CN102229909A - Method for inducing bovine induced pluripotent stem cells - Google Patents
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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
Technical field
The present invention relates to the cell mutation technical field, specifically is a kind of ox inductive pluripotent stem cells induction method.
Background technology
Since (Takahashi K and Yamanaka S in 2006,2006) report that for the first time making mouse skin inoblast reprogrammed by exogenous importing four kinds of qualifications factor (Oct4, Sox2, c-Myc, Klf4) is inductive pluripotent stem cells (induced pluripotent stem cells, iPS) after, up to the present, from comprise a plurality of species, broad variety somatocyte such as people, monkey, rat and pig, successfully induced and produced inductive pluripotent stem cells (Yu J et al., 2007; Liu H et al., 2008; Liao J et al., 2009; Esteban MA et al., 2009).And, along with further developing of research, the mouse and the problems such as people's inductive pluripotent stem cells inefficiency and security of having attempted using some method to solve and inducing generation at first.And two research groups of China report that all they induce the mouse inductive pluripotent stem cells of generation to obtain the intravital mouse that is prepared by inductive pluripotent stem cells fully by tetraploid embryo complementary technology recently, convincingly demonstrate inductive pluripotent stem cells and had real totipotency (Zhao XY et al., 2009; Kang L etal., 2009).Therefore, using and limiting the direct reprogrammed somatocyte of the factor is that inductive pluripotent stem cells is the important breakthrough that stem cell biological is learned the field, is with a wide range of applications in fields such as genetic engineering and regenerative medicines.
Different with the embryo of animal-origins such as mouse, rat, monkey, isolated embryonic stem cell ties up in the culturing process very unstablely from cloven-hoofed big livestock embryo, will soon lose its characteristic.Although the existing trial for many years of various countries scientist, the present report that the versatility embryonic stem cell line that successfully separates ox is not arranged as yet.If therefore successfully induce generation to have totipotent ox inductive pluripotent stem cells by limiting the factor, so just can effectively utilize the scientific research that its cells and characteristic of stem is correlated with, and utilize its characteristic similar to be used for the production application of breeding transgenic livestock animal to embryonic stem cell.Yet the inductive technology about the ox inductive pluripotent stem cells at home and abroad there is no bibliographical information at present.
Summary of the invention
The invention provides a kind of ox inductive pluripotent stem cells induction method, utilize external source to limit the factor and enhanced green fluorescence protein (Enhanced green fluorescent protein, EGFP) construction of fusion protein slow virus expression vector is used for inducing of ox inductive pluripotent stem cells, cultivate and go down to posterity expressing bovine fetal fibroblast that external source limits factor fusion protein, progressively separation and Culture goes out colony edge boundary cell clone clearly, the cell colony growth conditions is stable, caryogram is normal, the alkaline phosphatase test positive, immunocytochemistry detects and shows Oct4, Nanog, the SSEA-1 protein expression is positive, can be differentiated to form the teratoma that contains three germinal layers in the body, the result confirms that the cell clone of separation and Culture has embryonic stem cell sample feature, and successful separation and Culture obtains the ox inductive pluripotent stem cells.
Technical scheme of the present invention is:
A kind of ox inductive pluripotent stem cells induction method is characterized in that, may further comprise the steps:
(1), limit the structure of factor slow virus expression vector:
According to the mRNA sequence of pig Sox2, c-Myc, Klf4 gene and the dna sequence dna of people Oct4, design synthesizes the upstream and downstream primer of pSox2, pKlf4, pc-Myc, hOct4 gene;
From pig blastocyst, extract total RNA, the pSox2 that utilizes design to synthesize, on pKlf4 and the pc-Myc gene, downstream primer amplifies pig through RT-PCR and limits gene DNA, the dna sequence dna of people Oct4 is on the hOct4 gene, downstream primer directly increases from people Oct4 gene and obtains, the dna sequence dna that pig is limited gene DNA and people Oct4 carries out enzyme and cuts, be connected with retroviral vector pLEGFP-N1 then, construct the retrovirus fusion protein expression vector, from the retrovirus fusion protein expression vector, amplify the CMV promotor together with limiting gene and EGFP gene, cut by enzyme simultaneously and from the pLL3.7 plasmid, remove the U6 promotor, CMV promotor and GFP sequence, at last the CMV promotor is recombinated together with the pLL3.7 that limits after gene and EGFP gene and enzyme are cut, construct slow virus and limit gene fusion albumen reconstruct expression vector;
The upstream and downstream primer sequence of described pSox2, pKlf4, pc-Myc, hOct4 gene is:
PSox2 upstream: 5 '-TTTAAGCTTGCCACCATGTACAACATGATGGAGAC-3 ';
Downstream: 5 '-AAAGGATCCGGCATGTGAGAGAGAGGCAGTGTAC-3 ';
PKlf4 upstream: 5 '-TATAAGCTTGCCACCATGGCTGTCAGCGACGCAC-3 ';
Downstream: 5 '-GGCGGATCCGGAAAATGCCTCTTCATGTGTAAGGC-3 ';
Pc-Myc upstream: 5 '-GCCAAGCTTGCCACCCTGGATTTCCTTCGGATAG-3 ';
Downstream: 5 '-AAAGGATCCGCTGGGCAAGAGTTCCGTAGCTG-3 ';
HOct4 upstream: 5 '-AATGTCGACGCCACCATGGCGGGACACCTGGCTTC-3 ';
Downstream: 5 '-CGCGGATCCGCTCAGTTTGAATGCATGGGAGAGC-3 '.
