CN101705247B - Method for transfecting bovine somatic cells into inducted pluripotent stem cells by adopting transcription factors - Google Patents

Abstract

The invention discloses a method for transfecting bovine somatic cells into inducted pluripotent stem cells (iPSCs) by adopting transcription factors, concretely comprising the following steps: 1) isolation and culture of bovine dermal fibroblasts; 2) cloning of bovine transcription factor genes maintaining pluripotency and construction of retroviral vectors of the transcription factor genes; 3) retrovirus packaging and preparation; and 4) infection of bovine dermal fibroblasts by retrovirus and acquisition of bovine inducted pluripotent stem cells. The invention first adopts the acellular human amniotic membranes as support to replace the mouse embryonic fibroblasts (MEFs) to maintain in vitro proliferation of the bovine iPSCs and keep the bovine iPSCs in undifferentiated pluripotent states for long time. The method which adopts gene inducing to generate the PSCs not only solves the technical problem that the PSCs of the animals such as cattle, sheep, pigs and other livestock are difficult to obtain but also avoids the problem of heterogeneous animal cell pollution caused by the feeder layer which applies the MEFs as the 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 animal varieties, transcription factor kind and quantity, transcription factor array mode and foreign gene introduction method or the like many aspects.Yamanaka in 2007 ^[2]Utilize same transcription factor successfully to obtain human iPSCs again; The same year, Yu (Yu Junying) and Thomson ^[3]Utilize Deng the people that (Nanog Lin28) has also obtained human iPS cell for Oct3/4, Sox2 with other four kinds of transcription factors.Rhesus monkey is arranged again subsequently ^[4], rat ^[5], pig ^[6]Report (Li et al., 2009 Deng animal iPSCs achieving success; 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 MEC (MEF) provides multiple growth factor 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 MEC (MEF) as culture layer, just can keep ESCs and be in undifferentiated state.In the ESCs culture system, use MEC, directly introduced the heterogenous animal cell, the result has directly influenced the clinical application of 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 explain that then domestic animal will obtain embryonic stem cell from the body early embryo material sepd and also exist many technical and theoretic restraining factors to the livestock embryo stem cell.Therefore; Utilize Principles of Gene Engineering; The versatility key gene is imported somatocyte, and making its reprogrammed is the stem cell with versatility, and having solved not only that stem cell builds is technical barrier; Having enriched that reprogramming of somatic cells and stem cell build is theory, and to build for other stem cell animals be that theoretical foundation and experiment basis are provided.

Reference

[1]Takahashi，K.，and?Yamanaka，S.(2006).Induction?of?pluripotent?stemcells?from?mouse?embryonic?and?adult?fibroblast?cultures?by?defined?factors.Cell126，663-676.

[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).Induced?pluripotent?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?StemCells?from?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).Challenges?and?prospects?for?the?establishment?of?embryonic?stem?cell?lines?ofdomesticated?ungulates.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?linesderived?from?human?blastocysts.Science?282，1145-1147.

Summary of the invention

Building to the domestic animal stem cell is that a middle above-mentioned prior art difficult problem that exists is with not enough; The object of the present invention is to provide the method for a kind of acquisition inductive pluripotent stem cells (iPSCs); With this method obtain cell at cell colony form, growth characteristics, stem cell surface mark, express dryness gene, 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 a kind of method of utilizing the transcription factor transfecting bovine somatic cells to become 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 SF (NDFs) obtain a large amount of adult ox-hide skin inoblast and new born bovine SF respectively through conventional the cultivation to grow up;

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

From 50-60 age in days ox fetus, separate sex-ridge, extract total RNA, obtain cDNA through reverse transcription reaction; With cDNA is template; Reaction obtains ox Oct4, Sox2, Nanog and four gene amplification products of Lin28 and correctly then goal gene is inserted among the retroviral vector pMSCVneo through the sequence verification sequence through PCR, makes up the retrovirus expression vector of these 4 kinds of genes;

