CN101812480A - Method utilizing transcription factor to transfect bovine body cell into induced pluripotent stem cell - Google Patents

Abstract

The invention discloses a method utilizing a transcription factor to transfect bovine body cells into induced pluripotent stem cells (iPSCs). The method specifically comprises the following steps: 1) separating and culturing bovine skin fibroblasts; 2) cloning bovine maintenance pluripotent gene transcription factors and constructing retroviral vectors thereof; 3) packaging and preparing retroviruses; and 4) infecting bovine skin fibroblasts by retroviruses and obtaining bovine iPS cells. In the invention, human amniotic membrane after cell removal is utilized as a holder to replace mouse embryonic fibroblasts for the first time to sustain the in-vitro propagation of bovine iPSCs and maintain the undiferentiated pluripotent state for a long time. The strategy, which adopts gene induction to generate pluripotent stem cells, solves the technical problem that livestock particularly bovine pluripotent stem cells are difficult to obtain and avoids the problem of heterogenetic animal cell pollution brought by the way that mouse embryo fibroblasts are used as the feeding layer of stem cells.

Description

Utilize the transcription factor transfecting bovine somatic cells to become the method for inductive pluripotent stem cells

Technical field

The invention belongs to gene engineering technology field, be specifically related to a kind of method of utilizing the transcription factor transfecting bovine somatic cells to become inductive pluripotent stem cells.

Background technology

2006, Japanese scholar Yamanaka ^[1]Reported first is utilized mouse transcription factor Oct4, and Sox2, c-Myc, Klf4 make up common transfection mouse somatocyte, obtained mouse inductive pluripotent stem cells (inducted pluripotent stem cells, iPSCs).Subsequently, started the research boom of inducing pluripotent stem cell in the world wide.All done research from a lot of aspects of animal varieties, transcription factor kind and quantity, transcription factor array mode and foreign gene introduction method or the like.Yamanaka in 2007 ^[2]Utilize same transcription factor successfully to obtain human iPS cell again; The same year, Yu (Yu Junying) and Thomson ^[3]Utilize Deng the people that (Nanog Lin28) also successfully obtains human iPS cell for Oct3/4, Sox2 with other four kinds of transcription factors.Rhesus monkey is arranged subsequently ^[4], rat ^[5], pig ^[6]Report (Li et al., 2009 Deng the success of animal iPS cell; Liao et al., 2009; Liu et al., 2008).Because iPSCs and embryonic stem cell (ESCs) have closely similar biological characteristics, so culture condition of all having used for reference the ES cell in the process of setting up at existing iPSCs, for example use the nutrient solution of ESCs, using mouse embryo fibroblasts (MEF) provides multiple somatomedin to keep the versatility of iPSCs as feeder layer.But domestic animal, for example animals such as pig, ox, sheep also do not have the generally acknowledged embryonic stem cell that is of building ^[7], from body early embryo, separate very difficulty of these embryonic stem cells, and culture system is not definite fully.Existing embryonic stem cell culture condition all is to adopt mouse embryo fibroblasts (MEF) as culture layer, just can keep ESCs and be in undifferentiated state.Use mouse embryo fibroblasts in the ESCs culture system, directly introduced the heterogenous animal cell, the clinical application that this has just directly influenced the ESCs cell has also increased workload and the difficulty in the culturing process.Since mouse embryo stem cell in 1981 ^[8]With hESC in 1998 ^[9]Build be since, over and done with so far two more than ten years, but to be difficult to so far build be to illustrate that then domestic animal will obtain embryonic stem cell from the body early embryo material separation and also exist many technology and theoretic restraining factors to the livestock embryo stem cell.Therefore, utilizing transcription factor transfection somatocyte to obtain ox iPS cell has great importance.

Reference

[1]Takahashi，K.，and?Yamanaka，S.(2006).Induction?of?pluripotent?stem?cellsfrom?mouse?embryonic?and?adult?fibroblast?cultures?by?defined?factors.Cell?126，663-676.

[2]Takahashi，K.，Tanabe，K.，Ohnuki，M.，Narita，M.，Ichisaka，T.，Tomoda，K.，and?Yamanaka，S.(2007).Induction?of?pluripotent?stem?cells?from?adult?humanfibroblasts?by?defined?factors.Cell?131，861-872.

[3]Yu，J.Y.，Vodyanik，M.A.，Smuga-Otto，K.，Antosiewicz-Bourget，J.，Frane，J.L.，Tian，S.，Nie，J.，Jonsdottir，G.A.，Ruotti，V.，Stewart，R.，et?al.(2007).Inducedpluripotent?stem?cell?lines?derived?from?human?somatic?cells.Science?318，1917-1920.

[4]Liu，H.S.，Zhu，F.F.，Yong，J.，Zhang，P.B.，Hou，P.P.，Li，H.G.，Jiang，W.，Cai，J.，Liu，M.，Cui，K.，et?al.(2008).Generation?of?Induced?Pluripotent?Stem?Cellsfrom?Adult?Rhesus?Monkey?Fibroblasts.Cell?Stem?Cell?3，587-590.

[5]Li，W.L.，Wei，W.，Zhu，S.，Zhu，J.，Shi，Y.，Lin，T.，Hao，E.，Hayek，A.，Deng，H.，and?Ding，S.(2009).Generation?of?Rat?and?Human?Induced?PluripotentStem?Cells?by?Combining?Genetic?Reprogramming?and?Chemical?Inhibitors(vol?4，pg?16，2009).Cell?Stem?Cell?4，370-370.

