3'

Downstream: 5'-AAAGGATCCGCTGGGCAAGAGTTCCGTAGCTG-3'

Upstream of hOct4: 5'-AATGTCGACGCCACCATGGCGGGACACCTGGCTTC-3'

Downstream: 5'-CGCGGATCCGCTCAGTTTGAATGCATGGGAGAGC-3'

Vector modification primer: upstream: 5'-AAAGGGCCCGCGGGACTCTGGGGTTCGTAATA-3'

Downstream: 5'-GCGCAATTGTTACTTGTACAGCTCGTCC-3'

Extract total RNA from pig blastocysts, and amplify pig-specific gene DNA by RT-PCR. Human Oct4 DNA sequence is directly amplified from human Oct4 gene, digested with restriction enzymes, and retroviral vector pLEGFP-N1 was connected to construct a retroviral fusion protein expression vector. Amplify the CMV promoter together with the defined gene and EGFP gene from the recombinant retroviral fusion protein expression vector, and remove the U6 promoter, CMV promoter and GFP sequence from the pLL3. The amplified promoter and defined gene fusion protein sequences in the recombinant retroviral vector were recombined with the digested pLL3.7 to construct a recombinant expression vector for the lentiviral defined gene fusion protein (Figure 3). The specific operation steps are as follows: first, digest pLL3.7 with Apa I, T4 DNA polymerase to smooth its ends, and then perform single digestion with EcoR I, that is, remove the U6 promoter and CMV from the pLL3.7 plasmid Promoter and GFP sequences. Design vector modification primers to amplify the CMV promoter, target fragment and EGFP sequence from the constructed retroviral vector plasmid pLEGFP--hOCT4/pSOX2/pMYC/pKLF4, then use Mun I for single enzyme digestion, after purification, use T4 The ligase connects the two to construct the recombinant expression vector of the lentiviral-defined gene fusion protein.

(2), induction of bovine fetal fibroblasts:

Materials and Reagents:

Isolation and cultivation of fetal bovine fibroblasts: a 2.5-month-old Holstein cow fetal fibroblast cell line was established by tissue block culture method, and the culture medium contained 10% fetal bovine serum (Hyclone) by volume, 100 U/ml penicillin, 0.1 High glucose DMEM (Hyclone) with mg/ml streptomycin.

Establishment of mouse embryo fibroblasts (MEFs) line: Pregnancy 12.5dICR fetal mice, after removing the head, limbs and viscera, mouse embryo fibroblasts were isolated and cultured by trypsinization. The culture medium is high-sugar DMEM (Hyclone) containing 10% newborn bovine serum (Sijiqing), 100 U/ml penicillin, and 0.1 mg/ml streptomycin.

Preparation of the feeder layer: select the 2-4 generation mouse embryonic fibroblasts that cover the bottom of the dish, treat with 10ug/ml mitomycin C (Sigma) for 2.5h, and inoculate with conventional trypsinization to cover 0.01g /L gelatin culture dish, inoculate the cell density at 2×10 ⁵ cells/ml. The culture medium is the same as the culture medium of mouse embryonic fibroblasts.

Bovine induced pluripotent stem cell induction and maintenance medium (ES medium): containing 2mM glutamine (Gibco), 2mM sodium pyruvate (Gibco), 1% non-essential amino acid (Gibco) by volume, 0.1mM β- High glucose with mercaptoethanol (Sigma), 1000 U/ml LIF (ESGRO, Chemicon International), 4 ng/ml bFGF (Sigma), 15% by volume FBS (Hyclone), 1% by volume penicillin (Gibco) DMEM (Hyclone).

The 293T cell culture medium is the same as the bovine fetal fibroblast culture medium, and all cells are cultured in a CO ₂ incubator at 37.5°C, 5% CO ₂ , and saturated humidity.