(2), inducing of bovine fetal fibroblast:
Adopt tissue mass cell culture to set up bovine fetal fibroblast system, adopt calcium phosphate method transfection slow virus to limit four factor plasmid PLL-hOCT4/pSOX2/pMYC/pKLF4 of gene fusion albumen reconstruct expression vector, simultaneously the slow virus packing is produced the helper plasmid pMDLg that virus needs, pRSV REV and pVSVg are packaged into the 293T cell, change 293T cell fresh medium behind the 12-16h, collect behind the virus packing 48h and contain virus-culturing fluid, behind ultrafiltration and concentration, add to and contain in the bovine fetal fibroblast culture plate, infect after 18-20 days, behind the preliminary metamorphosis cell that infects is digested through Dispase II appears in cell, be seeded on the mouse fetal inoblast feeder layer that ametycin handled and cultivate, infect after 23-25 days, ox embryonic stem cell sample clone obviously as seen.
The concrete operations step that described slow virus limits gene fusion albumen reconstruct expression vector establishment comprises: at first pLL3.7 is carried out enzyme with Apa I and cut, the T4 archaeal dna polymerase is with its terminal smoothing, carry out single endonuclease digestion with EcoR I again, promptly from the pLL3.7 plasmid, remove U6 promotor, CMV promotor and GFP sequence; Design vector is transformed primer, from the retrovirus fusion protein expression vector that builds, transform primer amplification and go out the CMV promotor together with limiting gene and EGFP sequence through carrier, carry out single endonuclease digestion with Mun I then, the CMV promotor that will amplify from the retrovirus fusion protein expression vector with the T4 ligase enzyme behind the purifying is connected together with the pLL3.7 after limiting gene and EGFP gene and enzyme being cut recombinates, and builds slow virus and limits gene fusion albumen reconstruct expression vector;
The dna sequence dna that described design vector is transformed primer is:
Upstream: 5 '-AAAGGGCCCGCGGGACTCTGGGGTTCGTAATA-3 ';
Downstream: 5 '-GCGCAATTGTTACTTGTACAGCTCGTCC-3 '.
Described virus packing is converged the 293T cell the day before yesterday, and the 293T cell culture fluid is: contain foetal calf serum, the 95-105U/ml penicillin of 8-12%, the high sugared DMEM of 0.08-0.12mg/ml Streptomycin sulphate, carry out virus when cell converges 50-60% and pack; Described virus packing adopts calcium phosphate method transfection slow virus to limit four factor plasmid PLL-hOCT4/pSOX2/pMYC/pKLF4 of gene fusion albumen reconstruct expression vector, helper plasmid pMDLg, pRSV REV and pVSVg that simultaneously the slow virus packing being produced virus needs are packaged into the 293T cell, change 293T cell fresh medium behind the 12-16h, collect behind the 48h and contain virus-culturing fluid and behind the membrane filtration of 0.45 μ m, transfer in the 100KD ultrafiltration pipe of Millipore, get concentrating virus liquid at the 3-5 ℃ of centrifugal 20-40min of following 4000rpm.
Be injected into bovine fetal fibroblast in the bovine fetal fibroblast culture plate the day before yesterday of described virus beginning infected cattle fetal fibroblast, and cell density is 1 * 10
5/ hole, the bovine fetal fibroblast nutrient solution is: the foetal calf serum that contains 8-12%, 95-105U/ml penicillin, 0.08-0.12mg/ml the high sugared DMEM of Streptomycin sulphate, change into after 24 hours and be added with concentrating virus liquid and concentration is the bovine fetal fibroblast nutrient solution of 10 μ g/mL Polybrene, virus begins to infect the bovine fetal fibroblast nutrient solution that changes anosis venom after 12 hours, second day of beginning to infect of virus changes into and be added with concentrating virus liquid and concentration is that the bovine fetal fibroblast nutrient solution of 10 μ g/mLPolybrene infects once again, change the bovine fetal fibroblast nutrient solution of anosis venom after 12 hours again, through promptly viral the 3rd day of beginning to infect after 2 slow virus infections the nutrient solution in the bovine fetal fibroblast culture plate is replaced by the ox inductive pluripotent stem cells and induces and keep nutrient solution, after 18-20 days, on 12.5 days mouse fetal inoblast feeder layers handling to ametycin with Dispase II digestive inoculation, add new ox inductive pluripotent stem cells and induce and keep nutrient solution at 37.5 ℃, 5%CO
2Proceed under the saturated humidity to cultivate.
Described bovine fetal fibroblast is that the concrete steps of cultivating comprise: get 2.5 monthly age holstein cow fetal skin tissues, adopt tissue mass cell culture to set up fibroblast, nutrient solution is for containing the 8-12% foetal calf serum, 95-105U/ml penicillin, the high sugared DMEM of 0.08-0.12mg/ml Streptomycin sulphate; The preparation method of described mouse fetal inoblast feeder layer is for selecting to be paved with the mouse embryo fibroblasts in the 2-4 generation at the bottom of the ware, ametycin through 9-11ug/ml is handled 2-3h, conventional trysinization is inoculated in the culture dish that the shop over-richness is the 0.008-0.012g/L gelatin and cultivates, and inoculating cell density is 2 * 10
5Individual/ml.
