CN103667179A - Method for inducing fetal bovine fibroblast to reprogram - Google Patents
Method for inducing fetal bovine fibroblast to reprogram Download PDFInfo
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Abstract
The invention belongs to the field of cells, and discloses a method for inducing fetal bovine fibroblast to reprogram. According to the invention, the fetal bovine fibroblast is subject to permeabilized treatment by a digitonin solution, and is subject to co-incubation with xenopus oocytes extract; whole medium containing 2mMcaCl2 is adopted to block reversible pores in the cell membrane, extract treated medium is adopted to cultivate and induce the fetal bovine fibroblast to reprogram to obtain fetal bovine fibroblast pluripotent stem cells. The method discloses by the invention is efficient, stable, and safe; the alkaline phosphatase staining of the fetal bovine fibroblast pluripotent stem cells disclosed by the invention shows a positive result; the acetylation degree of histone H3K9 is lowered; Oct-4 protein and pluripotent marker gene Oct-4, Nanog are expressed; therefore the fetal bovine fibroblast undergoes reprogramming, the gene expression mode of cells is changed to show characteristics of stem cell, partial growing totipotent is restored, and the fetal bovine fibroblast can be used as nuclear donor cells to prepare clone embryo for body cell clone.
Description
Technical field
The invention belongs to cell field, be specifically related to a kind of method of inducing reprogramming of somatic cells, relate in particular to a kind of method of inducing bovine fetal fibroblast reprogrammed to become.
Background technology
Since ancient times, ox is exactly that the mankind like the domestic animal of raising.Ox of many uses: meat and breast can supply food, and skin is process hides raw material, and angle, bone are also industrial raw material, and ox horn also can be done medicinal material and artwork, the cow-bezoar in inner is the valuable ingredients of traditional Chinese medicine especially, and ox also can be agriculture production etc. labour power and fertilizer are provided.People expect to obtain always and have certain required characteristic or character cattle breeds for a long time, the beef breed of high meat yield for example, the dairy bread that high milk yield, lactation interval is long and disease resistance is strong etc.Traditional method for breeding the most so can be turned out the cattle breeds with some specific desired characteristic, but these characteristics are accompanied by some unwanted character conventionally, and traditional method for breeding is consuming time, costliness and genetic stability unreliable.
The Cloning Mammals from Somatic Cells developing history of existing nearly 20 years, it refers to by laboratory means such as the fusion of micrurgy stoning, nuclear donor cell, reconstructed embryo activation, the somatocyte of certain type and nuclear receptor (being generally mature oocyte) are carried out to external fusion reconstruct to form clone embryos, again clone's embryo is carried out to embryo transfer to the dam of replace-conceive, reach the mammiferous a kind of biotechnology of a large amount of production heredity homogeneity.Somatic cell clone technique has four large advantages: the one, and can sex control; The 2nd, cost is low; The 3rd, overcome the restriction introducing good strains of seeds abroad; The 4th, can update fast, expand numerous improved seeds.Somatic cell clone technique has important application prospect in the aquaculture of ox, and to accelerating, poultry kind is improved, raising dairy quality has important effect.Application somatic cell clone technique can be preserved some individual good character without restriction, and promptly expands the quantity of defect individual.Somatic cell clone technique is combined with transgenic technology, can greatly improve genetically modified efficiency, accelerates to realize the transformation of animal heredity.Somatic cell clone technique is combined with animal mammary gland reactor technology, can directly from dairy products, produce pharmaceutical protein or modified dairy quality.
Although somatic cell clone technique succeeds at many animals such as mouse, rat, ox, goat, pig, cat, horse, ferret, wolf, buffalos, but the efficiency of somatic cell clone is still very low, the growth of clone embryos expires rate less than 5%, cloned animal exists high abortion ratio and newborn fetus high mortality, only have only a few cloned animal healthy survival extremely to grow up, these are all seriously restricting widespread use and the development of clone technology.Think that at present causing the inefficient major cause of somatic cell clone is that well differentiated somatocyte reprogrammed in ovocyte matter is incomplete, epigenetic modification caused by abnormal.And the lower cell of differentiation degree seems stronger remoldability, the replacement that is easier to send out protedogenous.Therefore somatic cell clone is combined with reprogramming of somatic cells, inductor cellular-restoring is to lower differentiation state, and the cell that the differentiation degree of usining is lower carries out somatic cell clone to improve cloning efficiency as nuclear donor.
Reprogramming of somatic cells refers to that the somatocyte having broken up is reversed back original multipotency state under given conditions or transdifferentiation is other types cell.The method of induction reprogramming of somatic cells mainly contains body-cell neucleus transplanting, cytogamy, specific transcription factor transduction etc.
Body-cell neucleus transplanting refers to donorcells is moved in enucleation oocyte, in ovocyte matter, under various factor effects, dedifferentes recovery totipotency, forms embryo, develops into the process of new individuality after immigration acceptor.Within 1997, the world the first one-tenth somatic cell clone sheep Dolly is born, and has confirmed that well differentiated somatocyte has the ability of recovering totipotency and developing into new individuality, has opened the upsurge of body-cell neucleus transplanting research.Therefore, somatic cell nuclear transfer technique provides wide thinking for studying the molecular mechanism of reprogramming of somatic cells.Although body-cell neucleus transplanting succeeds multiple Mammals, its mechanism is still indefinite, and donor nuclei exists the phenomenons such as reprogrammed is incomplete, epigenetic modification is abnormal, and cloning efficiency is very low, even on ox, total efficiency is also lower than 10%; And this technology is also subject to the intervention of ethics and national legislation, seriously restricting the development of this technology.
Cytogamy refers under spontaneous or artificial induction, and two or more cells are merged into a double-core or coenocytic process.Experimental results show that in most of hybrid cells, the lower cell of differentiation degree is dominate often.1976, Miller and Ruddle confirmed that cytogamy can make reprogramming of somatic cells recover versatility first.They are by mouse chest cell and embryo cells hybridization, and the tetraploid of generation is expressed multipotency cell sign, hybrid cell is injected to nude mice and can form the teratoma that comprises 3 germinal layer tissues.Somatocyte has also obtained similar result to the heterozygosis that embryonic genital cell, embryonic stem cell carry out in recent years.Although cell-fusion techniques has been avoided the problems such as ovocyte source is difficult, genetic manipulation is complicated, the existence of polyploid causes heterozygote genomic instability, has potential risk; And the further growth of tetraploid state retardance cell, therefore after cytogamy, need to remove the karyomit(e) that derives from ES, EC, EG cell, but not yet find at present the unnecessary chromosomal method of effective removal, the application of cell-fusion techniques in reprogramming of somatic cells has larger limitation.