The bovine fetal fibroblast cell line was established by separating and culturing the fetal skin of 2.5-month-old Holstein dairy cows by tissue block culture method. Fetal bovine fibroblasts were infected from passage 7. The specific reprogramming scheme is shown in Figure 4. Transfect the four-factor plasmid PLL-hOCT4/pSOX2/pMYC/pKLF4 and the packaging components pMLg, pRSV REV and pVSVg into 293T cells by the calcium phosphate method, change the fresh culture medium after 12-16 hours, and collect the virus-containing culture medium after 48 hours of virus packaging , the virus-containing culture solution was filtered through a 0.45 μm filter membrane and transferred to a Millipore 100KD ultrafiltration tube, and centrifuged at 4000 rpm for 20-40 minutes at 4°C to obtain a concentrated virus solution. The day before the virus begins to infect the bovine fetal fibroblasts, inject the bovine fetal fibroblasts into a 6-well plate, the cell density is 1×10 ⁵ /well, and the culture medium contains 10% fetal bovine serum, 100U/ml penicillin, 0.1 mg/ml streptomycin high-sugar DMEM, and the next day, replace it with bovine fetal fibroblast culture medium supplemented with concentrated virus liquid and Polybrene at a concentration of 10 μg/mL. After 12 hours, replace with fresh bovine fetal fibroblast culture medium. On the third day, replace with bovine fetal fibroblast culture medium with concentrated virus liquid and Polybrene at a concentration of 10 μg/mL for another infection, and then change to fresh bovine fetal fibroblast culture medium 12 hours later, and the virus begins to infect porcine fetal fibroblasts The day of the cells was recorded as day 0. After two lentivirus infections, the bovine induced pluripotent stem cell induction and maintenance medium was replaced on the 3rd day. After about 19 days, the morphology of fibroblasts began to change, that is, a small number of fetal bovine fibroblasts Changed from a long spindle to a spherical shape, and grew like a colony, digested with Dispase II (Roche) and inoculated on the feeder layer of 12.5d mouse fetal fibroblasts, added bovine induced pluripotent stem cell induction and maintenance medium at 37.5 Cultivation was continued at 5% CO ₂ saturated humidity, and round embryonic stem cell-like colonies with clear borders were clearly visible on day 23 after infection. The clones digested with Dispase II were inoculated on a new feeder layer to continue the expansion culture, and the ratio of 1:1 was firstly passaged, and then the ratio of 1:4 was passaged every 4 days or so.

(3) Identification of bovine induced cells:

For RT-PCR detection, Trizol reagent (Invitrogen) was used to treat bovine induced pluripotent stem cells, then the total RNA was extracted, and cDNA was synthesized according to the instructions of cDNA first-strand synthesis kit (Takara Company). The corresponding genes were amplified with the following identification primers, and the primer sequences were as follows:

Upstream of bovine Oct4: 5'-TTGAAGCTTGCCACCATGGCGGGACACCTCGCTT-3'

Downstream: 5'-GCAGGATCCTCAGTTTGCATGCATAGGGGAGCCC-3'

Bovine Nanog upstream: 5'-ATTAAGCTTGCCACCATGAGTGTGGGCCCAGCTT-3'

Downstream: 5'-CGCGGATCCTTACAAAATCTTCAGGCTGTA-3'

Bovine Klf4 upstream: 5'-ATTAAGCTTGCCACCATGGCTGTCAGCGACGCGC-3'

Downstream: 5'-GCGGGATCCTTAAAAGTGCCTCTTCATGTGTAAGGCG-3'

Upstream of bovine c-Myc: 5'-TATAAGCTTGCCACCATGCCCCTCAACGTCAGCT-3'

Downstream: 5'-GCGGGATCCTTAGGCGCAAGAGTTCCGTA-3'

Upstream of GAPDH: 5'-TTGGTATCGTGGAAGGACTCTA-3'

Downstream: 5'-TGTCATATTTGGCAGGTT-3'

Karyotype analysis: bovine induced pluripotent stem cells were treated with 0.1 μg/ml colchicine (Sigma) for 2.5 h, digested with trypsin and resuspended in 0.075 mol/L KCL, incubated at 37°C for 25 min, fixed solution (methanol: glacial acetic acid = 3: 1) Fix for 10 min, centrifuge and fix for 3 times. Cells were harvested, sliced, stained with Gimsa, air-dried, and mounted. Chromosome pairing was observed under a microscope and related software was used to analyze chromosome pairing.