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 vector transfection that makes up is got into packing cell PT67 cell; Using preceding 1 hour of liposome packaging virus particle; The nutrient solution DMEM substratum of packing cell PT67 changes serum-free medium into; Retroviral plasmid DNA and the 10-20 μ l liposome that 4-8 μ g is included transcription factor gene joins 500 μ l Opti-MEM-I+GlutaMAX-I transfections respectively to be optimized liquid and dilutes, and mixing was hatched under the room temperature 5 minutes gently; The diluent that will contain liposome lipofectamine2000 then slowly splashes in the diluent that contains retroviral plasmid; Mixing gently, hatch 20 minutes again under the room temperature after, above-mentioned mixed solution is dropwise added in the PT67 packing born of the same parents petridish; After the transfection 24 hours, change every day contain virion nutrient solution once, for three days on end, collect viral suspension, under 4 ℃ of conditions through 25000 rev/mins, after centrifugal 90 minutes, 10 times of concentrating virus supernatants; To include Oct4, Sox2, Nanog and Lin28 gene viruses supernatant carry out balanced mix, and add the combination of 8 μ g/mlPolybrene composition special genes, and-80 ℃ of preservations are subsequent use;

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 SF NDFs cultivates in the 60mm plastic culture dish according to ordinary method, treats in the petridish that cell grows into the retrovirus supernatant that will comprise Oct4, Sox2, Nanog and Lin28 gene transcription factor when 70-80% merges and joins respectively in inoblast BDFs and the NDFs petridish and infect inoblast; Cell goes down to posterity by the 1:3 routine after covering with petridish, visual cell's growing state 6-10 days 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, and the personnel selection amnion replaces MEF to cultivate 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, observed continuously 20-28 days, till having cell colony to occur, was designated as for 0 generation, is ox iPSCs.

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 Stimulina, 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 growth factor (bFGF), 100 μ l concentration are 10 ⁵U/ml LIF (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 is every to go down to posterity once at a distance from 3-5 days.Liquid nitrogen cryopreservation, the back growth vigor that thaws is better, and the 85%-90% survival rate is arranged approximately.

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

A further object of the invention provides the recombinant retroviral expression vector pMSCV-Lin28 of Lin28 gene and the application in genetically engineered.

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

1, among the present invention 4 of application autonomous clened cows kinds keep stem cell versatility key gene; Utilize the several genes array mode; Lead like ox-hide skin inoblast with the stem cell retrovirus-mediated method, make its reprogrammed for having the multipotent stem cells of embryonic stem cell biological nature fully.Many strains multi-functional stem cell of setting up through this method surpassed for 25 generations external having gone down to posterity, and growth characteristics are all acted normally.Frozen and thawing test shows that they still have good versatility.

2, the present invention adopts first in culture condition and takes off cell people amnion (HAM) and replace MEC (MEF) to keep ox iPSCs in-vitro multiplication as upholder and keep undifferentiated versatility state for a long time, and has avoided using the heterogenous animal cell contamination problem that feeder layer brought of rat embryo fibroblast cell as stem cell.

3, the present invention adopts the method for gene induced generation multipotent stem cells, has 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 SF (BDFs) 50 * microscope figure that grows up.

Newborn bull SF (NDFs) 50 * microscope of Fig. 2 figure.

Fig. 3 PT67 packing cell 50 * microscope figure.

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

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

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

Fig. 7 TRA-1-60 bright field microscope figure.

Fig. 8 TRA-1-60 dyeing (+++) microscope figure.

Fig. 9 Nanog bright field microscope figure.

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

Figure 11 blister cavities appearance 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-FP that the endoderm cell is (the stained positive microscope figure of α-fetoprotein).

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

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

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

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

The PCR detected result gel electrophoresis figure of each tissue of Figure 19 mosaic goat.

Embodiment

Below the acquisition ox iPSCs that provides through the applicant concrete grammar and all have and the embryonic stem cell similar biological aspect many in colony form, growth characteristics, stem cell surface sign, inside and outside differentiation capability and mosaic formation etc. through multiple evidence ox iPSCs.