Yamanaka and Takahashi import l cell by 4 to the vital factor Oct4 of cell reprogrammed, Sox2, Klf4 and c-Myc, obtain the induction multipotency stem cell of embryonic stem cell-like character, there is powerful updating ability and differentiation potential, this landmark discovery, has opened the new page of specific transcription factor induction reprogramming of somatic cells research.Specific transcription factor induction reprogramming of somatic cells, has realized the direct transformation of cell fate, has abandoned the ethics morals problem that stem cell and ovocyte bring, and aspect cell therapy and regenerative medicine, is having broad application prospects.But cell induction efficiency is low at present, and faces the threat of bringing out cancer, is seriously restricting its application.Therefore, further probe into reprogrammed mechanism, the method for setting up efficient, stable, safe induction reprogramming of somatic cells is current scientific circles problem demanding prompt solutions.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of method of efficient, stable, safe induction bovine fetal fibroblast reprogrammed.
For realizing object of the present invention, the present invention adopts following technical scheme:
Induce a method for bovine fetal fibroblast reprogrammed, bovine fetal fibroblast is hatched altogether with xenopus leavis oocytes extract after digitonin solution is changed processing thoroughly, with containing 2mM CaCl
2full substratum closing cell film on reversible duct, then extract is processed culture medium culturing induction bovine fetal fibroblast reprogrammed and is obtained bovine fetal fibroblast multipotential stem cell.
Preferably, described bovine fetal fibroblast is obtained through full culture medium culturing after rinsing through 75% alcohol with in containing 1% dual anti-PBS by 40 age in days ox fetus trunk tissues in T25 culturing bottle.
Preferably, described digitonin strength of solution is 7 μ g/mL.
Preferably, describedization is treated under 0 ℃ of condition and processes 2min.
Preferably, described xenopus leavis oocytes extract preparation method is that xenopus oocyte goes egg jelly after high speed centrifugation fragmentation, to collect ovocyte crude extract by cell pyrolysis liquid cracking again.
Preferably, described hatching altogether as hatch together 1h under 23 ℃ of conditions.
Preferably, the time of described sealing is 2h.
Preferably, the time of described cultivation is 6-7d.
The bovine fetal fibroblast multipotential stem cell that the present invention also provides aforesaid method reprogrammed to obtain.
The invention provides a kind of method of inducing bovine fetal fibroblast reprogrammed, described method is that bovine fetal fibroblast is hatched altogether with xenopus leavis oocytes extract after digitonin solution is changed processing thoroughly, with containing 2mM CaCl
2full substratum closing cell film on reversible duct, then extract is processed culture medium culturing induction bovine fetal fibroblast reprogrammed and is obtained bovine fetal fibroblast multipotential stem cell.The method of the invention is efficient, stable, safety.The bovine fetal fibroblast multipotential stem cell alkaline phosphatase staining obtaining through the method for the invention induction reprogrammed is positive; histone H 3 K9 degree of acetylation reduces; there is the expression of Oct-4 albumen and versatility marker gene Oct-4, Nanog; show that reprogrammed has occurred bovine fetal fibroblast; cellular gene expression mode changes; performance stem cell characteristic, recuperation section totipotency, can be used as nuclear donor and prepares clone embryos and carry out somatic cell clone.
Accompanying drawing explanation
Fig. 1 shows bovine fetal fibroblast and the control cells (100 *) that embodiment 5 xenopus leavis oocytes extracts are processed, wherein, in figure A be in pending cell, figure B be in cell during extract is processed, figure C be in cell after extract is processed, figure D be after extract is processed 6d cell to form E in " clone bunch ", figure be that in the 6d extract unprocessed control cells of processing culture medium culturing, figure, F is the unprocessed control cells that 6d conventional medium is cultivated;
Fig. 2 shows bovine fetal fibroblast and the unprocessed control cells H3K9 immuning fluorescent dyeing analysis result figure that embodiment 6 xenopus leavis oocytes extracts are processed, wherein experimental group is the bovine fetal fibroblast that xenopus leavis oocytes extract is processed, and control group is unprocessed control cells;
Fig. 3 shows bovine fetal fibroblast and the unprocessed control cells alkaline phosphatase staining figure (100 *) that embodiment 7 xenopus leavis oocytes extracts are processed, wherein, in figure A be after extract is processed 6d cell form in clone bunch, figure B for C in clone's bunch alkaline phosphatase enzyme positive, figure be that untreatment control cell alkaline phosphatase is negative;
Fig. 4 shows bovine fetal fibroblast and the unprocessed control cells Oct4 immunofluorescence dyeing figure (100 *) that embodiment 8 xenopus leavis oocytes extracts are processed, wherein, in figure, A-C is that extract is processed after 6d " clone bunch " cell Oct4 immunofluorescence dyeing is positive, D-F does not form " clone bunch " Oct4 immunofluorescence dyeing to be negative after untreatment control cell 6d in figure; In figure, A and D are that in ordinary light source, figure, B and E are that in excitation wavelength 450-490nm, figure, C and F are excitation wavelength 510-560nm;
Fig. 5 shows the bovine fetal fibroblast of embodiment 9 xenopus leavis oocytes extracts processing and the Western Blot detection figure of control cells Oct4 expression amount, wherein, swimming lane A is the bovine fetal fibroblast that xenopus leavis oocytes extract is processed, and swimming lane B is unprocessed control cells;
Fig. 6 shows the bovine fetal fibroblast of embodiment 10 xenopus leavis oocytes extracts processing, the electrophorogram of processing the expression of versatility marker gene Oct4, Sox2, Nanog in rear conventional medium (DMEM+10%FBS) culturing cell with untreated cell (extract processing culture medium culturing) and the extract of same source, cultivation simultaneously, and GAPDH gene is reference gene; Wherein, swimming lane A is that extract is processed stem cell media cultivation, and swimming lane B is that extract is processed ordinary culture medium cultivation, and swimming lane C is that untreated stem cell media is cultivated, and swimming lane D is that untreated ordinary culture medium is cultivated.
Embodiment
The embodiment of the invention discloses a kind of method of inducing bovine fetal fibroblast reprogrammed.Those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, and related personnel obviously can change method as herein described or suitably change and combination within not departing from content of the present invention, spirit and scope, realizes and apply the technology of the present invention.