Immunofluorescence detection: Cells were fixed with 4% paraformaldehyde, blocked with 1% bovine serum albumin (BSA) at room temperature for 30 min, and primary antibodies were rabbit anti-Oct4 (Abcam), goat anti-Nanog (R&D), mouse anti-SSEA-1 ( Chemicon), mouse anti-SSEA-3 (R&D), mouse anti-SSEA-4 (R&D), mouse anti-TRA-1-60 (Chemicon), mouse anti-TRA-1-81 (Chemicon), 4 Incubate overnight at °C. Wash 3 times with PBS, dilute CY3 red fluorescently labeled secondary antibody at 1:200, incubate at room temperature in the dark for 1 h, wash 3 times with PBS, stain cell nuclei with 1 μg/mL DAPI (Roche) for 1 min, and observe the results under a fluorescent microscope.

AP staining: fix the cells with 4% paraformaldehyde, add alkaline phosphatase (Alkaline phosphatase, AP) staining solution for staining, and the cytoplasm is stained as reddish brown or brown particles as positive.

In vivo differentiation test: Dispase II digested bovine induced pluripotent stem cells, resuspended cells in serum-free DMEM and injected subcutaneously into the dorsal side of immunodeficiency mice (BALB/C-nu). After 8 weeks of injection, the nude mice were killed, and more than 4% of the tumor tissue was collected. Hematoxylin and eosin staining was performed after paraformaldehyde fixation.

(4), the result:

The defined factor lentivirus packaged by calcium phosphate method infected porcine and bovine fetal fibroblasts. After 18-19 days in the stem cell culture medium, the morphology of bovine fetal fibroblasts began to change, and a small amount of bovine fetal fibroblasts gradually changed from long shuttle The shape turns into a spherical shape and grows in clusters. Dispase II digested spherical cells were cultured on feeder cells with stem cell culture medium containing 1000U/ml LIF and 4ng/ml bFGF, and colony-like cell clones with clear borders gradually formed on the 23rd to 24th day after infection. A single cell in a colony under a high-power microscope exhibits a larger nucleus and a higher ratio of nucleoplasm to cytoplasm. In the case of 1:4 subculture, the cell clones were subcultured approximately every 3-4 days, and the cell colony growth and passage were stable (Figs. 5, 6, 7). The karyotype analysis of the bovine induced pluripotent stem cells at passage 13 showed that the pig induced pluripotent stem cells had 60 chromosomes and a normal XY karyotype (Figure 8). RT-PCR detection of expression of pluripotency genes in bovine induced pluripotent stem cells induced by defined factor fusion protein, bovine induced pluripotent stem cells express Oct4, c-Myc, Klf4 and Nanog genes, bovine fetal fibroblasts under the same conditions Related genes such as Nanog, Oct4, c-Myc, and Klf4 were not expressed (Fig. 9). AP is highly expressed in stem cells of early embryos, but its activity is significantly reduced in differentiated cells, or even not expressed. The expression of AP is one of the important characteristics of ES cells and related stem cells. AP staining results showed that the expression of AP in bovine induced pluripotent stem cells induced by the defined factor fusion protein was strongly positive ( FIG. 10 ). Cellular immunofluorescence detection showed that induced pluripotent stem cells expressed Oct4, Nanog, and SSEA-1 proteins (Fig. In cells with strong expression of Oct4 protein, cells with strong expression of EGFP are strongly positive for Oct4 protein, and cells with weak or silent expression of EGFP are generally positive for Oct4 protein expression (Figure 11). Under the same conditions, the expression level of Nanog protein Expression levels did not differ between cells expressing EGFP or not expressing EGFP in cell colonies. The teratomas formed by the porcine induced pluripotent stem cells induced by the fusion protein of defined factors were subcutaneously formed in BALB/C nude mice for 8 weeks. The results of HE staining showed that there were three germ layers in the teratoma tissue: nerve, muscle and gland. cells and tissues (Figure 14, 15, 16).