[6]Esteban，M.A.，Xu，J.，Yang，J.，Peng，M.，Qin，D.，Li，W.，Jiang，Z.，Chen，J.，Deng，K.，Zhong，M.，et?al.(2009).Generation?of?Induced?Pluripotent?Stem?CellLines?from?Tibetan?Miniature?Pig.Journal?of?Biological?Chemistry?284，17634-17640.

[7]Keefer，C.L.，Pant，D.，Blomberg，L.，and?Talbot，N.C.(2007).Challengesand?prospects?for?the?establishment?of?embryonic?stem?cell?lines?of?domesticatedungulates.Animal?Reproduction?Science?98，147-168.

[8]Martin，G.R.(1981).Isolation?of?a?pluripotent?cell?line?from?early?mouseembryos?cultured?in?medium?conditioned?by?tera-tocarcinoma?stem?cells.Proc?NatlAcad?Sci?USA?78，7634-7638.

[9]Thomson，J.A.，Itskovitz-Eldor，J.，Shapiro，S.S.，Waknitz，M.A.，Swiergiel，J.J.，Marshall，V.S.，and?Jones，J.M.(1998).Embryonic?stem?cell?lines?derived?fromhuman?blastocysts.Science?282，1145-1147.

Summary of the invention

At above-mentioned problems of the prior art and deficiency, the object of the present invention is to provide a kind of method of utilizing the transcription factor transfecting bovine somatic cells to become inductive pluripotent stem cells, inductive pluripotent stem cells (iPSCs) with this method preparation, its cell colony form, growth characteristics, stem cell surface mark, dryness genetic expression, main dryness gene promoter sequence zone methylation patterns, embryoid forms and vitro differentiation ability, teratoma form ability, and it is all closely similar with the embryonic stem cell characteristic to participate in eight aspects such as mosaic formation.

Realize that foregoing invention purpose technical scheme is to utilize the transcription factor transfecting bovine somatic cells to produce the method for inductive pluripotent stem cells, specifically comprises the following steps:

1) fibroblastic separation of ox-hide skin and cultivation

Ox-hide skin inoblast BDFs and new born bovine skin flbroblast NDFs obtain a large amount of adult ox-hide inoblast skin cell and new born bovine skin flbroblast respectively through the routine cultivation to grow up;

2) ox is kept the clone and the Expressed by Retrovirus Vector thereof of versatility gene transcription factor

From 50-60 age in days ox fetus, separate sex-ridge, extract total RNA, obtain cDNA through reverse transcription reaction; With cDNA is template, obtain ox Oct4, Sox2 and three gene amplification products of Klf4 and correctly then goal gene is inserted among the retroviral vector pMSCVneo through PCR reaction, make up the retrovirus expression vector of these 3 kinds of genes through the sequence verification sequence;

3) retroviral packing and preparation

The PT67 packing cell is cultivated in containing the DMEM substratum of 15% foetal calf serum according to ordinary method, used liposome the retroviral plasmid transfection that makes up is entered packing cell PT67 cell; Using preceding 1 hour of liposome packaging virus particle, change the nutrient solution DMEM substratum of packing cell PT67 into serum-free medium, 4-8 μ g is included the retroviral plasmid DNA of transcription factor gene and 10-20 μ l liposome to join 2 part of 500 μ l Opti-MEM-I+GlutaMAX-I transfection respectively and optimizes liquid and dilute, mixing gently, hatched under the room temperature 5 minutes, the diluent that will contain liposome lipofectamine2000 then slowly splashes in the diluent that contains retroviral plasmid, mixing gently, hatched again under the room temperature 20 minutes, and then above-mentioned mixed solution was dropwise added in the PT67 packing born of the same parents culture dish; After the transfection 24 hours, replacing in per 24 hours contains the virion nutrient solution once, for three days on end, collects viral suspension, under 4 ℃ of conditions through 25000 rev/mins after centrifugal 90 minutes, 10 times of concentrating virus supernatant liquors; To include three gene viruses supernatant liquors of Oct4, Sox2 and Klf4 and carry out balanced mix, and add the specific three factor genes combination of 8 μ g/ml Polybrene composition ,-80 ℃ of preservations are standby;

4) acquisition of the iPS cell of retroviral infection ox-hide skin inoblast and ox

To grow up ox-hide skin inoblast BDFs and newborn bull skin flbroblast NDFs cultivates in the 60mm plastic culture dish according to ordinary method, treat that cell grows into the about 5ml of retrovirus supernatant liquor mixed solution that will comprise Oct4, Sox2 and Klf4 gene transcription factor when 70-80% merges in the culture dish, join respectively in inoblast BDFs and the NDFs culture dish and infect inoblast; Cell covers with the back and goes down to posterity by the 1:3 routine behind the culture dish, visual cell's growing state at 6-10d with cell with 1 * 10 ⁵The density of cells/well is transferred on 6 well culture plates that are covered with people's amnion (HAM) in advance, cultivates metainfective ox cell as feeder layer, and nutrient solution is replaced with the stem cell nutrient solution simultaneously; Measure and change liquid every day half in the culture hole, observes 20-28d continuously, till having cell colony to occur, was designated as for 0 generation, is ox iPSCs cell.

