CN104357483A - Method for increasing pig cell reprogramming capability and application - Google Patents
Method for increasing pig cell reprogramming capability and application Download PDFInfo
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- CN104357483A CN104357483A CN201410613163.7A CN201410613163A CN104357483A CN 104357483 A CN104357483 A CN 104357483A CN 201410613163 A CN201410613163 A CN 201410613163A CN 104357483 A CN104357483 A CN 104357483A
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Abstract
The invention discloses a method for increasing the pig cell reprogramming capability and application, and belongs to the technical field of cell transplantation and embryonic development. The method provided by the invention comprises the following steps: obtaining and culturing a pig oocyte and a pig fetus fibroblast, then conducting somatic cell nuclear transplantation, and finally conducting iPs induction and alkaline phosphatase staining. According to the method, a micromolecular medicine GSK126 is adopted for treatment, and the micromolecular medicine GSK126 is used in the following three ways: 1) the pig fetus fibroblast is treated with the micromolecular medicine GSK126 before nuclear transplantation; 2) a fused embryo is treated with the micromolecular medicine GSK126 after the somatic cell nuclear transplantation of the pig fetus fibroblast; 3) the pig fetus fibroblast for iPS induction is treated after being cultured to be mature and before being inoculated through the utilization of the micromolecular medicine GSK126. The method provided by the invention can remarkably increases the developmental rate of cloned embryos and the iPS induction efficiency by 44.7% and 111.3% respectively.
Description
Technical field
The present invention relates to a kind of method and the application that improve pig cell reprogrammed ability, belong to Transplanted cells and fetal development technical field.
Background technology
Somatic cell nuclear transfer technique is a kind of cell reprogrammed technology, and since clone sheep " Dolly " is born, this technology successively obtains successfully on multiple Mammals, but cloning efficiency is general very low, on average between 0.1%-2.0%.For improving nuclear transplantation efficiency, researchist has carried out large quantifier elimination in the screening and pre-treatment etc. of nuclear transfer technology program, oocyte maturation quality, embryo in vitro culture condition, donor cell, although achieve certain progress, cloning efficiency still has much room for improvement.Think that the low main reason of cloning efficiency is that somatic cell nuclear reprogrammed is incomplete at present.Somatic cell nuclear reprogrammed refers to that donorcells core moves into the gene expression program of stopping after ovocyte itself, reverts to fetal development necessary specific gene expression program.This process mainly ovocyte is write again to donorcells epigenetic modification, reinvents, DNA methylation is rebuild, histone modification changes and imprinted gene expression regulation etc. comprising karyomit(e).If in body-cell neucleus transplanting process, the tissue-specific epigenetic modification of donorcells can not be effectively canceled, and will affect the normal development of embryo.Therefore promote that somatic cell nuclear epigenetic reprogrammed will contribute to improving nuclear transplantation efficiency.
At present; have been found that some drug treating donorcellses or clone embryos can improve cloning efficiency to a certain extent; as methylated or deacetylation inhibitor (U-18496 (5-AZA-dC), Trichostatin A (TSA)).5-AZA-dC and TSA is by promoting that the reprogrammed methylated with Acetylation Level improves cloning efficiency.But these medicines all thoroughly do not solve the low problem of cloning efficiency.
Employing methylates or deacetylation inhibitor process donorcells or clone embryos; by regulating the reason that effectively can not improve cloning efficiency with Acetylation Level that methylates be: the nuclear transplantation reprogrammed causing clone embryos developmental rate low is many-sided extremely; incessantly comprise and methylating and Acetylation Level exception; although methylate or the pre-treatment of deacetylation inhibitor can improve some aspect of nuclear transplantation reprogrammed; but want to allow cloning efficiency reach a higher level, people must carry out exploring the method promoting nuclear transplantation reprogrammed from more perspective.
H3K27me3 is a kind of important histone modification, has restraining effect to genetic expression.In pig in-vitro fertilization embryo (IVF), H3K27me3 is arranged in female pronucleus at 1-cell, and after 2-cell, level sharply declines, and 4-cell generally can't detect it later to be existed, until late period, blastaea just started again to occur in trophocyte.People it is generally acknowledged that the rapid erasing of H3K27me3 embryo is in early days conducive to the genetic expression in body early embryo (particularly zygotic gene activation) period.But in porcine clone embryos, the distribution situation of H3K27me3, namely whether H3K27me3 is not also in the news by effective reprogrammed.