Described ox inductive pluripotent stem cells is induced and is kept nutrient solution: the nonessential amino acid, 0.08-0.12mM beta-mercaptoethanol, 990-1100U/ml LIF, the FBS of 3.5-4.5ng/ml bFGF, 13-17% and the high sugared DMEM of 0.8-1.2% mycillin that contain 1.5-2.5mM glutamine, 1.5-2.5mM Sodium.alpha.-ketopropionate, 0.8-1.2%.
% average of the present invention accounts for the volume percent of cumulative volume.
The mRNA sequence information of described pig Sox2, c-Myc, Klf4 gene is from the U.S. state-run biotechnology information center database.
The upstream and downstream primer of described pSox2, pKlf4, pc-Myc gene is meant the specific dna sequence that can amplify corresponding gene from the dna fragmentation of species pig.The upstream and downstream primer of hOct4 gene is meant the specific dna sequence that amplifies the hOct4 gene from direct people Oct4 gene.
Described carrier transformation primer is meant and amplifies the CMV promotor together with the specific dna sequence that limits gene and EGFP sequence from the retroviral vector plasmid pLEGFP--hOCT4/pSOX2/pMYC/pKLF4 that builds, primer generally is oneself design, and special biotech firm is synthetic.
The purpose of described replacing 293T cell fresh medium is that unnecessary calcium phosphate precipitation that does not enter cell and products of cellular metabolism are removed, change the nutrient solution that normal 293T cell cultures needs into, help the nutrient solution that contains virus that the 293T cell is grown and 293T cell packing produces in follow-up time like this and collect.
The invention has the advantages that:
(1), the present invention utilizes external source to limit the factor and EGFP construction of fusion protein slow virus expression vector is used for inducing of ox inductive pluripotent stem cells, cultivate and go down to posterity expressing bovine fetal fibroblast that external source limits factor fusion protein, progressively separation and Culture goes out colony edge boundary cell clone clearly, the cell colony growth conditions is stable, caryogram is normal, the alkaline phosphatase test positive, immunocytochemistry detects and shows Oct4, Nanog, the SSEA-1 protein expression is positive, can be differentiated to form the teratoma that contains three germinal layers in the body, the result confirms that the cell clone of separation and Culture is a multipotent stem cells, can be referred to as the ox inductive pluripotent stem cells; Successfully induce first and produce the ox inductive pluripotent stem cells;
(2) compare with common inductive pluripotent stem cells inductive technology, what the present invention adopted is that the fusion rotein that limits the factor and EGFP establishment carries out inducing of ox inductive pluripotent stem cells, fusion rotein (fusionprotein) is to adopt the DNA recombinant technology on gene level the coding region of two kinds of protein or protein domain to be connected from beginning to end according to single open reading frame, is controlled the gene expression product that constitutes by same regulating and controlling sequence; Fusion protein technology because of its expression at target protein, make up to have aspect the researchs such as having bifunctional target protein and have irreplaceable effect, at medicine, agricultural has important purposes in the production of environment etc., the research; EGFP is use always in the fluorescin family a kind of, fluorescence has high stability, limit mechanism and the characteristics that the factor plays a role for external source in cell reprogrammed process, the fusion rotein that external source limits the factor and EGFP establishment can detect, follows the trail of and grasp the characteristics that external source limits the factor effectively, helps to carry out in the future the mechanism research of ox inductive pluripotent stem cells;
(3) the ox inductive pluripotent stem cells that adopts of the present invention induce and the nutrient solution prescription of keeping its cells and characteristic of stem to future separation and Culture set up real ox embryonic stem cell line and have certain reference value.
Utilize the external source qualification factor and EGFP to set up fusion rotein and be applied to inducing of ox inductive pluripotent stem cells, traceable grasp external source limits mechanism and the characteristics of the factor in the reprogrammed process, from the present invention as can be known, external source qualification factor great majority in ox inductive pluripotent stem cells colony are in does not express or low expression level, a few cell then is in the high expression level state, foreign gene was not expressed after the complete reprogrammed formation of somatocyte inductive pluripotent stem cells had been described, not exclusively the somatocyte foreign gene of reprogrammed still is in the high expression level state.(Zhao Y et al. such as Zhao, 2008) lure in the imported inductive pluripotent stem cells as fluorescent mark with EGFP and find, what the complete reprogrammed that the silence that EGFP expresses is indicating inductive pluripotent stem cells, the expression of EGFP showed inductive pluripotent stem cells still is in incomplete reprogrammed.It is consistent with the expression level of people ES cell Oct4 that Yu etc. (Yu J et al., 2009) detect people's inductive pluripotent stem cells of finding complete reconstruct by gene chip.Maherali etc. (Maherali N et al., 2007) have confirmed to form the back external source by the mouse inductive pluripotent stem cells when adopting external source to limit the abduction delivering of the factor to limit the expression of the factor unessential by the growth of inductive pluripotent stem cells.Numerous results of study has shown that inductive pluripotent stem cells forms back activated endogenous versatility gene and is enough to keep growing of stem cell, and the exogenous qualification factor is in quiet state in inductive pluripotent stem cells.Utilize the qualification factor and EGFP to set up the reprogrammed that fusion rotein the present invention technology is used for bovine somatic cells, can successfully induce and produce the ox inductive pluripotent stem cells, and do not see as yet both at home and abroad that for ox inductive pluripotent stem cells technology bibliographical information is arranged at present, therefore the present invention initiates both at home and abroad, the inductive pluripotent that furthered is dried to move towards the distance that livestock industry is used, and has a far reaching significance.Secondly, utilize EGFP to have stablize the characteristics of green fluorescence, traceable grasp external source limits mechanism and the characteristics that the factor plays a role in cell reprogrammed process, utilize ox inductive pluripotent stem cells related work to lay the foundation for furtheing investigate in the future.Once more, because of not setting up the ox embryonic stem cell line at present both at home and abroad as yet, that adopts in the technology of the present invention induces and keeps ox inductive pluripotent stem cells nutrient solution prescription and set up the ox embryonic stem cell line for research in the future reference can be provided.