Described stem cell nutrient solution consists of: contain in every 100ml solution: the knockout-DMEM of 82.69ml, 15ml FBS, 1ml concentration are that 200mM glutamine, 1ml concentration are that 10mM non-essential amino acid, 200 μ l concentration are that 55mM beta-mercaptoethanol, 10 μ l concentration are that 400ng/mL people's recombination basic fibroblast somatomedin (bFGF), 100 μ l concentration are 10 ⁵U/ml leukaemia inhibitory factor (LIF).

When ox iPSCs goes down to posterity, use the digestion of 0.05%trypsin+0.02%EDTA Digestive system, through neutralization, centrifugal and resuspended being passaged to again on the new HAM upholder, at 37-38 ℃, 5% left and right sides CO ₂, amplification cultivation under the saturated humidity condition obtains ox iPS cell strain.Ox iPSCs went down to posterity once every 3-5 days, went down to posterity at present 120 days, surpassed for 25 generations.Liquid nitrogen cryopreservation, the back growth vigor that thaws is better, nearly 80%-90% survival rate.

A further object of the invention provides the recombinant retroviral expression vector pMSCV-Oct4 of Oct4 gene and the application in the gene genetically engineered.

A further object of the invention provides the recombinant retroviral expression vector pMSCV-Sox2 of Sox2 gene and the application in the gene genetically engineered.

The present invention obtains inductive pluripotent stem cells and its preparation method compared with prior art, has the following advantages:

1, among the present invention 3 of application autonomous clened cows kinds keep stem cell versatility gene, by the stem cell retrovirus-mediated method, induce ox-hide skin inoblast, make its reprogrammed for having the ox iPSCs of embryonic stem cell biological nature fully.

2, the present invention adopts first in culture condition that to take off cell-free human amniotic membrane be that upholder replaces mouse embryo fibroblasts MEF to keep ox iPSCs in-vitro multiplication and keeps undifferentiated versatility state for a long time as HAM, and has avoided the heterogenous animal cell contamination problem that feeder layer brought of application mouse embryo fibroblasts (MEF) as stem cell.

3, the present invention adopts the method for gene induced generation multipotent stem cells, has not only solved the particularly ox multipotent stem cells technical barrier that is difficult to obtain of domestic animal, and to build for other stem cell animals be that theoretical foundation and experiment basis are provided.

Description of drawings

Fig. 1 cow skin flbroblast (BDFs) 50x microscope figure that grows up.

Newborn bull skin flbroblast (NDFs) 50x of Fig. 2 microscope figure.

Fig. 3 PT67 packing cell 50x microscope figure.

100,000 times of Electronic Speculum figure of retroviral particle under Fig. 4 Electronic Speculum.

Fig. 5 colony alkaline phosphatase staining 100x microscope figure.

Fig. 6 colony alkaline phosphatase staining 200x microscope figure.

Fig. 7 SSEA-3 bright field microscope figure.

Fig. 8 SSEA-3 dyeing (+++) microscope figure.

Fig. 9 Sox2 bright field microscope figure.

Figure 10 Sox2 dyeing (+++) microscope figure.

Figure 11 blister cavities sample embryoid structure microscope figure.

Figure 12 represents the β III-Tubulin of the ectoderm cell system positive microscope figure that dyes.

Figure 13 represents the α-actin stained positive microscope figure of mesoblastema system.

Figure 14 represents the alpha-fetoprotein that the endoderm cell is (the stained positive microscope figure of α-fetoprotein).

Figure 15 body of gland spline structure (entoderm) 400x microscope figure.

Figure 16 structure of skeletal muscles (mesoderm) 400x microscope figure.

Figure 17 archineuron structure (ectoderm) 400x microscope figure.

The RT-PCR detected result gel electrophoresis figure of Figure 18 EB.

The PCR of Figure 19 mosaic goat tissue detects electrophorogram.

Embodiment

Below the concrete acquisition ox iPSCs that provides by the applicant method and through multiple evidence it all has and the on all four biological characteristics of embryonic stem cell in all many-sides such as colony form, growth characteristics, surface marker, inside and outside differentiation capability and mosaic formation.

Embodiment fibroblastic separation of 1 ox-hide skin and cultivation

Growing up, (bovine dermal fibroblasts is BDFs) from the female Holstein milk the ears of an ox or cow edge skin of growing up for ox-hide skin inoblast.Be cut into the fine tissue piece after will organizing cleaning and sterilizing, under conventional culture condition, with cultivating among the high sugared DMEM (Gibco company product) that contains 15%-20% foetal calf serum (FBS) (Gibco company product).Every 2-3d changes and supports liquid 1 time, just has inoblast to shift out from the tissue block edge behind the 7-10d, and a large amount of inoblasts closely are arranged in around the tissue block behind the 12-16d.With going down to posterity after trypsinase and the digestion of EDTA solution, promptly obtain a large amount of adult ox inoblast skin cellss, see Fig. 1.(newborn bovine dermal fibroblast NDFs) from the newborn bull ear of Holstein kind edge skin, sees Fig. 2 to newborn bull skin flbroblast, and separation is identical with above-mentioned cow skin flbroblast method with cultural method.In order to guarantee cell viability, the present invention adopt 2～6 generations with interior cell as inducing preceding target cell.