For realizing object of the present invention, the present invention adopts following technical scheme:
Induce a method for bovine fetal fibroblast reprogrammed, bovine fetal fibroblast is hatched altogether with xenopus leavis oocytes extract after digitonin solution is changed processing thoroughly, with containing 2mM CaCl
2full substratum closing cell film on reversible duct, then extract is processed culture medium culturing induction bovine fetal fibroblast reprogrammed and is obtained bovine fetal fibroblast multipotential stem cell.
Extract induction reprogramming of somatic cells is to adopt the cell extracts such as ovocyte, stem cell and somatocyte to hatch altogether, and inductor cell generation reprogrammed, changes gene expression pattern, causes that cell dedifferentes, recovery section distribution and totipotency.
Wherein, described bovine fetal fibroblast is obtained through full culture medium culturing after rinsing through 75% alcohol with in containing 1% dual anti-PBS by 40 age in days ox fetus trunk tissues in T25 culturing bottle.
Described 1% dual anti-PBS refers to the phosphate buffered containing the pH7.4 of 1v/v% penicillin and Streptomycin sulphate, each 0.5v/v% of each penicillin and Streptomycin sulphate, and penicillin final concentration is 100U/mL, Streptomycin sulphate final concentration is 100 μ g/mL.
Described full substratum is conventional medium, specifically refers to DMEM+10%FBS.
Being specially after rinsing rear torso tissue shear becomes to organize fragment and evenly spreading upon in T25 culturing bottle through full culture medium culturing in T25 culturing bottle of described bovine fetal fibroblast, careful upset culturing bottle, add full substratum, be inverted in incubator, after 6-8h, carefully turn over bottle, continue to cultivate, after 1-2d, observe and add full substratum, until cell, reach 80% while converging, go down to posterity or frozen.
In certain embodiments, described bovine fetal fibroblast is thoroughly changed processing and also cell is cleaned, and is specially when cell sucks cell culture medium in hole when 50%-60% converges and cleans rear exhaustion liquid twice with the PBS of precooling.
Digitonin, English name digitonin, is a kind of glycoside obtaining from the leaf of pale reddish brown foxglove and seed.Digitonin is a kind of nonionic washing agent, and the digitonin solution of lower concentration is assembled the cholesterol on cytolemma, can make cytolemma occur duct, and not destroy cellularstructure.The method of the invention is usingd digitonin solution and is processed bovine fetal fibroblast to improve the permeability of cell membrane of bovine fetal fibroblast as the saturating agent of cytolemma.Wherein, described digitonin solution is that digitonin is dissolved in the solution obtaining in PBS liquid.
The present invention is with the digitonin solution-treated bovine fetal fibroblast of different concns, filter out the optimal concentration of the saturating agent of cytolemma, result shows with 7 μ g/mL digitonin solution-treated bovine fetal fibroblasts, there is 55% saturatingization of cell generation, and cell state is good, through follow-up culturing cell mortality ratio, be less than 10%, cell permeabilization rate and mortality ratio are the highest.Therefore, described digitonin strength of solution is preferably 7 μ g/mL.
Preferably, describedization is treated under 0 ℃ of condition and processes 2min.In certain embodiments for acting on 2min on ice, and frequently jiggle, then suck fast digitonin solution, the PBS of precooling cleans 3-5 time.
The method of the invention is hatched xenopus leavis oocytes extract and the cell through saturatingization after digitonin solution is changed processing thoroughly altogether at bovine fetal fibroblast.
Wherein, described hatch to be altogether preferably under 23 ℃ of conditions hatch together 1h.
The preparation method of described xenopus leavis oocytes extract is that xenopus oocyte goes egg jelly after high speed centrifugation fragmentation, to collect ovocyte crude extract by cell pyrolysis liquid cracking again in certain embodiments.Described cell pyrolysis liquid refers to containing 2.5mM MgCl
2.6H
2o, 50mM KCl, 10mM Hepes(4-hydroxyethyl piperazine ethanesulfonic acid) and 250mM Sucrose(sucrose) the aqueous solution.
Preferably, before cell pyrolysis liquid cleavage step and the broken step of high speed centrifugation, add respectively proteinase inhibitor, to prevent proteolysis.Described proteinase inhibitor is preferably Trypsin inhibitor,Trasylol or leupeptin.
Trypsin inhibitor,Trasylol also claims trypsin inhibitor, in the internal organs such as ox parotid, ox pancreas or lung, extract and alkali polypeptide, be a kind of natural broad-spectrum protease inhibitor.Every 1 unit Trypsin inhibitor,Trasylol can make 0.8 μ g methyl trypsin inactivation.Trypsin inhibitor,Trasylol can suppress its activity with an activity centre lysyl of competition of multiple protein enzyme; can suppress cellulase, trypsinase, Chymotrypsin, kallikrein, zymoplasm and factor IV-XII, stop self activating of the activation of other activated protein proenzymes in pancreas and trypsinogen etc.
Leupeptin belongs to the trypsinase that rose streptomycete produces and presses down agent, and it is a kind of in many proteinase inhibitor.Leupeptin can suppress Serine and L-Cysteine HCL Anhydrous, trypsinase, papoid, organize egg enzyme B, plasminogen, kallikrein etc., and its restraining effect and substrate are competition and suppress.To the inhibition IC50 of plasminogen, be 8 μ g/mL, but do not suppress Chymotrypsin.
Preparation method at xenopus leavis oocytes extract described in some specific embodiments specifically comprises:
Step 1, xenopus oocyte separate collection, in beaker, are outwelled unnecessary water, with the useless ovum of plastic suction pipe sucking-off, retain clear-cut, the unified ovum of form;
Step 2, remove after unnecessary water and useless ovum, add freshly prepared 2% halfcystine, soft stirring removed egg jelly;
Step 3, outwell above-mentioned solution, 0.25 * MMR washes ovum three times, and 1 * MMR washes ovum once;
Once, this stage chooses except bad ovum with plastic suction pipe again for step 4, cell pyrolysis liquid rinsing, and cell pyrolysis liquid post rinse once;
Step 5, ovum is transferred in the cell pyrolysis liquid containing 1mM DTT and 100 μ L proteinase inhibitor, after stirring gently, outwell cell pyrolysis liquid and ovum is transferred in 15mL centrifuge tube, after ovum precipitation, with plastic suction pipe, removing centrifuge tube upper strata redundant solution;
Step 6, the centrifugal 10-15s of 800rpm, further remove centrifuge tube upper strata redundant solution;
Step 7, add Trypsin inhibitor,Trasylol or leupeptin to ovum top, 4 ℃, the centrifugal 15min of 10000rpm, centrifugal rear xenopus oocyte fragmentation, liquid present clearly demarcated three layers;
Step 8, thrust lower floor top draw crude extract with 2mL syringe, crude extract is transferred in the centrifuge tube of a precooling and is added glycerine, and 4 ℃ mix, packing, liquid nitrogen freezing, be stored in-80 ℃ standby.