Embodiment fibroblastic separation of 1 ox-hide skin and cultivation

Growing up, (female bovine dermal fibroblasts BDFs) separates from female Holstein milk the ears of an ox or cow edge skin 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 products) that contains 15%-20% foetal calf serum (FBS) (Gibco Company products).Changed in every 2-3 days and support liquid 1 time, just have inoblast to shift out from the tissue block edge after 7-10 days, a large amount of inoblasts closely are arranged in around the tissue block after 12-16 days.With trypsinase and EDTA mixture slaking liquid had digestive transfer culture, promptly obtain a large amount of adult ox-hide skin cells, see Fig. 1.(male newborn bovine dermal fibroblasts NDFs) from the newborn bull ear of Holstein kind edge skin, sees Fig. 2 to newborn bull SF, and separation is identical with above-mentioned cow SF 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 (Holstein) kind milk cow, 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 open frame (ORF) complete sequence of reading of pcr amplification Oct4, Sox2, Nanog and four kinds of transcription factor genes of Lin28, gene clone the primer sequence is seen shown in the table 1 respectively.

Table 1 amplification ox Oct4, Sox2, Nanog and four gene RT-PCR of Lin28 amplimer information

Wherein Oct4 gene PCR amplification parameter is: 94 ℃ of preparatory 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 preparatory 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.

Nanog gene amplification parameter is: 94 ℃ of preparatory sex change 4min, and 94 ℃ become 35s, 58 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 35 circulations, 72 ℃ are extended 10min again, and expection PCR product length should be 903bp.

Lin28 gene amplification parameter is 94 ℃ of preparatory sex change 4min, 94 ℃ of sex change 30s, and 56 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 30 circulations, 72 ℃ are extended 10min again, and expection PCR product length should be 618bp.

Get each 5 μ l of above-mentioned PCR product, to identify its size, remain ℃ preservation of PCR product-20 at 1% agarose gel electrophoresis.Electrophoresis detection result shows that 4 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 4 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.Through the LB that contains Ampicillin (80 μ g/ml), X-gal and IPTG dull and stereotyped last 37 ℃ of screening and culturing 14-16 hours.Picking 6-10 white colony inoculated and shaken bacterium in the LB nutrient solution that contains penbritin (Ampicillin) in a small amount respectively from 4 kinds of cultures, and according to plasmid extraction test kit specification sheets step extracting trace plasmid.Comprise Oct4, Sox2, Nanog, the plasmid of 4 kinds of genes such as Lin28 is identified through double digestion, single endonuclease digestion and plasmid PCR.Wherein 3 kinds of plasmids are through BglII+EcoRI double digestion and EcoRI single endonuclease digestion and plasmid PCR evaluation, to obtain pMD-18T-Oct4, pMD-18T-Sox2, the positive colony of three kinds of plasmids of pMD-18T-Lin28.Because the restriction endonuclease kind of introducing in the primer is different, the plasmid that comprises the Nanog gene is through HapI+Bgl II double digestion and Bgl II single endonuclease digestion and plasmid PCR evaluation, to obtain pMD-18T-Nanog plasmid positive colony.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 of DNA start 7.10 softwares and the GenBank.Analytical results shows, Oct4, and Sox2, Nanog and Lin28 gene order and reference sequences homology are respectively 99.4%, 99.6%, and 100% and 100%.

The pMD-18T-Oct4 that above-mentioned order-checking is correct; PMD-18T-Sox2; After reclaiming purifying, three kinds of plasmids of pMD-18T-Lin28 pass through Bgl II+EcoRI double digestion respectively with retrovirus pMSCVneo carrier; Plasmid pMD-18T-Nanog and retrovirus process BglII+HapI double digestion, purifying and the target gene fragment and the linearizing pMSCVneo carrier that reclaim after enzyme is cut.Retrovirus vector pMSCVneo after the linearizing is connected at 16 ℃ with DNALigation Kit Version 2.0 with the purpose fragment of recovery spends 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 4 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 and the sequencing analysis evaluation through the two enzymic digestion evaluations of BglII+EcoRI (perhaps BglII+HapI), plasmid pcr amplification.Four kinds of recombinant retrovirus plasmids can obtain the target gene fragment and the retroviral vector fragment of known dimensions through behind the double digestion, explain that our constructed recombinant retroviral expression vector structure is correct.Detect through order-checking; The sequence and the pcr amplification sequence of 4 goal gene that comprised in all recombinant retrovirus plasmids are in full accord; Also with GenBank in reference sequences in full accord; Be illustrated in this patent goal gene in constructed 4 kinds of recombinant retrovirus plasmid processes and do not read the situation such as frameshit and base mutation of frame, explain that applied 4 kinds of gene orders are entirely true.4 kinds of constructed in this patent recombinant retroviral expression vectors are distinguished called after: pMSCV-Oct4, pMSCV-Sox2, pMSCV-Nanog and pMSCV-Lin28.