2 Ns of clone and Expressed by Retrovirus Vector thereof of keeping versatility key gene transcription factor of embodiment

In the fetus body of 50-60 age in days He Sitan kind milk cow (Holstein), separate sex-ridge, extract total RNA with TRIzol LS Reagent behind the tissue homogenate, handle to remove genomic dna through DNaseI (RNase free) and pollute.Obtain cDNA through reverse transcription reaction.Be template then with cDNA, through pcr amplification Oct4, Sox2 and open frame (ORF) complete sequence of reading of three kinds of transcription factor genes of Klf4, gene clone the primer sequence is shown in Table 1 respectively.

The primer of table 1 amplification ox Oct4, Sox2 and Klf43 gene

Wherein the amplification parameter of Oct4 gene PCR amplification is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 45sec, 60 ℃ of annealing 45sec, 72 ℃ are extended 1min, circulate 35 times, and 72 ℃ are extended 10min again, and expection PCR product length should be 1083bp.

The amplification parameter of Sox2 gene is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 45sec, 54 ℃ of annealing 30sec, 72 ℃ are extended 1min, circulate 35 times, and 72 ℃ are extended 10min again, and expection PCR product length should be 963bp.

The amplification parameter of Klf4 gene is: 94 ℃ of pre-sex change 4min, and 94 ℃ become 30s, 60 ℃ of annealing 1min, 72 ℃ are extended 1min30sec, totally 35 circulations, 72 ℃ are extended 10min again, and expection PCR product length should be 1434bp.

Above-mentioned PCR product is respectively got 5 μ l, to identify its size, remains ℃ preservation of PCR product-20 at 1% agarose gel electrophoresis.Electrophoresis detection result shows that 3 kinds of transcription factor gene sizes of pcr amplification product length and expection milk cow are in full accord, the Pass Test design requirements.Then 3 kinds of PCR product remainders are passed through dna fragmentation Gel Extraction kit purifying, recovery respectively; Reclaim product and be connected with solution I mixing with cloning vector pMD18-T, 16 ℃ of connections are spent the night.To connect product is transformed in the competent cell TG I and clones.Dull and stereotyped last 37 ℃ of screening and culturing 14-16 hours of LB through containing Ampicillin (80 μ g/ml), X-gal and IPTG.Picking 6-10 white colony inoculated and shaken bacterium in the LB nutrient solution that contains penbritin (Ampicillin) in a small amount respectively from 3 kinds of cultures, and according to plasmid extraction test kit specification sheets step extracting trace plasmid.The plasmid that comprises Oct4, Sox2 and Klf43 kind gene is identified through double digestion, single endonuclease digestion and plasmid PCR.Wherein plasmid is through Bgl II+EcoRI double digestion and EcoRI single endonuclease digestion and plasmid PCR evaluation, to obtain pMD-18T-Oct4, pMD-18T-Sox2, three kinds of plasmid positive colonies of pMD-18T-Klf4.Selecting and recommending the positive plasmid of qualification result checks order to biotech firm.Reference sequences carries out the homology comparative analysis among sequencing result application DNAstart 7.10 softwares and the GenBank.Analytical results shows that the gene order of Oct4, Sox2 and Klf4 and reference sequences homology are respectively 99.4%, 99.9%, 99.9%.

Three kinds of plasmids of the pMD-18T-Oct4 that above-mentioned order-checking is correct, pMD-18T-Sox2 and pMD-18T-Klf4 pass through Bgl II+EcoRI double digestion respectively with retrovirus pMSCVneo carrier after reclaiming purifying, then purifying with reclaim enzyme and cut after 3 kinds of target gene fragment and linearizing pMSCVneo plasmid vector.The purpose fragment of linearizing retrovirus vector pMSCVneo and recovery is used DNA Ligation KitVersion 2.0 to be connected at 16 ℃ and to spend the night, after 14-16 hour, connect product and be transformed into the JM109 competent cell, through filtering out the positive colony of retroviral plasmid on penbritin (Ampicillin, 50 μ g/mL) the LB flat board.From 3 culture plates, each recombinant plasmid picking 6-10 bacterium colony respectively shakes bacterium in a small amount, and micro-method is extracted the retroviral plasmid of reorganization.Identify through the two enzymic digestion evaluations of Bgl II+EcoRI, the evaluation of plasmid pcr amplification and sequencing analysis.Three kinds of recombinant retrovirus plasmids can obtain the target gene fragment and the retroviral vector fragment of known dimensions through behind the double digestion, illustrate that the constructed recombinant retroviral expression vector structure of the present invention is correct.Detect by order-checking, the sequence and the pcr amplification sequence of 3 goal gene that comprised in all recombinant retrovirus plasmids are in full accord, also with GenBank in reference sequences in full accord, show that goal gene in constructed in the present invention 3 kinds of recombinant retrovirus plasmid processes do not read situations such as the frameshit of frame and base mutation, illustrate that gene order is entirely true in applied 3.3 kinds of constructed among the present invention recombinant retroviral expression vectors are distinguished called after: pMSCV-Oct4, pMSCV-Sox2, pMSCV-Kl f4.