Description of drawings
Fig. 1 is a pLL3.7 plasmid map synoptic diagram.
Fig. 2 limits factor fusion protein retroviral vector construct collection of illustrative plates synoptic diagram.
Fig. 3 limits factor fusion protein lentiviral vectors plasmid pLL-hOCT4/pSOX2/pMYC/pKLF4-GFP to make up the collection of illustrative plates synoptic diagram.
Fig. 4 is that the ox inductive pluripotent stem cells is induced the process synoptic diagram.
Fig. 5 is the aspect graph of bovine fetal fibroblast before the virus infection.
Fig. 6 be behind the virus infection 2 times under stem cell nutrient solution culture condition the aspect graph of the 19th day bovine fetal fibroblast.
Fig. 7 is an ox inductive pluripotent stem cells clone aspect graph.
Fig. 8 is the 13rd a generation ox inductive pluripotent stem cells metacinesis phasor.
Fig. 9 is that RT-PCR detects the versatility gene map, and 1 represents bovine fetal fibroblast among the figure, and 2-3 represents the ox inductive pluripotent stem cells, and 4 represent mouse ES cells.
Figure 10 is that the ox inductive pluripotent stem cells is expressed the painted microscope figure of embryonic stem cell AP.
Figure 11 is the microscope figure that the ox inductive pluripotent stem cells is expressed the proteic expression of embryonic stem cell Oct4.
Figure 12 is the microscope figure that the ox inductive pluripotent stem cells is expressed the proteic expression of embryonic stem cell Nanog.
Figure 13 is the microscope figure that the ox inductive pluripotent stem cells is expressed the proteic expression of embryonic stem cell SSEA-1.
Figure 14 is the microscope figure of ox inductive pluripotent stem cells at the intravital neural sample tissue of nude mice (ectoderm).
Figure 15 is the microscope figure of ox inductive pluripotent stem cells at the intravital muscle sample of nude mice tissue (mesoderm).
Figure 16 is the microscope figure of ox inductive pluripotent stem cells at all tissues of the intravital glandular tube of nude mice (entoderm).
Embodiment
Specific embodiment: the holstein cow fetal fibroblast is induced and is the inductive pluripotent stem cells method:
(1), limit the structure of factor slow virus expression vector:
According to the mRNA sequence of NCBI last pig Sox2 (NM 001123197), c-Myc (NM 001005154), Klf4 (NM001031782) gene, the synthetic upstream and downstream of design primer, primer sequence is as follows:
PSox2 upstream: 5 '-TTTAAGCTTGCCACCATGTACAACATGATGGAGAC-3 '
Downstream: 5 '-AAAGGATCCGGCATGTGAGAGAGAGGCAGTGTAC-3 '
PKlf4 upstream: 5 '-TATAAGCTTGCCACCATGGCTGTCAGCGACGCAC-3 '
Downstream: 5 '-GGCGGATCCGGAAAATGCCTCTTCATGTGTAAGGC-3 '
Pc-Myc upstream: 5 '-GCCAAGCTTGCCACCCTGGATTTCCTTCGGATAG-3 '
Downstream: 5 '-AAAGGATCCGCTGGGCAAGAGTTCCGTAGCTG-3 '
HOct4 upstream: 5 '-AATGTCGACGCCACCATGGCGGGACACCTGGCTTC-3 '
Downstream: 5 '-CGCGGATCCGCTCAGTTTGAATGCATGGGAGAGC-3 '
Carrier is transformed primer: upstream: 5 '-AAAGGGCCCGCGGGACTCTGGGGTTCGTAATA-3 '
Downstream: 5 '-GCGCAATTGTTACTTGTACAGCTCGTCC-3 '
From pig blastocyst, extract total RNA, amplify pig through RT-PCR and limit gene DNA, the dna sequence dna of people Oct4 directly increases from people Oct4 gene and obtains, through digestion with restriction enzyme, pLEGFP-N1 is connected with retroviral vector, constructs the retrovirus fusion protein expression vector.From the retrovirus fusion protein expression vector of reconstruct, amplify the CMV promotor together with limiting gene and EGFP gene, cut by enzyme simultaneously and from the pLL3.7 plasmid, remove U6 promotor, CMV promotor and GFP sequence, at last the promotor that increases in the recombinant retroviral vector is recombinated with the pLL3.7 that limits after gene fusion protein sequence and enzyme are cut, construct slow virus and limit gene fusion albumen reconstruct expression vector (Fig. 3).The concrete operations step is as follows: at first pLL3.7 is carried out enzyme with Apa I and cut, the T4 archaeal dna polymerase carries out single endonuclease digestion with EcoR I again with its terminal smoothing, promptly removes U6 promotor, CMV promotor and GFP sequence from the pLL3.7 plasmid.Design vector is transformed primer and amplify CMV promotor, purpose fragment and EGFP sequence from the retroviral vector plasmid pLEGFP--hOCT4/pSOX2/pMYC/pKLF4 that builds, carry out single endonuclease digestion with Mun I then, with the T4 ligase enzyme both connections are promptly built slow virus behind the purifying and limit gene fusion albumen reconstruct expression vector.