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 transfection of 4 kinds of retroviral vectors difference is got into packing cell PT67 cell, carry out the virus packing to obtain to have infectious retroviral particle.Preceding 1 hour of transfection; The DMEM substratum changes serum-free medium into; 4-8 μ g retroviral plasmid DNA and 10-20 μ l liposome join 2 part of 500 μ l Opti-MEM-I+GlutaMAX-I (Gibco Company products) respectively optimizes in the transfection liquid soft mixing, incubated at room; Then with DNA and the soft mixing of liposome, the incubated at room again of diluting.After 20 minutes, mixed solution is dropwise added in the PT67 Tissue Culture Dish.Cell is at 37 ℃, 5%CO ₂With cultivate 4-6h under the saturated humidity condition.Afterwards, 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 is through the membrane filtration in 0.45 μ m aperture.Collected once, collected continuously 3 days in per 24 hours.Viral suspension under 4 ℃, through 25,000 rev/mins, centrifugal 90 minutes, 10 times of concentrating virus supernatants ,-80 ℃ of preservations.Oct4, Sox2, Nanog and four kinds of gene viruses supernatants of Lin28 are carried out balanced mix, and add 8 μ g/ml polybrene, prepare transfection ox-hide skin inoblast.

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

To grow up ox-hide skin inoblast (BDFs) and newborn bull SF (NDFs) cultivated in the 60mm plastic culture dish according to ordinary method, treats in the petridish that cell grows into the mixed solution that will comprise the retrovirus supernatant of Oct4, Sox2, Nanog and Lin28 gene transcription factor when 70-80% merges and joins respectively in inoblast BDFs and the NDFs petridish and infect inoblast; Cell goes down to posterity by 1: 3 routine after covering with petridish, visual cell's growing state 6-10 days with cell with 1 * 10 ⁵The density of cells/well is transferred to and is covered with in advance on people's amnion 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, observed continuously 20-28 days, till having ES cell appearance colony to occur, was designated as for 0 generation, is ox iPSCs.

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.Ox iPSCs is every to go down to posterity once at a distance from 3-5 days.Liquid nitrogen cryopreservation, the back growth vigor that thaws is better, nearly 85%-90% survival rate.

The evaluation of the ox iPSCs system that Test Example 1 is set up

Embryonic stem cell has very similarly biological characteristics on ox iPSCs that sets up in order to identify among the present invention and the traditional sense, and the contriver identifies the iPS cell of the ox that the present invention set up in a plurality of different aspect designs,

1. as the ox-hide skin inoblast form of target cell, 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 appearance embryoid (Embryoid Body, EB) structure is seen Figure 11, wherein A is shown as spheric embryoid structure, what B showed is blister cavities appearance embryoid structure.

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

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

It is subcutaneous that ox iPSCs is expelled to the nude mice buttocks, grows through the 6-9 time-of-week, then can be in the nude mice injection site subcutaneous formation tumour, be teratoma.Be prepared into tissue slice with the teratoma collection, after fixing, dyeing back microscopic examination can be observed 3 germinal layer cellular fories and exist in the teratoma simultaneously and cut into slices, and explains that ox iPSCs has the totipotency of growth.Can observing clearly through the teratoma tissue preparation section microscopically after the H.E. dyeing, three germinal layer cells exist simultaneously.Shown in observations following Figure 15-17 (arrow).In addition, to the total RNA of teratoma tissue extraction, use three germinal layer cells and represent gene primer to carry out the RT-PCR augmentation detection, the result has also shown the existence of three germinal layer cells, and the ox iPSCs that same explanation is set up has totipotency.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:Actin alpha 2 (mesoderm); Swimming lane 6:Keratin-14, swimming lane 7:PDX-1, swimming lane 8:AFP (entoderm), the primer is seen 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