Embodiment 3 retroviral packing and preparations

The PT67 packing cell is cultivated in the DMEM substratum according to ordinary method, when treating that cell reaches 70%-80% and converges, use liposome the retroviral vector transfection is entered packing cell PT67 cell, carry out the virus packing to obtain to have infectious retroviral particle.Preceding 1 hour of transfection, the DMEM substratum that will contain foetal calf serum changes serum-free medium into, 4-8 μ g retroviral plasmid DNA and 10-20 μ l liposome join 500 μ l Opti-MEM-I+GlutaMAX-I (Gibco company product) respectively and optimize in the transfection liquid, soft mixing, incubated at room, with the plasmid DNA and the soft mixing of liposome of dilution, incubated at room dropwise added mixed solution in the PT67 Tissue Culture Dish after 20 minutes more then.Cell is at 37 ℃, 5%CO ₂After cultivating 4-6 hour under the saturated humidity condition, transfection liquid changes the new DMEM nutrient solution that contains 15%FBS into.After transfection in 24 hours, collect PT67 cell culture fluid supernatant, just comprise infectious retroviral particle in this supernatant.Supernatant liquor is through the membrane filtration in 0.45 μ m aperture.Every 24h collects once, collects 3d continuously.Viral suspension under 4 ℃ of conditions, through 25,000 rev/mins, centrifugal 90 minutes, 10 times of concentrating virus supernatant liquors.To include three kinds of gene viruses supernatant liquors of Oct4, Sox2 and Klf4 and carry out balanced mix, and add 8 μ g/mlPolybrene and form specific three factor genes combination, transfection ox-hide skin inoblast is prepared in-80 ℃ of preservations.

The acquisition of the iPS cell of embodiment 4 retroviral infection ox-hide skin inoblasts and ox and going down to posterity

Grow up ear edge skin flbroblast (BDFs) and the new-born calve skin flbroblast (NDFs) of cow, two kinds of cells are all preceding 1 day of virus infection, 2 * 10 ⁵Individual cell inoculation is in the 60mm culture dish.Cultivate after 24 hours, general cell has 70-80% and merges, and the retrovirus supernatant mixed solution that will contain Oct4, Sox2 and three genes of Klf4 adds respectively in inoblast BDFs and the NDFs culture dish and infects inoblast; Cell goes down to posterity by 1: 3 routine after covering with culture dish, visual cell's growing state at 6-10d with cell with 1 * 10 ⁵The density of cells/well is transferred to and is covered with in advance on people's amnion (HAM) 6 well culture plates, replaces MEF to cultivate metainfective ox cell as feeder layer with HAM, and nutrient solution is replaced with the stem cell nutrient solution simultaneously; Measure and change liquid every day half in the culture hole, observes 20-28d continuously, up to there being ES cell sample colony 0 generation to occur being designated as, is ox iPS cell.

When ox iPSCs goes down to posterity, use the digestion of 0.05%trypsin+0.02%EDTA Digestive system, through neutralization, centrifugal and resuspended being passaged to again on the new HAM upholder, at 37-38 ℃, 5% left and right sides CO ₂, amplification cultivation under the saturated humidity condition obtains ox iPS cell strain.Ox iPSCs went down to posterity once every 3-5 days, went down to posterity at present 120 days, surpassed for 25 generations.Liquid nitrogen cryopreservation, the back growth vigor that thaws is better, nearly 85%-90% survival rate.

The stem cell nutrient solution consists of: contain in every 100ml solution: the knockout-DMEM of 82.69ml, 15ml FBS, 1ml concentration are that 200mM glutamine, 1ml concentration are that 10mM non-essential amino acid, 200 μ l concentration are that 55mM beta-mercaptoethanol, 10 μ l concentration are that 400ng/mL people's recombination basic fibroblast somatomedin (bFGF), 100 μ l concentration are 10 ⁵U/ml leukaemia inhibitory factor (LIF).

The evaluation of the ox iPS clone that test example 1 is set up

The ox inductive pluripotent stem cells of setting up in order to identify among the present invention (bovine induced pluripotentstem cells, iPSCs) with traditional sense on embryonic stem cell have very similarly biological characteristics, the contriver identifies the iPS cell of the ox that the present invention set up in a plurality of different aspect designs

1. as target cell milk cow skin inoblast form, see Fig. 1-2.

2.PT67 the retroviral particle form after packing cell and the packing is seen Fig. 3-4.

3.iPSCs colony form and AP stained positive (redness) are identified, are seen Fig. 5-6.

4.iPSCs the immunofluorescence cell chemical staining of colony is identified, is seen Fig. 7-10.

5. the vitro differentiation that is formed at of embryoid is identified.

Utilize the external suspension culture of ox iPSCs just can form after 7 days a plurality of spheries and blister cavities sample embryoid (EmbryoidBody, EB) structure is seen Figure 11, wherein A is shown as spheric embryoid structure, what B showed is blister cavities sample embryoid structure.

The embryoid (EB) that utilizes ox iPSCs to form passed through adherent culture 14-21 days again, and made its directed differentiation by the method for adding inducing culture, formed the different cellular fories of representing 3 germinal layers.3 germinal layers are represented clone, see that Figure 12-14 shows.

6. the differentiation of teratoma formation and three germinal layer cells

With ox iPSCs (about 1.3 * 10 ⁶Cell) it is subcutaneous to be expelled to nude mice, through the party of 6-9 time-of-week in the nude mice injection site subcutaneous formation tumour, be teratoma.Teratoma is prepared into tissue slice, through H.E. dyeing back microscopic examination, can observe three germinal layer cellular fories and exist simultaneously in the teratoma, shown in observations following Figure 15-17 (arrow), illustrates that ox iPSCs has the totipotency of growth.In addition, to the total RNA of teratoma tissue extraction, use the representative gene primer of three germinal layers and carry out the RT-PCR detection, the result has also shown the existence of three germinal layer cells, illustrates that also the ox iPSCs that is set up has the growth versatility.On behalf of the Gene RT-PCR detected result, three germinal layer cells of embryoid see Figure 18, and swimming lane 1 among the figure: β-Actin is confidential reference items; Swimming lane 2:Nestin, swimming lane 3: β-tubulin (ectoderm); Swimming lane 4:GATA4, swimming lane 5:Actinalpha 2 (mesoderm); Swimming lane 6:Keratin-14, swimming lane 7:PDX-1, swimming lane 8:AFP (entoderm), the primer sees Table 2.