(2), inducing of bovine fetal fibroblast:
Material and reagent:
Fibroblastic separation of tire ox and cultivation: the employing tissue mass cell culture is set up 2.5 monthly age holstein cow fetal fibroblasts and is, nutrient solution accounts for volume percent 10% foetal calf serum (Hyclone) for containing, 100U/ml penicillin, the high sugared DMEM (Hyclone) of 0.1mg/ml Streptomycin sulphate.
The tire mouse fibroblast cell (mouse embryo fibroblasts, the foundation that MEFs) is: pregnant 12.5dICR strain tire mouse, adopt trypsin digestion separation and Culture mouse embryo fibroblasts behind removal head, four limbs and the internal organ.Nutrient solution accounts for volume percent 10% new-born calf serum (folium ilicis chinensis) for containing, 100U/ml penicillin, the high sugared DMEM (Hyclone) of 0.1mg/ml Streptomycin sulphate.
The preparation of feeder layer: select to be paved with the mouse embryo fibroblasts in the 2-4 generation at the bottom of the ware, handle 2.5h through the ametycin (Sigma) of 10ug/ml, conventional trysinization is inoculated in the culture dish of spreading the 0.01g/L gelatin, and inoculating cell density is 2 * 10
5Individual/ml.Nutrient solution is with Mouse Embryo Fibroblasts Culture in Vitro liquid.
The ox inductive pluripotent stem cells is induced and is kept nutrient solution (ES nutrient solution): contain 2mM glutamine (Gibco), 2mM Sodium.alpha.-ketopropionate (Gibco), account for volume percent 1% nonessential amino acid (Gibco), 0.1mM beta-mercaptoethanol (Sigma), 1000U/ml LIF (ESGRO, Chemicon International), 4ng/ml bFGF (Sigma), account for volume percent 15%FBS (Hyclone), account for the high sugared DMEM (Hyclone) of volume percent 1% mycillin (Gibco).
The 293T cell culture fluid is with the bovine fetal fibroblast nutrient solution, and all cells is all at 37.5 ℃, 5%CO
2, CO under the saturated humidity
2Cultivate in the incubator.
Set up bovine fetal fibroblast system by tissue mass cell culture from 2.5 monthly age holstein cow fetal skin separation and Culture, tire ox inoblast infected since the 7th generation, and concrete reprogrammed scheme is seen Fig. 4.Adopt calcium phosphate method transfection four factor plasmid PLL-hOCT4/pSOX2/pMYC/pKLF4 and packing composition pMLg, pRSV REV and pVSVg to the 293T cell, renew bright nutrient solution behind the 12-16h, collect behind the virus packing 48h and contain virus-culturing fluid, contain virus-culturing fluid and behind the membrane filtration of 0.45 μ m, transfer in the 100KD ultrafiltration pipe of Millipore, get concentrating virus liquid at 4 ℃ of centrifugal 20-40min of following 4000rpm.Be injected into bovine fetal fibroblast in 6 orifice plates the day before yesterday of virus beginning infected cattle fetal fibroblast, and cell density is 1 * 10
5/ hole, nutrient solution is for containing 10% foetal calf serum, l00U/ml penicillin, 0.1mg/ml the high sugared DMEM of Streptomycin sulphate, changed in second day and be added with concentrating virus liquid and concentration is the bovine fetal fibroblast nutrient solution of 10 μ g/mL Polybrene, renew bright bovine fetal fibroblast nutrient solution after 12 hours, changing in the 3rd day and being added with concentrating virus liquid and concentration is that the bovine fetal fibroblast nutrient solution of 10 μ g/mL Polybrene infects once again, renew bright bovine fetal fibroblast nutrient solution after 12 hours again, be designated as 0 day the same day of virus beginning infected pigs fetal fibroblast, change the ox inductive pluripotent stem cells through 2 slow virus infection 3d and induce and keep nutrient solution, after about 19 days, fibroblastic form begins to change, be that a small amount of tire ox inoblast becomes spheroidal by spindle shape, and be the growth of colony sample, be inoculated on the 12.5d mouse fetal inoblast feeder layer after Dispase II (Roche) digestion, interpolation ox inductive pluripotent stem cells is induced and is kept nutrient solution at 37.5 ℃, 5%CO
2Proceed under the saturated humidity to cultivate, after infection the 23rd day, circular clear-cut embryonic stem cell sample clone obviously as seen.Be inoculated on the new feeder layer with Dispase II digestion clone and continue amplification cultivation, the ratio that has just begun in 1: 1 goes down to posterity, and goes down to posterity in 1: 4 ratio subsequently, goes down to posterity once every about 4d.