First the preparation method of above-mentioned xenopus leavis oocytes extract collects clear-cut, the unified ovum of form adds the soft stirring of film damping fluid 2% halfcystine that removes photoresist to remove egg jelly, the agitation of removing egg jelly does not surpass 5min, after glued membrane is removed completely, ovum flocks together conventionally, and washing lotion is muddy and have a precipitation.After stirring removal egg jelly, outwelling the film damping fluid that removes photoresist utilizes MMR to wash ovum.Wherein 2% halfcystine compound method is that 2g cysteine hydrochloride is dissolved in 100mL1 * MMR, with NaOH, adjusts pH to 7.8.Described MMR comprises 5mM pH7.8HEPES(4-hydroxyethyl piperazine ethanesulfonic acid), 0.1mM EDTA, 100mM NaCl, 2mM KCl, 1mM MgSO
4, 2mM CaCl
2.Described MMR washes ovum and first through 0.25 * MMR, washes ovum and use 1 * MMR to wash ovum again, and MMR wash ovum process will be as quickly as possible, to avoid ovum overstand in halfcystine and MMR.The result of preparing for extract due to the quality of ovum has a significant impact, therefore the ovum of washing through MMR is with choosing except animal pole vegetative pole fuzzy, floating, fluffy bad ovum with plastic suction pipe after cell pyrolysis liquid rinsing, cell pyrolysis liquid post rinse once, is slightly out of shape ovum afterwards.The method of said extracted ovocyte crude extract is transferred to ovum in the cell pyrolysis liquid containing 1mM DTT and 5 μ g/mL proteinase inhibitor after cell pyrolysis liquid rinsing, after stirring gently, outwell cell pyrolysis liquid and ovum is transferred in 15mL centrifuge tube, after ovum precipitation, with plastic suction pipe, remove centrifuge tube upper strata redundant solution, the centrifugal 10-15s of 800rpm, further removes centrifuge tube upper strata redundant solution afterwards.Owing to removing redundant solution after recentrifuge, both needed unnecessary liquid removal totally, needed again carefully to avoid losing ovum, therefore preferably adopted pipettor or injector operations.After removing redundant solution, high speed centrifugation makes xenopus oocyte broken, and ovocyte extract discharges.In order to prevent proteolysis, before centrifugal, add proteinase inhibitor, final concentration to the 5 μ g/mL of described proteinase inhibitor, described proteinase inhibitor is preferably Trypsin inhibitor,Trasylol or leupeptin.After xenopus oocyte centrifugal breaking, liquid presents clearly demarcated three layers, and upper strata is yellow lipid, and middle level is brown cell crude extract, and lower floor is pullous cell debris.With 2mL syringe, thrust lower floor top and draw crude extract, transfer in the centrifuge tube of a precooling and add glycerine, 4 ℃ mix, packing, liquid nitrogen freezing, be stored in-80 ℃ standby.Syringe should be noted that and avoids sneaking into impurity while drawing crude extract.And the final concentration of the glycerine mixing with crude extract is 5%.In order to obtain more extract, can also repeat to add ultracentrifugal step after proteinase inhibitor.
Conventionally impel in certain embodiments Xenopus laevis superovulation to obtain more xenopus oocyte, and then obtain more xenopus leavis oocytes extract.Described Xenopus laevis superovulation is preferably injection Hormone Induction Ovulation.Described hormone is preferably pregnant mare serum gonadotrop(h)in (PMSG) and/or human chorionic gonadotrophin.
Pregnant mare serum gonadotrop(h)in (PMSG) is PMSG(Pregnant Mare Serum Gonadotropin), also referred to as horse chorionic-gonadotropin hormone, it is a kind of hormone of finding in conceived mare serum, and knownly at pregnant equus (donkey, zebra etc.), have generation, be a kind of glycoprotein hormones.PMSG molecule is the same with pituitary gonadotropic hormone, and also Shi YouαHe β subunit forms, and Erβ subunit is its active major portion of performance.PMSG has the double activity of similar pituitary follicular stimulating hormone (FSH) and lutropin (LH), but with the master that act as of FSH, therefore has the effect of significantly short follicular development, the function that simultaneously has certain induced ovulation and corpus luteum to form.
Human chorionic gonadotrophin (HCG) is that it is comprised of α and the dimeric glycoprotein of β, also has the function of FSH and LH, the function that induced ovulation and corpus luteum form by a kind of glycoprotein of trophocyte's secretion of placenta.
Described Africa xenopus superovulation is in certain embodiments specially selectivity adult female Africa xenopus, in the shallow table in saccus lymphaticus place, back inject 100 μ L, concentration is the freshly prepared pregnant mare serum gonadotrop(h)in (PMSG) of 1IU/ μ L, 100 μ L are injected in receipts ovum same position the day before yesterday, concentration is 5IU/ μ L human chorionic gonadotrophin, after the rear 16-22h of injection, take out Africa xenopus, outwell excessive moisture, collect xenopus oocyte.Wherein, after injection pregnant mare serum gonadotrop(h)in (PMSG) and human chorionic gonadotrophin, Xenopus laevis need be positioned in 0.1M salt solution, in case claw stop toad infects.After injection pregnant mare serum gonadotrop(h)in (PMSG), again injecting human chorionic gonadotrophin need carry out at receipts ovum the day before yesterday, normally injects after pregnant mare serum gonadotrop(h)in (PMSG) 3-30 days.
The method of the invention is used containing 2mM CaCl after xenopus leavis oocytes extract and the bovine fetal fibroblast of processing through saturatingization are hatched altogether
2full substratum closing cell film on reversible duct, then extract is processed culture medium culturing induction bovine fetal fibroblast reprogrammed and is obtained bovine fetal fibroblast multipotential stem cell.