Table 2. detects noble cells and represents the gene PCR the primer

Test example 2 utilizes ox iPSCs to set up and " ox-goat " mosaic

58 days Sas of application gestation can be done the acceptor sheep by milk goat, adopt laparotomy ventrotomy to touch fetus through Uterus wall, wherein a strain ox iPSCs (about 1.1x10 ⁶Individual cell) is expelled in the fetus abdominal cavity.Behind the goat full-term pregnancy, common property is given birth to 4 lambs, and wherein 1 male lamb whole body fur finds that big area is chimeric, is judged to be ox-sheep mosaic from the body surface feature, and regrettably this lamb was born back 2 hours because cardiorespiratory failure and death.Corpse is not found the organs abnormality phenomenon through after cuing open inspection.Goat fetus abdominal injection ox iPS cell is made the mosaic goat, and there is the black decorative pattern mosaic lamb fur many places of giving birth to, and these decorative patterns and Holstein milk cow are closely similar.This chimeric lamb has irregular sheet and streak black fur at a plurality of positions of body surface, comprising chest, back, lip, nose, eye face, auricle, four hoof, eyeball, tail point, positions such as scrotum.The D-LOOP hypervariable region sequences Design primer of using the Mitochondrial DNA of ox carries out Mitochondrial DNA to the mosaic goat and detects, and chimeric have the tissue of ox iPS cell chimerism to comprise: a plurality of tissues such as the heart, liver, spleen, lung, kidney, blood, blood vessel, testis.But pancreas, small intestine and osseous tissue do not detect chimerism.These test-results confirm that this lamb is a cattle and sheep cytochimera goat.Inject ox-hide skin inoblast (BDFs) and (NDFs) contrast tire sheep simultaneously and the chimerism of ox cell do not occur.

Among PCR product gel electrophoresis Figure 19, the 1-30 swimming lane is represented each histoorgan of mosaic goat: 1 heart, 2 blood vessels, 3 visceral pericardium, 4 lymphoglandula, 5 blood, 6 livers, 7 pancreas (-), 8 spleens, 9 oesophaguses, 10 stomaches, 11 small intestines (-), 12 large intestines, the 13BDF cell, 14BDF-iPSO cell, 15 acceptor fetus mothers (-), 16 brains, 17 spinal cords, 18 corneas, 19 lungs, 20 tracheaes, 21 kidneys, 22 testis, 23 casting skins, 24 pale skins, 25 skeletal muscle, 26 cartilages (-), 27 unstriated muscle, the 28BDF cell, 29BDF-iPSO cell, 30 acceptor fetus mothers (-).

＜110〉Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology

＜120〉utilize the transcription factor transfecting bovine somatic cells to become the method for inductive pluripotent stem cells