(3), the evaluation of ox inducing cell:
RT-PCR detects and uses Trizol reagent (Invitrogen) processing ox inductive pluripotent stem cells, extracts cell total rna then, according to the synthetic cDNA of the cDNA first chain synthetic agent box specification sheets (Takara company).With following primers designed amplification corresponding gene, primer sequence is as follows:
Ox Oct4 upstream: 5 '-TTGAAGCTTGCCACCATGGCGGGACACCTCGCTT-3 '
Downstream: 5 '-GCAGGATCCTCAGTTTGCATGCATAGGGGAGCCC-3 '
Ox Nanog upstream: 5 '-ATTAAGCTTGCCACCATGAGTGTGGGCCCAGCTT-3 '
Downstream: 5 '-CGCGGATCCTTACAAATCTTCAGGCTGTA-3 '
Ox Klf4 upstream: 5 '-ATTAAGCTTGCCACCATGGCTGTCAGCGACGCGC-3 '
Downstream: 5 '-GCGGGATCCTTAAAAGTGCCTCTTCATGTGTAAGGCG-3 '
Ox c-Myc upstream: 5 '-TATAAGCTTGCCACCATGCCCCTCAACGTCAGCT-3 '
Downstream: 5 '-GCGGGATCCTTAGGCGCAAGAGTTCCGTA-3 '
GAPDH upstream: 5 '-TTGGTATCGTGGAAGGACTCTA-3 '
Downstream: 5 '-TGTCATATTTGGCAGGTT-3 '
Karyotyping: 0.1 μ g/ml colchicine (Sigma) is handled ox inductive pluripotent stem cells 2.5 h, trysinization also is resuspended in 0.075 mol/LKCL, hatch 25min for 37 ℃, stationary liquid (methyl alcohol: fixing 10min Glacial acetic acid=3: 1), centrifugal fixedly repetition 3 times.Harvested cell drips sheet, Gimsa dyeing, dry air, mounting.Microscopically is observed and is used related software and analyzes chromosome pairing.
Immunofluorescence detects: cell is fixed through 4% Paraformaldehyde 96, and 1% bovine serum albumin (BSA) room temperature sealing 30min adds the anti-anti-Oct4 of rabbit (Abcam), the anti-Nanog of a goat (R﹠amp of being; D), mouse-anti SSEA-1 (Chemicon), mouse-anti SSEA-3 (R﹠amp; D), mouse-anti SSEA-4 (R﹠amp; D), mouse-anti TRA-1-60 (Chemicon), mouse-anti TRA-1-81 (Chemicon), by 1: 200 the dilution back 4 ℃ of overnight incubation.PBS washes 3 times, and two of CY3 red fluorescence mark anti-is hatched 1h by 1: 200 dilution back room temperature lucifuge, and PBS washes 3 times, carries out the nucleus observations under the fluoroscope that dyes behind the 1min with the DAPI (Roche) of 1 μ g/mL.
AP dyeing: with 4% Paraformaldehyde 96 fixed cell, (Alkaline phosphatase, AP) dye liquor dyes the interpolation alkaline phosphatase, is colored as reddish-brown or coffee-like particle is positive with endochylema.
Differentiation detects in the body: Dispase II digestion ox inductive pluripotent stem cells, it is subcutaneous to be expelled to immune deficiency mouse (BALB/C-nu) dorsal part behind the serum-free DMEM re-suspended cell, put to death nude mice behind the injection 8w, get and carry out hematoxylin eosin stain after tumor tissues 4% Paraformaldehyde 96 is fixed.
(4), result:
The qualification factor slow virus infection pig bovine fetal fibroblast of calcium phosphate method packing, after under the stem cell nutrient solution culture condition 18-19 days, the form of bovine fetal fibroblast begins to change, and a small amount of bovine fetal fibroblast is transformed into spheroidal by spindle shape gradually, is the aggregation growth.The spheroidal cell of Dispase II digestion is used to contain under the stem cell nutrient solution culture condition of 1000U/ml LIF and 4ng/ml bFGF on feeder layer cells, 23-24d after infection, and border tangible colony shape cell clone forms gradually.It is bigger that individual cells under the powerful microscope in the colony presents nucleus, and N/C is higher.Cell clone approximately went down to posterity once every 3-4 days under situation about going down to posterity at 1: 4, cell colony growth go down to posterity stable (Fig. 5,6,7).The ox inductive pluripotent stem cells that reached for the 13rd generation is carried out karyotyping show that pig inductive pluripotent stem cells chromosome number is 60, XY normal karyotype (Fig. 8).The sub-fusion rotein inductive of the RT-PCR detectability determining cause ox inductive pluripotent stem cells versatility expression of gene of being correlated with, the ox inductive pluripotent stem cells is expressed Oct4, c-Myc, Klf4 and Nanog gene, and bovine fetal fibroblast is not expressed genes involveds (Fig. 9) such as Nanog, Oct4, c-Myc, Klf4 under the same terms.AP has than high expression level in embryo's the stem cell in early days, and actively in the cell that has broken up obviously reduces, and even expresses, and the expression of AP is one of key character of a kind of ES cell and relevant stem cell.The AP coloration result shows that limiting factor fusion protein inductive ox inductive pluripotent stem cells AP is expressed as strong positive (Figure 10).Cellular immunofluorescence detects the ox inductive pluripotent stem cells that shows inducing culture and expresses Oct4, Nanog, SSEA-1 albumen (Figure 11,12,13), for the proteic detection of expression of Oct4, the cell that carries the external source qualification factor presents Oct4 albumen strongly expressed in whole cell clone, EGFP be strongly expressed cell then the Oct4 Protein Detection be strong positive, EGFP express more weak or quiet cell then the Oct4 protein expression level present the general positive (Figure 11), under the same conditions, the proteic expression level of Nanog does not have difference between expressing EGFP or not expressing between the cell of EGFP in cell colony.Get and limit the teratoma that factor fusion protein inductive pig inductive pluripotent stem cells forms behind the subcutaneous 8w of BALB/C nude mice, the HE coloration result is presented at cell and the tissue (Figure 14 that has three germinal layer sources such as nerve, muscle, body of gland in the teratoma tissue, 15,16).