Wherein, containing 2mM CaCl
2full substratum closing cell film on off-period in reversible duct be preferably 2h.And the time of extract processing culture medium culturing is preferably 6-7d.And described extract processing substratum refers to containing 5v/v%FBS, 1v/v% penicillin and Streptomycin sulphate, 0.1mM2-mercaptoethanol, the nonessential amino acid of 1v/v%, 1mM glutamine, the DMEM low sugar nutrient solution of 0.3 μ M Nucleotide.
Step D of the present invention sucks extract after the xenopus leavis oocytes extract that comprises ATP-regeneration system and F4 are hatched altogether for bovine fetal fibroblast, and full substratum cleans 3-5 time, with containing 2mM CaCl
2full culture medium culturing cell 2h, with the reversible duct on closing cell's film.Then remove above-mentioned substratum, add extract to process culture medium culturing, renew every other day fresh extract and process substratum and remove dead cell, continue to cultivate 6-7d.
The present invention carries out H3K9 premunition fluorescent dye analysis of cells acetylize changing conditions after extract is processed culture medium culturing 24h.At extract, process after culture medium culturing 6d, carry out respectively the RT-PCR augmentation detection of the western blot detection of alkaline phosphatase staining experiment, Oct4 phase immuning fluorescent dyeing analysis, Oct4 albumen and Oct4, Sox2, Nanog, detecting cell dryness, versatility factor Oct4 expression and versatility marker gene Oct4, Sox2, Nanog express.Result shows; alkaline phosphatase staining is positive; histone H 3 K9 degree of acetylation reduces; and the expression of Oct-4 albumen detected; the expression of versatility marker gene Oct-4, Nanog detected simultaneously; show that induction bovine fetal fibroblast reprogrammed of the present invention becomes the method for multipotential stem cell can cause that somatic cell gene phraseology changes, performance stem cell characteristic, recuperation section totipotency.
Therefore the bovine fetal fibroblast multipotential stem cell that the present invention also provides the method reprogrammed of described induction bovine fetal fibroblast reprogrammed to obtain.
In order further to understand the present invention, below in conjunction with embodiment, method provided by the invention is elaborated.Wherein reagent used and test kit are commercially available prod and can are bought and be obtained or prepare voluntarily by existing bibliographical information by commercial channel.
Embodiment 1: the superovulation of Africa xenopus
Selectivity adult female Africa xenopus, it is 1IU/ μ L that the shallow table in saccus lymphaticus place, back is injected the freshly prepared PMSG(concentration of 100 μ L).After injection, Xenopus laevis is positioned in 0.1M salt solution, prevents that Xenopus laevis from infecting.It is 5IU/ μ L that 100 μ LHCG(concentration are injected in the same position of receipts ovum the day before yesterday (normally after PMSG Injection 3-30d)).After injection, Xenopus laevis is positioned over separately in 0.1M salt solution.After 16-22h, take out Africa xenopus, outwell excessive moisture, collect xenopus oocyte.
Embodiment 2: xenopus leavis oocytes content extracts
1. xenopus oocyte separate collection, in beaker, is outwelled unnecessary water, with the useless ovum of plastic suction pipe sucking-off, retains clear-cut, the unified ovum of form.
2. remove after unnecessary water and useless ovum, add freshly prepared 2% halfcystine, soft stirring removed egg jelly, and process does not surpass 5min.After glued membrane is removed completely, ovum flocks together conventionally, and washing lotion is muddy and have a precipitation.
3. outwell the film damping fluid that removes photoresist, 0.25 * MMR washes ovum three times, and 1 * MMR washes ovum once, and washing ovum process will be as quickly as possible, to avoid ovum overstand in halfcystine and MMR.
4. once, this stage chooses except bad ovum (animal pole vegetative pole fuzzy, floating, fluffy ovum) with plastic suction pipe again in cell pyrolysis liquid rinsing, and cell pyrolysis liquid post rinse once, is slightly out of shape ovum.
5. ovum is transferred in the cell pyrolysis liquid containing 1mM DTT and 5 μ g/mL proteinase inhibitor.After stirring gently, outwell cell pyrolysis liquid and ovum is transferred in 15mL centrifuge tube, after ovum precipitation, with plastic suction pipe, removing from suction pipe upper strata redundant solution.
The centrifugal 10-15s of 6.800rpm, further removes centrifuge tube upper strata redundant solution.This step had both needed unnecessary liquid removal clean, needed again carefully to avoid losing ovum, preferably adopted pipettor or injector operations.
7. add Trypsin inhibitor,Trasylol or leupeptin to ovum top (according to volume final concentration to the 5 μ g/mL of ovum), 4 ℃, the centrifugal 15min of 10000rpm.Centrifugal rear xenopus oocyte is broken, liquid presents clearly demarcated three layers, and upper strata is yellow lipid, and middle level is brown cell crude extract, and lower floor is pullous cell debris.
8. with 2mL syringe, extract and thrust lower floor's top absorption crude extract, note avoiding sneaking into impurity.
9. crude extract is transferred in the centrifuge tube of a precooling, added Trypsin inhibitor,Trasylol or leupeptin to ovum top (according to volume final concentration to the 5 μ g/mL of ovum), 4 ℃, the centrifugal 15min of 10000rpm.
10. same method is drawn extract, with suction pipe, extract is transferred in the clean centrifuge tube of 4 ℃ of precoolings, adds glycerine, and 4 ℃ mix.
11. by extract packing, liquid nitrogen freezing, be stored in-80 ℃ standby.
Embodiment 3: fetal fibroblast is cultivated
Slaughterhouse is fetched 40 age in days ox fetuses and is cleaned up, and is transferred in Bechtop and removes amnion, with tweezers and scissors, removes and ox fetal head, tail, four limbs and internal organ, retains trunk tissue.Remaining trunk is organized successively through 75% alcohol with in containing 1% dual anti-PBS to rinsing 3 times.With the scissors of sterilization, the tissue after cleaning is cut into about 1mm
2left and right organize fragment.Organize in fragment and add a little serum, with pasteur pipet, draw fragment, evenly spread upon in T25 culturing bottle.Careful upset culturing bottle, adds the full substratum of 1mL, is inverted in incubator.After 6-8h, carefully turn over bottle, continue to cultivate.After 1-2d, observe and add liquid, until cell, reach 80% while converging, going down to posterity, it is frozen to be cultured to F4 generation.