Use 58 days Sas of gestation and can do the acceptor sheep by milk goat, adopt laparotomy ventrotomy to touch fetus, a strain ox iPSCs (about 1.1 * 10 through Uterus wall 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 characteristic, 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 the mosaic lamb fur of giving birth to has many places black decorative pattern, and these decorative patterns and Holstein milk cow are closely similar.This chimeric lamb has in flakes at a plurality of positions of body surface, the black fur of slivering, 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; There is 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; Pancreas, small intestine and bone do not detect chimeric, confirm that this lamb is a cattle and sheep cytochimera goat.Inject Niu Chengti skin cells (BDFs) and (NDFs) contrast sheep simultaneously and the chimerism of ox cell do not occur.

Among 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 Skelettmuskel, 26 cartilages (-), 27 unstriated muscle, 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

<160>4

<210>1

<211>1083bp

<212>DNA

<213>OCT4

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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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<213>Sox2

<400>2

ATGTACAACATGATGGAGACGGAGCTGAAGCCGCCGGGCCCGCAGCAAACTTCGGGGGGC 60

GGCGGCGGCGGCGGCGGCAACTCCACCGCGGCGGCGGCGGGCGGCAACCAGAAGAACAGC 120

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>903bp

<212>DNA

<213>NANOG

<400>3

ATGAGTGTGGGCCCAGCTTGTCCCCAAAGCCTGCTTGGCCCCGAAGCATCCAACTCTAGG 60

GAATCTTCACCCATGCCTGAAGAAAGTTACGTGTCCTTGCAAACGTCATCTGCTGACACC 120

CTCGACACGGACACTGTCTCTCCTCTTCCCTCCTCCATGGATCTGCTTATTCAGGACAGT 180

CCTGATTCTTCCACAAGCCCCAGAGTGAAACCACTGTCCCCGTCTGTGGAGGAGAGCACA 240

GAGAAGGAAGAGACGGTCCCGGTCAAGAAACAAAAGATTAGAACTGTGTTCTCGCAGACC 300

CAGCTGTGTGTGCTCAATGACAGATTTCAGAGGCAGAAATACCTCAGTCTCCAGCAAATG 360

CAAGAACTTTCCAACATCTTGAACCTCAGCTACAAGCAGGTGAAGACCTGGTTCCAGAAC 420

CAGAGAATGAAATGTAAGAAATGGCAGAAAAACAACTGGCCGAGGAATAGCAATGGCATG 480

CCTCAGGGGCCAGCAATGGCAGAATACCCAGGCTTCTATTCCTACCACCAGGGGTGTTTG 540

GTGAACTCTCCTGGAAACCTGCCCATGTGGGGTAACCAGACCTGGAATAACCCCACGTGG 600

AGCAACCAGAGCTGGAACAGTCAGTCTTGGAGCAACCACTCCTGGAACAGTCAGGCCTGG 660

TGCCCCCAAGCCTGGAATAACCAGCCTTGGAACAATCAGTTCAACAACTACATGGAGGAA 720

TTCCTGCAGCCCGGGATCCAGCTCCAGCAGAATTCTCCCGTCTGTGATCTGGAGGCCACC 780

CTGGGAACTGCTGGGGAAAATTATAACGTTATACAGCAAACTGTCAAGTATTTCAATTCC 840

CAGCAGCAAATCACTGATTTATTCCCAAACTACCCTCTCAACATACAGCCTGAAGATTTG 900

TAA 903

<210>4

<211>618bp

<212>DNA

<213>LIN28

<400>4

ATGGGCTCTGTGTCAAACCAGCAGTTCGCAGGTGGCTGCGCTAAGGCGCCGGAGGAGGCG 60

CCGGAGGACGCGGCCCGCGCGGCGGAGGAGCCGCAGCTGCTGCACGGTGCGGGCATCTGT 120

AAGTGGTTCAACGTGCGCATGGGGTTCGGCTTCCTGTCCATGACCGCCCGCGCAGGGGTC 180

GCGCTCGACCCCCCGGTGGATGTCTTTGTGCACCAGAGTAAGCTGCACATGGAGGGCTTC 240

CGGAGCCTGAAGGAGGGGGAGGCCGTGGAGTTCACCTTTAAGAAGTCCGCCAAAGGCCTG 300

GAATCTATCCGAGTCACCGGCCCTGGGGGGGTGTTCTGTATTGGGAGTGAAAGGCGGCCC 360

AAAGGGAAGAATATGCAGAAACGCAGATCAAAGGGAGACAGGTGCTACAACTGTGGAGGT 420

CTAGACCATCATGCCAAGGAGTGCAAACTGCCACCGCAGCCCAAGAAGTGCCATTTCTGC 480

CAGAGCATCAACCATATGGTAGCTTCGTGCCCACTGAAGGCCCAGCAAGCTCCCAGCTCC 540

CAGGGAAAGCCAGCCTACTTTCGGGAGGAGGAAGAAGAGATCCATAGCTCTGCCATGCTC 600

CCAGAGGCCCAGAATTGA 618

Claims (1)