Claims (6)
1. an ox inductive pluripotent stem cells induction method is characterized in that, may further comprise the steps:
(1), limit the structure of factor slow virus expression vector:
According to the mRNA sequence of pig Sox2, c-Myc, K1f4 gene and the dna sequence dna of people Oct4, design synthesizes the upstream and downstream primer of pSox2, pK1f4, pc-Myc, hOct4 gene;
From pig blastocyst, extract total RNA, the pSox2 that utilizes design to synthesize, on pK1f4 and the pc-Myc gene, downstream primer amplifies pig through RT-PCR and limits gene DNA, the dna sequence dna of people Oct4 is on the hOct4 gene, downstream primer directly increases from people Oct4 gene and obtains, the dna sequence dna that pig is limited gene DNA and people Oct4 carries out enzyme and cuts, be connected with retroviral vector pLEGFP-N1 then, construct the retrovirus fusion protein expression vector, from the retrovirus fusion protein expression vector, amplify the CMV promotor together with limiting gene and EGFP gene, cut by enzyme simultaneously and from the pLL3.7 plasmid, remove the U6 promotor, CMV promotor and GFP sequence, at last the CMV promotor is recombinated together with the pLL3.7 that limits after gene and EGFP gene and enzyme are cut, construct slow virus and limit gene fusion albumen reconstruct expression vector;
The upstream and downstream primer sequence of described pSox2, pK1f4, pc-Myc, hOct4 gene is:
PSox2 upstream: 5 '-TTTAAGCTTGCCACCATGTACAACATGATGGAGAC-3 ';
Downstream: 5 '-AAAGGATCCGGCATGTGAGAGAGAGGCAGTGTAC-3 ';
PK1f4 upstream: 5 '-TATAAGCTTGCCACCATGGCTGTCAGCGACGCAC-3 ';
Downstream: 5 '-GGCGGATCCGGAAAATGCCTCTTCATGTGTAAGGC-3 ';
Pc-Myc upstream: 5 '-GCCAAGCTTGCCACCCTGGATTTCCTTCGGATAG-3 ';
Downstream: 5 '-AAAGGATCCGCTGGGCAAGAGTTCCGTAGCTG-3 ';
HOct4 upstream: 5 '-AATGTCGACGCCACCATGGCGGGACACCTGGCTTC-3 ';
Downstream: 5 '-CGCGGATCCGCTCAGTTTGAATGCATGGGAGAGC-3 '.
(2), inducing of bovine fetal fibroblast:
Adopt tissue mass cell culture to set up bovine fetal fibroblast system, adopt calcium phosphate method transfection slow virus to limit four factor plasmid PLL-hOCT4/pSOX2/pMYC/pKLF4 of gene fusion albumen reconstruct expression vector, simultaneously the slow virus packing is produced the helper plasmid pMDLg that virus needs, pRSV REV and pVSVg are packaged into the 293T cell, change 293T cell fresh medium behind the 12-16h, collect behind the virus packing 48h and contain virus-culturing fluid, behind ultrafiltration and concentration, add to and contain in the bovine fetal fibroblast culture plate, infect after 18-20 days, behind the preliminary metamorphosis cell that infects is digested through Dispase II appears in cell, be seeded on the mouse fetal inoblast feeder layer that ametycin handled and cultivate, infect after 23-25 days, ox embryonic stem cell sample clone obviously as seen.
2. ox inductive pluripotent stem cells induction method according to claim 1, it is characterized in that, the concrete operations step that described slow virus limits gene fusion albumen reconstruct expression vector establishment comprises: at first pLL3.7 is carried out enzyme with Apa I and cut, the T4DNA polysaccharase is with its terminal smoothing, carry out single endonuclease digestion with EcoR I again, promptly from the pLL3.7 plasmid, remove U6 promotor, CMV promotor and GFP sequence; Design vector is transformed primer, from the retrovirus fusion protein expression vector that builds, transform primer amplification and go out the CMV promotor together with limiting gene and EGFP sequence through carrier, carry out single endonuclease digestion with Mun I then, the CMV promotor that will amplify from the retrovirus fusion protein expression vector with the T4 ligase enzyme behind the purifying is connected together with the pLL3.7 after limiting gene and EGFP gene and enzyme being cut recombinates, and builds slow virus and limits gene fusion albumen reconstruct expression vector;
The dna sequence dna that described design vector is transformed primer is:
Upstream: 5 '-AAAGGGCCCGCGGGACTCTGGGGTTCGTAATA-3 ';
Downstream: 5 '-GCGCAATTGTTACTTGTACAGCTCGTCC-3 '.