Embodiment 4: cell permeabilization concentration screening
Get 4 generation well-grown and bovine fetal fibroblast, go down to posterity and be inoculated in 24 orifice plates.When fetal fibroblast grows to 50%-60% and converges, beginning is processed, and sucks the nutrient solution in culture dish, and cell cleans twice with the PBS of precooling.To every hole, add fast the saturating agent digitonin of cytolemma of 200 μ L different concns, culture dish acts on rapidly 2min on ice, during frequently jiggle.After saturatingization 2min, suck fast the digitonin solution in culture dish, with the PBS of precooling, clean 3-5 time, to remove residual digitonin solution.Adopt 10 μ g/mL PI dye liquor cell dyeing 5min, fluorescence microscopy Microscopic observation dyeing situation.Every kind of concentration repeats 3 times, chooses at random 3 visuals field at every turn and carries out counting statistics, is calculated as follows rate, thoroughly rate=staining cell number/total cell count.And adopt SAS8.0 software to carry out the analysis of the ANOVA significance of difference to the saturating rate obtaining under different digitonin concentration.
First test selects agent digitonin concentration is that 1,5,10,15,20 μ g/mL carry out preliminary screening, by PI dye (PI can enter the damaged cell of cytolemma or dead cell but can not enter viable cell) find, when digitonin concentration reaches 10 μ g/mL, more than 85% cell is colored, cytolemma occurs changes thoroughly, but after processing with this concentration, cell state is poor, surpasses 80% necrocytosis after cultivating, and the results are shown in Table 1.
Further dwindle screening scope, selecting digitonin concentration is that 5,6,7,8,9 μ g/mL screen, result shows when concentration is 7 μ x/mL, there be 55% cell generation saturatingization (table 2), and cell state is good, through follow-up culturing cell mortality ratio, is less than 10%.Digitonin concentration is that under 7 μ g/mL, cell permeabilization rate and mortality ratio are the highest, so the method for the invention adopts agent digitonin concentration, is 7 μ g/mL.
The different digitonin concentration of table 1 is changed efficiency ratio (1-20 μ g/mL) thoroughly
| Concentration (μ g/mL) | 1 | 5 | 10 | 15 | 20 |
| Saturating rate (%) | 3.69±0.95 a | 8.06±2.52 a | 85.26±3.99 b | 90.58±3.98 c | 96.34±0.40 d |
Note: different alphabetical subscripts replace significant difference (p<0.05)
The different digitonin concentration of table 2 is changed efficiency ratio (5-9 μ g/mL) thoroughly
| Concentration (μ g/mL) | 5 | 6 | 7 | 8 | 9 |
| Saturating rate (%) | 8.06±2.52 a | 39.62±4.84 b | 55.44±2.30 c | 73.55±4.32 d | 80.42±2.40 e |
Note: different alphabetical subscripts replace significant difference (p<0.05)
Embodiment 5: extract is processed bovine fetal fibroblast
Get with embodiment 4 saturatingization process identical sources 4 generation well-grown bovine fetal fibroblast as experiment cell, be inoculated in 24 orifice plates.When cell grows to 50%-60% and converges, suck substratum in culture dish, with the PBS of precooling, clean twice, liquid in exhaustion culture dish.Add fast 200 μ L7 μ g/mL digitonin, act on rapidly 2min on ice, during frequently jiggle.After saturatingization 2min, suck fast digitonin solution, the PBS of precooling cleans 3-5 time, to remove residual digitonin solution.To every hole in culture dish, add the xenopus leavis oocytes extract that comprises ATP-regeneration system that 250 μ L embodiment 2 make, under 23 ℃ of conditions, hatch altogether 1h with cell.After hatching altogether, suck extract, full substratum cleans 3-5 time, with containing 2mM CaCl
2full nutrient solution culturing cell 2h, with the reversible duct on closing cell's film.After cell sealing, removal sealing substratum adds extract to process culture medium culturing, changes every other day liquid and removes dead cell, continues observation and cultivates 6-7d.Microscopic examination during this time, the results are shown in Figure 1.
After being processed by the visible extract of Fig. 1 result, visible cell slightly bounces back but form is normal, by naked-eye observation cell, approaches 20%-30% death or come off (A-C in Fig. 1).Cell continues to cultivate, and after 2-3d, cell state recovers to start fast breeding, and 4-5d cell starts to assemble growth, and 6-7d cell forms " clone bunch " (D in Fig. 1).And as a control group, same source, the untreated cell (extract processing culture medium culturing) of cultivating and extract are processed rear conventional medium (DMEM+10%FBS) culturing cell normal growth simultaneously, have no cell aggregation, cluster growth (E-F in Fig. 1).
The fluorescent dye of embodiment 6:6H3K9 premunition
Extract is processed after bovine fetal fibroblast 24h, and the untreated cell (extract processing culture medium culturing) of select extract processing cell and same source, simultaneously cultivating carries out H3K9 premunition fluorescent dye analysis of cells acetylize changing conditions.Two groups of cells suck respectively nutrient solution, with the PBS of preheating, clean 3 times, remove cell debris, add 4% paraformaldehyde stationary liquid to each hole, and room temperature is 20min fixedly.After fixing end, suck stationary liquid, with the PBS+1%BSA of preheating, clean 3 times, each 5min.With hatching altogether 30min containing PBS and the cell of 0.5%TritonX-100, with penetrating cytolemma, discard liquid in hole after penetrating, with the PBS of preheating, clean 3 times, at every turn 5min.With 10% lowlenthal serum confining liquid closing cell 30min, with the non-specific binding of blocking antibody.After sealing finishes, discard confining liquid, primary antibodie is pressed 1:200 dilution with the PBS containing 1%BSA, and incubated at room 1h, discards primary antibodie diluent, with PBS, cleans 3 times, each 5min.Under lucifuge condition, with the PBS containing 1%BSA, press 1:200 dilution two and resist, under room temperature, lucifuge is placed 1h.Under lucifuge condition, discard two anti-diluents, with PBS, clean 3 times, each 5min.The PI dye liquor transfect cell core that adds 10 μ g/mL, room temperature effect 5min, at fluorescence microscopy Microscopic observation and take pictures.Experiment in triplicate, is chosen at random 3 visuals field at every turn and is taken pictures, Image-pro Plus software analysis H3K9 premunition fluorescent dye intensity.The results are shown in Figure 2.