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ATGGCGGGACACCTCGCTTCTGACTTCGCCTTCTCGCCCCCGCCGGGCGGTGGAGGCGAT 60

GGGCCGGGAGGGCCAGAGCCGGGCTGGGTTGATCCTCGGACCTGGATGAGCTTCCAAGGG 120

CCTCCCGGTGGGTCGGGGATCGGGCCGGGGGTTGTGCCTGGCGCCGAGGTGTGGGGGCTT 180

CCCCCGTGCCCCCCGCCCTATGACTTGTGTGGAGGGATGGCCTACTGTGCGCCGCAGGTT 240

GGAGTGGGGCCGGTGCCCCCAGGCGGCCTGGAGACCCCTCAGCCCGAGGGCGAGGCGGGA 300

GCCGGGGTGGAGAGCAACTCCGAGGGGGCCTCCCCGGACCCCTGCGCCGCACCCGCAGGC 360

GCCCCGAAACTGGACAAGGAGAAGCTGGAGCCGAACCCTGAGGAGTCCCAGGACATCAAA 420

GCTCTTCAGAAAGACCTTGAACAATTTGCCAAGCTCCTAAAGCAGAAGAGGATCACACTA 480

GGATATACCCAGGCCGATGTGGGGCTCACCCTGGGGGTTCTCTTTGGAAAGGTGTTCAGC 540

CAAACGACTATCTGCCGTTTTGAGGCTTTGCAGCTCAGTTTCAAGAACATGCGTAAGCTG 600

CGGCCCCTGCTGCAGAAGTGGGTGGAGGAAGCTGACAACAACGAGAATCTGCAGGAGATA 660

TGCAAGGCAGAGACCCTTGTGCAGGCCCGAAAGAGAAAGCGGACGAGTATCGAGAACCGA 720

GTGAGAGGCAACCTGGAGAGCATGTTCCTGCAGTGCCCGAAGCCCACCCTGCAGCAAATT 780

AGCCACATCGCCCAGCAGCTCGGGCTGGAGAAAGACGTGGTCCGAGTGTGGTTTTGCAAC 840

CGTCGCCAGAAGGGCAAACGATCAAGCAGTGACTACTCCCAACGTGAGGATTTTGAGGCT 900

GCTGGGTCTCCTTTCGCAGGGGGACCCGTATCCTTTCCTCTGGCGCCGGGGCCCCATTTT 960

GGTACCCCAGGCTACGGGGGCCCTCACTTCACTACTCTGTACTCTTCGGTCCCATTCCCT 1020

GAGGGTGAGGCCTTTCCCTCGGTGTCTGTCACCGCTCTGGGCTCCCCTATGCATGCAAAC 1080

TGA 1083

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CCGGACCGAGTCAAGCGGCCCATGAACGCCTTCATGGTGTGGTCCCGCGGGCAGCGGCGC 180

AAGATGGCCCAAGAGAACCCTAAGATGCACAACTCGGAGATCAGCAAGCGCTTGGGCGCC 240

GAGTGGAAACTTTTGTCCGAGACGGAGAAGCGGCCGTTCATCGACGAGGCCAAGCGGCTG 300

CGAGCGCTGCACATGAAGGAACACCCGGATTATAAATACCGGCCCCGGCGGAAAACCAAG 360

ACGCTCATGAAGAAGGATAAGTACACACTGCCGGGAGGGCTGCTCGCCCCGGGCGGCAAC 420

AGCATGGCGAGCGGGGTCGGGGTGGGCGCCGGCCTCGGCGCGGGCGTGAACCAACGCATG 480

GACAGCTACGCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGCAGGACCAG 540

CTGGGCTACCCGCAACACCCGGGCCTCAACGCGCACGGCGCCGCTCAGATGCAGCCCATG 600

CACCGCTACGACGTGAGCGCCCTGCAGTACAACTCTATGACCAGCTCGCAGACCTACATG 660

AACGGCTCGCCCACCTACAGCATGTCCTATTCTCAGCAGGGCACCCCTGGCATGGCGCTT 720

GGCTCCATGGGCTCGGTGGTGAAGTCCGAGGCCAGCTCCAGCCCCCCCGTGGTTACCTCT 780

TCTTCCCACTCCAGGGCGCCCTGCCAAGCCGGGGACCTCCGGGACATGATCAGCATGTAC 840

CTCCCCGGCGCCGAGGTGCCGGAGCCCGCCGCCCCCAGCAGACTTCACATGTCCCAGCAC 900

TACCAGAGCGGCCCGGTGCCCGGCACGGCCATTAACGGCACACTGCCCCTCTCGCACATG 960

TGA 963

<210>3

<211>1434bp

<212>DNA

<213>KLF4

<400>3

ATGGCTGTCAGCGACGCGCTGCTCCCGTCCTTCTCCACGTTCGCGTCCGGCCCGGCGGGA 60

AGGGAGAAGACACTGCGTCCAGCAGGTGCCCCAAATAACCGCTGGCGGGAGGAGCTCTCC 120

CACATGAAGCGACTTCCCCCGGTGCTTCCCGGCCGCCCCTACGACCTGGCGGCGGCGACC 180

GTGGCCACCGACCTGGAGAGTGGAGGAGTCGGCGCGGCTTGCGGCAGCAGCAACCCGGCT 240

CTCCTGCCCCGGAGGGAGACGGAGGAGTTCAATGATCTCCTGGACCTGGACTTTATCCTC 300

TCCAACTCGCTGTCCCATCAGGAGTCCGTGGCCGCCACGGTGTCCTCGTCGGCATCAGCC 360

TCATCCTCGTCCTCCCCGTCGAGCAGCGGTCCAGCCAGTGCGCCTTCCACCTGCAGCTTC 420

AGCTATCCAATCCGGGCCGGGGGCGACCCGGGCGTGGCGGCGCCGGGCGGCGCGGGCGGC 480

GGCCTCCTGTACGGCCGGGAGTCTGCACCGCCTCCGACGGCTCCCTTCAACCTGGCGGAC 540

ATCAACGATGTGAGCCCCTCCGGCGGCTTCGTGGCCGAGCTCCTGCGGCCTGAATTGGAC 600

CCAGTGTACATTCCGCCGCAGCAGCCGCAGCCGCCAGGTGGCGGGCTGATGGGCGAGTTC 660

GTGTTGAAGGCGTCGCCGAGTGCCCCTGGCAGCGAGTACGGCAGCCCGTCGGTCATCAGT 720

GTTAGCAAAGGCAGCCCGGACGGCAGCCACCCGGTGGTGGTGGCGCCCTACAGCGGCGGG 780

CCGCCACGCATGTGCCCCAAGATCAAGCAGGAGGCTGTCTCCTCGTGCACCGTCGGTCGG 840

CCCCTAGAGGCCCACTTGGGCACTGGACCTCCTCTCAGCAATGGCCACCGGCCGCCTGCT 900

CACGACTTTCCCTTGGGGCGGCAGCTCCCCAGCAGGACTACCCCGACCCTGGGTGCCGAG 960

GAACTGCTGAGCAGCCGGGACTGTCATCCTGCCCTGCCGCTCCCCCCGGGCTTCCATCCC 1020

CACCCCGGGCCCAACTACCCTCCCTTCCTGCCCGACCAGATGCAGCCGCAGGTCCCACCG 1080

CTCCATTACCAAGAGCTCATGCCACCCGGTTCCTGCATGCCGGAGGAGCCCAAACCAAAG 1140

AGGGGAAGACGGTCGTGGCCCCGGAAAAGGACGGCCACTCACACTTGTGATTATGCAGGC 1200

TGCGGCAAAACCTACACGAAGAGTTCTCATCTCAAGGCACACCTGCGCACCCACACAGGT 1260

GAGAAACCTTACCACTGTGACTGGGATGGTTGTGGGTGGAAGTTTGCCCGCTCAGATGAA 1320

CTGACCAGGCACTACCGCAAACACACCGGGCACCGCCCCTTCCAGTGCCAGAAGTGCGAC 1380

CGGGCATTCTCGAGGTCGGACCACCTCGCCTTACACATGAAGAGGCACTTTTAA 1434

Claims (7)