Claims (6)

1. A method for inducing bovine induced pluripotent stem cells, comprising the following steps: (1), construction of defined factor lentiviral expression vector: According to the mRNA sequence of pig Sox2, c-Myc, K1f4 gene and the DNA sequence of human Oct4, the upstream and downstream primers of pSox2, pK1f4, pc-Myc, hOct4 gene were designed and synthesized; The total RNA was extracted from pig blastocysts, and the pig-specific gene DNA was amplified by RT-PCR using the designed and synthesized upstream and downstream primers of pSox2, pK1f4 and pc-Myc genes. The downstream primers are directly amplified from the human Oct4 gene, and the DNA sequence of the porcine-defined gene and human Oct4 is digested, and then connected to the retroviral vector pLEGFP-N1 to construct a retroviral fusion protein expression vector, from which the retroviral The CMV promoter together with the defined gene and EGFP gene were amplified from the recording virus fusion protein expression vector, and the U6 promoter, CMV promoter and GFP sequence were removed from the pLL3. Gene and EGFP gene were recombined with the digested pLL3.7 to construct a lentiviral-limited gene fusion protein recombinant expression vector; The upstream and downstream primer sequences of the pSox2, pK1f4, pc-Myc, hOct4 genes are: Upstream of pSox2: 5'-TTTAAGCTTGCCACCATGTACAACATGATGGAGAC-3'; Downstream: 5'-AAAGGATCCGGCATGTGAGAGAGAGGCAGTGTAC-3'; Upstream of pK1f4: 5'-TATAAGCTTGCCACCATGGCTGTCAGCGACGCAC-3'; Downstream: 5'-GGCGGATCCGGAAAATGCCTCTTCATGTGTAAGGC-3'; Upstream of pc-Myc: 5'-GCCAAGCTTGCCACCCTGGATTTCCTTCGGATAG-3'; Downstream: 5'-AAAGGATCCGCTGGGCAAGAGTTCCGTAGCTG-3'; Upstream of hOct4: 5'-AATGTCGACGCCACCATGGCGGGACACCTGGCTTC-3'; Downstream: 5'-CGCGGATCCGCTCAGTTTGAATGCATGGGAGAGC-3'. (2), induction of bovine fetal fibroblasts: The bovine fetal fibroblast cell line was established by the tissue block culture method, and the four-factor plasmid PLL-hOCT4/pSOX2/pMYC/pKLF4, which was the recombinant expression vector of the lentivirus-defined gene fusion protein, was transfected by the calcium phosphate method, and the lentivirus was packaged to produce virus at the same time The required auxiliary plasmids pMDLg, pRSV REV and pVSVg were packaged into 293T cells, and the fresh culture medium of 293T cells was replaced after 12-16 hours. In the plate, after 18-20 days of infection, the initial morphological changes of the cells appeared, and the infected cells were digested by Dispase II, inoculated on the feeder layer of mouse fetal fibroblasts treated with mitomycin C, and cultured for 23-25 days after infection. Days later, bovine embryonic stem cell-like clones were clearly visible. 2. The method for inducing bovine induced pluripotent stem cells according to claim 1, characterized in that, the specific operation steps of the construction of the recombinant expression vector of the described lentiviral-defined gene fusion protein include: firstly using Apa I to pLL3.7 Carry out enzyme digestion, T4 DNA polymerase smoothes its end, and then perform single enzyme digestion with EcoR I, that is, remove the U6 promoter, CMV promoter and GFP sequence from the pLL3.7 plasmid; design vector transformation primers, from the constructed In the retrovirus fusion protein expression vector, the CMV promoter was amplified together with the defined gene and EGFP sequence through vector transformation primers, and then single enzyme digestion was carried out with Mun I, and after purification, T4 ligase was used to convert the retrovirus fusion protein expression vector The amplified CMV promoter together with the defined gene and EGFP gene were recombined with the digested pLL3.7 to construct the recombinant expression vector of the lentiviral defined gene fusion protein; The DNA sequence of the designed carrier transformation primer is: Upstream: 5'-AAAGGGCCCGCGGGACTCTGGGGTTCGTAATA-3'; Downstream: 5'-GCGCAATTGTTACTTGTACAGCTCGTCC-3'. 