From Fig. 2 result; bovine fetal fibroblast is after xenopus leavis oocytes extract is processed 24h; compare with the untreated control group (extract is processed culture medium culturing cell) of same source, cultivation simultaneously; histone H 3 K9 degree of acetylation reduces, but the two difference not significantly (p > 0.05).
Embodiment 7: alkaline phosphatase staining
Extract is processed bovine fetal fibroblast and is cultivated after 4-5d, and the bovine fetal fibroblast that extract is processed is assembled growth, and 6-7d forms " clone bunch ".After selecting extract to process, the untreated cell (extract processing culture medium culturing) of 6d bovine fetal fibroblast cell and same source, cultivation simultaneously carries out alkaline phosphatase staining experiment, to detect cell dryness.Two groups of cells suck respectively substratum, and PBS cleans 3 times; Add 4% paraformaldehyde stationary liquid, room temperature is 30min fixedly; Suck stationary liquid, PBS cleans cell 3-5 time, each 5min; After last washing, remove washings, add 500 μ L BCIP/NBT dyeing working fluids, room temperature lucifuge dyeing 30min; Remove staining fluid, PBS cleans 2-3 time, and Microscopic observation, takes pictures.Result as shown in Figure 3.
From Fig. 3 result, extract is processed after 6d, bovine fetal fibroblast cluster growth (A in Fig. 3).Alkaline phosphatase staining finds that " clone bunch " is intense violet color, and dyeing is positive (B in Fig. 3), and that other cell that does not form clone bunch does not almost have is painted, and dyeing is negative.The cell that same source, untreated cell (extract processing culture medium culturing) the alkaline phosphatase staining result of simultaneously cultivating and not forming cloned bunch is similar, coloration result be negative (C in Fig. 3).
Embodiment 8:Oct4 immunofluorescence dyeing
Extract is processed bovine fetal fibroblast and is cultivated after 4-5d, and extract is processed fetal fibroblast and assembled growth, and 6-7d forms " clone bunch ".After selecting extract to process, the untreated cell (extract processing culture medium culturing) of 6d cell and same source, cultivation simultaneously carries out Oct4 immuning fluorescent dyeing analysis, to detect versatility factor Oct4 expression.Two groups of cells suck nutrient solution in culture dish, with the PBS of preheating, clean 2 times, remove cell debris, add 4% paraformaldehyde stationary liquid to each hole, and room temperature is 30min fixedly; Suck stationary liquid, with PBS+1%BSA, clean 3 times, each 5min; With hatching altogether 10min containing PBS and the cell of 1%TritonX-100, penetrating cytolemma, discards liquid in hole after penetrating, and cleans 3 times with PBS, at every turn 5min; With 10% lowlenthal serum confining liquid closing cell 30min, with the non-specific binding of blocking antibody; After sealing finishes, discard confining liquid, primary antibodie is pressed 1:100 dilution, 4 ℃ of refrigerator overnight with the PBS containing 1%BSA.Discard every other day primary antibodie diluent, with PBS, clean 3 times, each 5min; Under lucifuge condition, with the PBS containing 1%BSA, press 1:500 dilution two and resist, under room temperature, lucifuge is placed 1h; Under lucifuge condition, discard two anti-diluents, with PBS, clean 3 times, each 5min; The PI dye liquor transfect cell core that adds 10 μ g/mL, room temperature effect 5min, at fluorescence microscopy Microscopic observation and take pictures.Result as shown in Figure 4.
From Fig. 4 result, extract is processed after 6d, the bovine fetal fibroblast cluster growth that extract is processed, the discovery of Oct4 immunofluorescence dyeing, " clone bunch " is green, and be positive (B in Fig. 4) dyes, and other does not form clone's bunch cell and does not have paintedly, dyeing is negative.Same source, untreated cell (extract processing culture medium culturing) the Oct4 immunofluorescence dyeing of simultaneously cultivating are not painted, and coloration result is negative.
The Western Blot of embodiment 9:Oct4 analyzes
Ox β-Actin protein antibodies of take is contrast, the untreated cell (extract processing culture medium culturing) of extract is processed to the cell that forms " clone bunch " after bovine fetal fibroblast is cultivated 6d and same source, simultaneously cultivating extracts albumen, adopts the content of Western Blot method detection Oct4 albumen.Concrete grammar is as follows:
The extraction of albumen: the cell of selecting extract to process to form after bovine fetal fibroblast is cultivated 6d " clone bunch " and same source, the untreated cell (extract processing culture medium culturing) of simultaneously cultivating, abandon cell culture fluid, with PBS solution cleaning 3 times.After trysinization, receive cell in clean Eppendorf pipe.Add 200 μ L protein lysates and 10 μ L protein inhibitors, vortex concussion, 4 ℃ of cracking 30min, the centrifugal 10min of 12000rpm, gets supernatant, packing ,-80 ℃ of preservations.
The foundation of standard protein concentration gradient: using proportional diluted method is the protein solution of different concns by the standard protein dilution of concentration known, is followed successively by 200 μ g/mL, 40 μ g/mL, 20 μ g/mL, 10 μ g/mL, 5 μ g/mL, 2.5 μ g/mL, 1 μ g/mL, 0.5 μ g/mL, the blank group of 0 μ g/mL().
The preparation of Micro BCA working fluid (WR): MA, MB in test kit and MC solution are mixed to lucifuge, matching while using in the ratio of 25:24:1.
In 96 orifice plates, add successively standard protein and testing protein (500 times of dilutions) 150 μ L, 3 repetitions are done in every hole; Every hole adds 150 μ L BCA working fluids, mixes gently; With masking foil, 96 orifice plates are wrapped up, lucifuge, hatches 2h for 37 ℃; Under 562nm wavelength, utilize microplate reader to measure the light absorption ratio in each hole, record data; Take protein concentration as ordinate zou, and light absorption ratio is X-coordinate, utilizes excel drawing standard graphic representation; Utilize gained formula and testing sample light absorption value, calculating mensuration is used testing protein concentration, testing sample concentration to be and is measured with testing sample concentration * 500.
The sex change of albumen: the albumen having extracted mixes in 5:1 ratio with albumen damping fluid, boils 10min, makes its abundant sex change, is put on ice rapidly.
Protein electrophorese: prepare running gel, carry out SDS-PAGE electrophoresis.