1. utilize the transcription factor transfecting bovine somatic cells to become the method for inductive pluripotent stem cells, it is characterized in that, comprise the steps:

1) fibroblastic separation of ox-hide skin and cultivation

Ox-hide skin inoblast BDFs and new born bovine skin flbroblast NDFs obtain a large amount of adult ox-hide skin inoblast and new born bovine skin flbroblast respectively through the routine cultivation to grow up;

2) ox is kept the clone and the Expressed by Retrovirus Vector thereof of versatility gene transcription factor

From 50-60 age in days ox fetus, separate sex-ridge, extract total RNA, obtain cDNA through reverse transcription reaction; With cDNA is template, obtain ox Oct4, Sox2 and three gene amplification products of Klf4 and correctly then goal gene is inserted among the retroviral vector pMSCVneo through PCR reaction, make up the retrovirus expression vector of these 3 kinds of genes through the sequence verification sequence;

3) retroviral packing and preparation

The PT67 packing cell is cultivated in containing the DMEM substratum of 15% foetal calf serum according to ordinary method, used liposome the retrovirus transfection that makes up is entered packing cell PT67 cell; 8 μ g are contained Oct4, Sox2 and three heterogeneic retrovirus expression vector plasmids of Klf4 and 3 part of 20 μ l transfection reagent liposome lipofectamine2000 to be diluted with 500 μ l Opti-MEM-I+GlutaMAX-I transfections optimization liquid respectively, mixing gently, hatched under the room temperature 5 minutes, the diluent that will contain liposome lipofectamine2000 then slowly splashes in the diluent that contains retroviral plasmid, mixing gently, after hatching 20 minutes again under the room temperature, then above-mentioned mixed solution is dropwise added in the PT67 packing born of the same parents culture dish; After the transfection 24 hours, every day, replacing contained the virion nutrient solution once, for three days on end, collected viral suspension, under 4 ℃ of conditions through 25000 rev/mins after centrifugal 90 minutes, 10 times of concentrating virus supernatant liquors; To contain three gene viruses supernatant liquors of Oct4, Sox2 and Klf4 and carry out balanced mix, and add the specific three factor genes combination of 8 μ g/ml Polybrene composition ,-80 ℃ of preservations are standby;

4) acquisition of the iPS cell of retroviral infection ox-hide skin inoblast and ox

To grow up ox-hide skin inoblast BDFs and newborn bull skin flbroblast NDFs cultivates in the 60mm plastic culture dish according to ordinary method, treats in the culture dish that cell grows into the about 5ml of retrovirus supernatant liquor mixed solution that will comprise Oct4, Sox2 and Klf4 gene transcription factor when 70-80% merges and joins respectively in inoblast BDFs and the NDFs culture dish and infect inoblast; Cell goes down to posterity by the 1:3 routine after covering with culture dish, visual cell's growing state at 6-10d with cell with 1 * 10 ⁵The density of cells/well is transferred on 6 well culture plates that are covered with people's amnion (HAM) in advance, cultivates metainfective ox cell as feeder layer, and nutrient solution is replaced with special stem cell nutrient solution simultaneously; Measure and change liquid every day half in the culture hole, observes 20-28d continuously, till having cell colony to occur, was designated as for 0 generation, is ox iPSCs cell.

2. method according to claim 1, it is characterized in that: using preceding 1 hour of liposome packaging virus particle, change the nutrient solution DMEM substratum of packing cell PT67 into serum-free medium, 4-8 μ g includes the retroviral plasmid DNA of transcription factor gene and 10-20 μ l liposome and joins two part of 500 μ l Opti-MEM-I+GlutaMAX-I respectively and optimize in the transfection liquid, soft mixing, incubated at room, then with the plasmid DNA and the soft mixing of liposome that dilute, incubated at room dropwise added the plasmid-lipidosome mixed solution in the PT67 Tissue Culture Dish after 20 minutes again.

3. method according to claim 1 is characterized in that: the consisting of of described stem cell nutrient solution: contain in every 100ml solution: the knockout-DMEM of 82.69ml, 15ml FBS, 1ml concentration are that 200mM glutamine, 1ml concentration are that 10mM non-essential amino acid, 200 μ l concentration are that 55mM beta-mercaptoethanol, 10 μ l concentration are that 400ng/ml people's recombination basic fibroblast somatomedin, 100 μ l concentration are 10 ⁵The U/ml leukaemia inhibitory factor.

4. the recombinant retroviral expression vector pMSCV-Oct4 of the described Oct4 gene of claim 1.

5. the recombinant retroviral expression vector pMSCV-Sox2 of the described Sox2 gene of claim 1.

6. the application of the described recombinant retroviral expression vector pMSCV-Oct4 of claim 4 in the gene genetically engineered.

7. the application of the described recombinant retroviral expression vector pMSCV-Sox2 of claim 5 in the gene genetically engineered.

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