3. ox inductive pluripotent stem cells induction method according to claim 1, it is characterized in that: may further comprise the steps: described virus is packed and is converged the 293T cell the day before yesterday, the 293T cell culture fluid is: contain foetal calf serum, the 95-105U/ml penicillin of 8-12%, the high sugared DMEM of 0.08-0.12mg/ml Streptomycin sulphate, carry out the virus packing when cell converges 50-60%; Described virus packing adopts calcium phosphate method transfection slow virus to limit four factor plasmid PLL-hOCT4/pSOX2/pMYC/pKLF4 of gene fusion albumen reconstruct expression vector, helper plasmid pMDLg, pRSV REV and pVSVg that simultaneously the slow virus packing being produced virus needs are packaged into the 293T cell, change 293T cell fresh medium behind the 12-16h, collect behind the 48h and contain virus-culturing fluid and behind the membrane filtration of 0.45 μ m, transfer in the 100KD ultrafiltration pipe of Millipore, get concentrating virus liquid at the 3-5 ℃ of centrifugal 20-40min of following 4000rpm.
4. ox inductive pluripotent stem cells induction method according to claim 1, it is characterized in that, may further comprise the steps: be injected into bovine fetal fibroblast in the bovine fetal fibroblast culture plate the day before yesterday of described virus beginning infected cattle fetal fibroblast, and cell density is 1 * 10
5/ hole, the bovine fetal fibroblast nutrient solution is: the foetal calf serum that contains 8-12%, 95-105U/ml penicillin, 0.08-0.12mg/ml the high sugared DMEM of Streptomycin sulphate, change into after 24 hours and be added with concentrating virus liquid and concentration is the bovine fetal fibroblast nutrient solution of 10 μ g/mL Polybrene, virus begins to infect the bovine fetal fibroblast nutrient solution that changes anosis venom after 12 hours, second day of beginning to infect of virus changes into and be added with concentrating virus liquid and concentration is that the bovine fetal fibroblast nutrient solution of 10 μ g/mLPolybrene infects once again, change the bovine fetal fibroblast nutrient solution of anosis venom after 12 hours again, through promptly viral the 3rd day of beginning to infect after 2 slow virus infections the nutrient solution in the bovine fetal fibroblast culture plate is replaced by the ox inductive pluripotent stem cells and induces and keep nutrient solution, after 18-20 days, on 12.5 days mouse fetal inoblast feeder layers handling to ametycin with Dispase II digestive inoculation, add new ox inductive pluripotent stem cells and induce and keep nutrient solution at 37.5 ℃, 5%CO
2Proceed under the saturated humidity to cultivate.
5. ox inductive pluripotent stem cells induction method according to claim 1, it is characterized in that, described bovine fetal fibroblast is that the concrete steps of cultivating comprise: get 2.5 monthly age holstein cow fetal skin tissues, adopt tissue mass cell culture to set up fibroblast, nutrient solution is for containing the 8-12% foetal calf serum, 95-105U/ml penicillin, the high sugared DMEM of 0.08-0.12mg/ml Streptomycin sulphate; The preparation method of described mouse fetal inoblast feeder layer is for selecting to be paved with the mouse embryo fibroblasts in the 2-4 generation at the bottom of the ware, ametycin through 9-11ug/ml is handled 2-3h, conventional trysinization is inoculated in the culture dish that the shop over-richness is the 0.008-0.012g/L gelatin and cultivates, and inoculating cell density is 2 * 10
5Individual/ml.
6. ox inductive pluripotent stem cells induction method according to claim 4 is characterized in that: described ox inductive pluripotent stem cells is induced and is kept nutrient solution and is: the nonessential amino acid, 0.08-0.12mM beta-mercaptoethanol, 990-1100U/ml LIF, the FBS of 3.5-4.5ng/ml bFGF, 13-17% and the high sugared DMEM of 0.8-1.2% mycillin that contain 1.5-2.5mM glutamine, 1.5-2.5mM Sodium.alpha.-ketopropionate, 0.8-1.2%.
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| CN111304157A (en) * | 2020-03-16 | 2020-06-19 | 吉林大学 | Method for obtaining bovine initial state induced pluripotent stem cells |
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| CN109975264A (en) * | 2019-04-18 | 2019-07-05 | 安徽农业大学 | A kind of method that slow-virus transfection blastaea trophocyte studies its gene function |
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| CN111304157A (en) * | 2020-03-16 | 2020-06-19 | 吉林大学 | Method for obtaining bovine initial state induced pluripotent stem cells |
| CN111304157B (en) * | 2020-03-16 | 2023-05-12 | 吉林大学 | Method for obtaining bovine initial state induced pluripotent stem cells |
| CN115074388A (en) * | 2021-06-29 | 2022-09-20 | 北京大学深圳医院 | Maternal factor induced 2C sample totipotent stem cell and transformation application thereof |
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