3. The method for inducing bovine induced pluripotent stem cells according to claim 1, characterized in that it comprises the following steps: the day before said virus packaging, 293T cells are confluent, and the 293T cell culture medium is: containing 8-12% fetal Bovine serum, 95-105U/ml penicillin, 0.08-0.12mg/ml streptomycin in high-sugar DMEM, when the cell confluency is 50-60%, virus packaging is carried out; the virus packaging is transfected with a limited gene of lentivirus by calcium phosphate method The four-factor plasmid PLL-hOCT4/pSOX2/pMYC/pKLF4 of the fusion protein reconstitutes the expression vector, and at the same time, the helper plasmids pMDLg, pRSV REV and pVSVg required for lentivirus packaging to produce viruses are packaged into 293T cells, and the 293T cells are replaced with fresh ones after 12-16 hours For the culture solution, the virus-containing culture solution was collected after 48 hours, filtered through a 0.45 μm filter membrane, transferred to a Millipore 100KD ultrafiltration tube, and centrifuged at 4000 rpm for 20-40 minutes at 3-5°C to obtain a concentrated virus solution. 4. The method for inducing bovine induced pluripotent stem cells according to claim 1, characterized in that it comprises the following steps: injecting bovine fetal fibroblasts into bovine fetal fibroblasts the day before said virus begins to infect bovine fetal fibroblasts In the fibroblast culture plate, the cell density is 1×10 ⁵ /well, and the bovine fetal fibroblast culture medium is: containing 8-12% fetal bovine serum, 95-105U/ml penicillin, 0.08-0.12mg/ml chain Mycin high-sugar DMEM, after 24 hours, replace with bovine fetal fibroblast culture medium supplemented with concentrated virus liquid and Polybrene with a concentration of 10 μg/mL, and replace with bovine fetal fibroblast culture without virus liquid 12 hours after the virus begins to infect On the second day after the virus started to infect, it was replaced with bovine fetal fibroblast culture medium with concentrated virus liquid and Polybrene at a concentration of 10 μg/mL for another infection, and then changed to bovine fetal fibroblast culture medium without virus liquid after 12 hours After 2 times of lentivirus infection, that is, on the third day after the virus began to infect, the culture medium in the bovine fetal fibroblast culture plate was replaced with the culture medium for induction and maintenance of bovine induced pluripotent stem cells. After 18-20 days, use Dispase II Digest and inoculate on the 12.5-day-old mouse fetal fibroblast feeder layer treated with mitomycin C, add new bovine induced pluripotent stem cell induction and maintenance medium, and continue to culture at 37.5°C, 5% CO ₂ saturated humidity . 5. The method for inducing bovine induced pluripotent stem cells according to claim 1, wherein the specific step of culturing the bovine fetal fibroblast cell line comprises: taking 2.5-month-old Holstein cow fetal skin tissue, using tissue The fibroblast cell line is established by the block culture method, and the culture medium is high-sugar DMEM containing 8-12% fetal bovine serum, 95-105U/ml penicillin, and 0.08-0.12mg/ml streptomycin; the mouse fetal fibroblast The preparation method of the cell feeder layer is to select the mouse embryonic fibroblasts of the 2-4 generations that cover the bottom of the dish, treat them with 9-11ug/ml mitomycin C for 2-3 hours, and inoculate them with routine trypsinization until the plated The culture is carried out in a petri dish with a concentration of 0.008-0.012g/L gelatin, and the seeding cell density is 2×10 ⁵ cells/ml. 6. The method for inducing bovine induced pluripotent stem cells according to claim 4, characterized in that: the bovine induced pluripotent stem cell induction and maintenance medium is: containing 1.5-2.5mM glutamine, 1.5-2.5 mM sodium pyruvate, 0.8-1.2% non-essential amino acids, 0.08-0.12mM β-mercaptoethanol, 990-1100U/ml LIF, 3.5-4.5ng/ml bFGF, 13-17% FBS and 0.8-1.2% green Streptomycin in high glucose DMEM.

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