Transferring film (half dry type transfer): electrophoresis finishes rear taking-up gel, is cut to suitable size, with transferring film damping fluid balance, 5min * 3 time; Cut out in advance the filter paper onesize with adhesive tape and pvdf membrane, immerse 10min in transferring film damping fluid; From transfer device negative pole, by sponge-filter paper (3 layers)-gel-pvdf membrane-filter paper (3 layers)-sponge, sequentially put well.Each step is removed bubble.Switch on power, constant current 100mA shifts 1h.After transfer finishes, take out pvdf membrane, carry out mark, put into TBST solution and clean 10min.With confining liquid, seal 2h.TBST solution rinsing film, 10min * 3 time.Primary antibodie is diluted to corresponding proportion by 1:200 with confining liquid, afterwards incubated at room 2h.TBST solution rinsing film, 10min * 3 time.By two, resist and be diluted to corresponding proportion by 1:500 confining liquid, afterwards incubated at room 2h.TBST solution rinsing film, 10min * 3 time.Chemoluminescence method develops, photographic fixing.The results are shown in Figure 5.
From Fig. 5 result, extract is processed bovine fetal fibroblast can express Oct4 multipotency albumen, and the untreated bovine fetal fibroblast of control group is not expressed Oct4 multipotency albumen.
The expression extract of embodiment 10:RT-PCR augmentation detection versatility marker gene Oct4, Sox2, Nanog is processed bovine fetal fibroblast and is cultivated after 4-5d, and extract is processed fetal fibroblast and assembled growth, and 6-7d forms " clone bunch ".The total RNA of 6d cell extraction after selecting extract to process, without DNA, pollute after testing, by total RNA reverse transcription, be cDNA, take same source, the untreated cell (extract processing culture medium culturing) of simultaneously cultivating and extract, to process conventional medium (DMEM+10%FBS) culturing cell be afterwards control group, take GAPDH as reference gene, by RT-PCR augmentation detection versatility marker gene Oct4, Sox2, Nanog expression.Concrete grammar is as follows:
Cell RNA extracts: untreated cell (extract processing culture medium culturing) and rear conventional medium (DMEM+10%FBS) culturing cell of extract processing of the cell of formation " clone bunch " and same source, cultivation simultaneously after selection extract processing bovine fetal fibroblast cultivation 6d, abandon cell culture fluid, with PBS solution, clean 3 times.After trysinization, receive cell in clean Eppendorf pipe.According to method shown in TIANGEN total RNA extraction reagent box (TAKARA company), extract respectively cell total rna, extract sample and adopt micro-spectrophotometer to measure total rna concentration.According to method shown in Takara reverse transcription test kit, by the RNA reverse transcription of extraction, be cDNA.
Cell RT-PCR detects: select versatility marker gene Oct4, Sox2, Nanog to detect gene as experiment, with reference to ox Oct4 gene order (NM_174580.2) in Genbank, Sox2 gene order (NM_001105463.1), Nanog gene order (NM_001024344.1) adopts primer5.0 software design primer, GAPDH reference gene primer is provided by laboratory, and design result is as table 3.
Each gene primer sequence of table 3
Pcr amplification system:
| Reagent composition | Volume (μ L) |
| 2×Taq?PCR?MasterMix | 10 |
| Upstream primer (10 μ M) | 0.5 |
| Downstream primer (10 μ M) | 0.5 |
| Template | 1 |
| ddH2O | 8 |
| Cumulative volume | 20 |
[0120]pcr amplification program:
Adopt 2% sepharose to carry out electrophoresis to PCR product, then with gel imaging instrument, detected result is taken a picture, the results are shown in Figure 6.
From Fig. 6 result, extract is processed bovine fetal fibroblast Oct4, Nanog genetic expression, and Sox2 gene has no expression (Fig. 6).Control group, all without above-mentioned genetic expression, illustrates that extract is processed and bovine fetal fibroblast process reprogrammed, has the versatility of stem cell, and expresses versatility genes involved.
The explanation of above embodiment is just for helping to understand method of the present invention and core concept thereof.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of the claims in the present invention.
Claims (9)
1. a method of inducing bovine fetal fibroblast reprogrammed, is characterized in that, bovine fetal fibroblast is hatched altogether with xenopus leavis oocytes extract after digitonin solution is changed processing thoroughly, with containing 2mM CaCl
2full substratum closing cell film on reversible duct, then extract is processed culture medium culturing induction bovine fetal fibroblast reprogrammed and is obtained bovine fetal fibroblast multipotential stem cell.
2. method according to claim 1, is characterized in that, described bovine fetal fibroblast is obtained through full culture medium culturing after rinsing through 75% alcohol with in containing 1% dual anti-PBS by 40 age in days ox fetus trunk tissues in T25 culturing bottle.
3. method according to claim 1, is characterized in that, described digitonin strength of solution is 7 μ g/mL.
4. method according to claim 1, is characterized in that, describedization is treated under 0 ℃ of condition and processes 2min.
5. method according to claim 1, is characterized in that, described xenopus leavis oocytes extract preparation method is that xenopus oocyte goes egg jelly after high speed centrifugation fragmentation, to collect ovocyte crude extract by cell pyrolysis liquid cracking again.
6. method according to claim 1, is characterized in that, described hatching altogether as hatch together 1h under 23 ℃ of conditions.
7. method according to claim 1, is characterized in that, the time of described sealing is 2h.
8. method according to claim 1, is characterized in that, the time of described cultivation is 6-7d.
9. the bovine fetal fibroblast multipotential stem cell that described in claim 1, method reprogrammed obtains.
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| EP3354724A1 (en) * | 2017-01-25 | 2018-08-01 | Institut National De La Recherche Agronomique | Method for reprogramming somatic cells of ruminants |
| CN115418343A (en) * | 2022-10-28 | 2022-12-02 | 深圳市俊元生物科技有限公司 | Method for extracting pluripotent stem cells from human retinal pigment epithelial cells |
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| EP3354724A1 (en) * | 2017-01-25 | 2018-08-01 | Institut National De La Recherche Agronomique | Method for reprogramming somatic cells of ruminants |
| WO2018138091A1 (en) * | 2017-01-25 | 2018-08-02 | Institut National De La Recherche Agronomique | Method for reprogramming ruminant somatic cells |
| CN107376025A (en) * | 2017-07-16 | 2017-11-24 | 陈强 | A kind of cytoskeleton composite material and preparation method thereof and application for cartilage damage reparation |
| CN115418343A (en) * | 2022-10-28 | 2022-12-02 | 深圳市俊元生物科技有限公司 | Method for extracting pluripotent stem cells from human retinal pigment epithelial cells |
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