1. a method of utilizing the transcription factor transfecting bovine somatic cells to become inductive pluripotent stem cells comprises the steps:

1) fibroblastic separation of ox-hide skin and cultivation

With ox-hide skin inoblast BDFs and the new born bovine SF NDFs of growing up, after conventional the cultivation, obtain a large amount of adult ox-hide skin cell and new born bovine SF respectively;

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

Origin comes from total RNA of 50-60 age in days ox fetus sex-ridge, obtains cDNA through reverse transcription reaction; With cDNA is template; Reaction obtains ox Oct4, Sox2, Nanog and four gene amplification products of Lin28 and correctly then goal gene is inserted among the retroviral vector pMSCVneo through the sequence verification sequence through PCR, makes up the retrovirus expression vector of these 4 kinds of genes;

3) retroviral packing and preparation

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 transfection of 4 kinds of retroviral vectors difference is got into packing cell PT67 cell, carry out the virus packing to obtain to have infectious retroviral particle; Preceding 1 hour of transfection; The DMEM substratum changes serum-free medium into; 4-8 μ g retroviral plasmid DNA and 10-20 μ l liposome join 2 part of 500 μ l Opti-MEM-I+GlutaMAX-I respectively and optimize in the transfection liquid soft mixing, incubated at room; Then with DNA and the soft mixing of liposome, the incubated at room again of diluting; After 20 minutes, mixed solution is dropwise added in the PT67 Tissue Culture Dish; Cell is at 37 ℃, 5%CO ₂With cultivate 4-6h under the saturated humidity condition, afterwards, 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 is through the membrane filtration in 0.45 μ m aperture; Collected once, collected continuously 3 days in per 24 hours; Viral suspension under 4 ℃, through 25,000 rev/mins, centrifugal 90 minutes, 10 times of concentrating virus supernatants ,-80 ℃ of preservations; Oct4, Sox2, Nanog and four kinds of gene viruses supernatants of Lin28 are carried out balanced mix, and add 8 μ g/ml polybrene, prepare transfection ox-hide skin inoblast;

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 SF NDFs cultivates in the 60mm plastic culture dish according to ordinary method, treats in the petridish that cell grows into the mixed solution that will comprise the retrovirus supernatant of Oct4, Sox2, Nanog and Lin28 gene transcription factor when 70-80% merges and joins respectively in inoblast BDFs and the NDFs petridish and infect inoblast; Cell goes down to posterity by 1: 3 routine after covering with petridish, visual cell's growing state 6-10 days with cell with 1 * 10 ⁵The density of cells/well is transferred to and is covered with in advance on people's amnion 6 well culture plates, and the personnel selection amnion replaces MEC MEF to cultivate 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, observed continuously 20-28 days, till having cell colony to occur, was designated as for 0 generation, is ox iPSCs;

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 Stimulina, 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 growth factor, 100 μ l concentration are 10 ⁵The U/ml LIF.

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