Inducing pluripotent stem cells (iPS) technology is another kind of cell reprogrammed technology beyond somatic cell nuclear transfer technique.It makes cell fate change by artificially importing several foreign gene in cell.But iPS technological guide efficiency is generally on the low side at present.Although the Technology Ways of iPS technology and nuclear transfer technology and principle have very large difference, the reprogrammed process of these two kinds of methods but has very large similarity, all needs the apparent modification of the differentiation state overcoming source cell to reach pluripotent state.So the method improving cloning efficiency also likely can improve iPS induced efficiency.
GSK126 is the micromolecular inhibitor of a kind of efficient and specific EZH2, can reduce the integral level of H3K27me3, and GSK126 can as the lymphadenomatous medicine for the treatment of EZH2 sudden change.But the never application in reprogrammed field, not yet finds that GSK126 can be used for improving the effect of fetal development or iPS induced efficiency at present.
Summary of the invention
For solving the problem, the invention provides a kind of method improving pig cell reprogrammed ability, the technical scheme taked is as follows:
A kind of method improving pig cell reprogrammed ability, to obtain and after cultivating porcine oocytes and porcine fetus fibroblasts, carry out body-cell neucleus transplanting, finally carry out iPS induction and carry out alkaline phosphatase staining, it is characterized in that, use small-molecule drug GSK126 in any one mode following:
1) porcine fetus fibroblasts utilizes small-molecule drug GSK126 process before nuclear transplantation;
2) after porcine fetus fibroblasts body-cell neucleus transplanting, small-molecule drug GSK126 process is utilized to merge embryo;
3) for the porcine fetus fibroblasts of iPS induction, after cultivation is ripe, small-molecule drug GSK126 process is utilized before inoculation.
1) utilizing small-molecule drug GSK126 process described in, is utilize the GSK126 process 40-50h of 0.5-3.0 μM.
Preferably, 1) utilizing small-molecule drug GSK126 process described in, is utilize the GSK126 process 48h of 0.5 μM.
2) utilizing small-molecule drug GSK126 process described in, is utilize the GSK126 process 18-30h of 0.05-0.15 μM.
Preferably, 2) utilizing small-molecule drug GSK126 process described in, is utilize the GSK126 process 24h of 0.1 μM.
Described method is for improving the developmental rate of clone embryos.
3) utilizing small-molecule drug GSK126 process described in, is utilize the GSK126 process 40-50h of 0.5-3.0 μM.
Preferably, 3) utilizing small-molecule drug GSK126 process described in, is utilize the GSK126 process 48h of 0.75 μM.
Described method is for improving the induced efficiency of induced multi-potent stem cells.
Beneficial effect of the present invention: for solving the inefficient problem of nuclear transplantation in prior art; overcome prior art can not promote nuclear transplantation reprogrammed comprehensively limitation by methyltransgerase or deacetylase inhibitor handling body cell or nuclear transfer embryo; the present invention is by comparing the distribution pattern of the fertilized embryo of pig cleavage stage and the H3K27me3 of clone embryos; find that the H3K27me3 in porcine clone embryos can not by normal reprogrammed, the H3K27me3 level of early stage clone embryos is higher than IVF embryo.Small-molecule drug GSK126 is the micromolecular inhibitor of a kind of efficient and specific EZH2, can reduce the integral level of H3K27me3, and GSK126 Chang Zuowei treats the lymphadenomatous medicine of EZH2 sudden change.Contriver is in the favorite outer discovery of research process, and small-molecule drug GSK126 has active effect to the raising developmental rate of clone embryos and the induced efficiency of iPS.Accordingly, the present invention, by with small-molecule drug GSK126 process donorcells or clone embryos, promotes the reprogrammed of H3K27me3, finally makes the developmental rate of porcine clone embryos improve 44.7%.The present invention is when inducing pig multipotent stem cells with GSK126 process, and make pig multipotent stem cells induced efficiency, increase rate reaches 111.3%.
Accompanying drawing explanation
Fig. 1 is porcine fetus fibroblasts (PEF) cell count and change of H3K27me3 level after different concns GSK126 process 48;
(a, GSK126 concentration is on the impact of PEF cell count; B, GSK126 concentration is on the impact of H3K27me3 level).
Fig. 2 is the H3K27me3 level of 1-cell, 2-cell clone embryos after the PEF nuclear transplantation of GSK126 process.
Fig. 3 is the H3K27me3 level of clone embryos after GSK126 process 24h.
Fig. 4 is alkaline phosphatase (AP) the positive colony number when the PEF of GSK126 process induces iPS.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not by the restriction of embodiment.
Agents useful for same, material, instruments and methods in following examples, without special instruction, be reagent, material, instruments and methods conventional in this area.
Embodiment 1
1. the maturation culture of porcine oocytes
Collect ovary by slaughterhouse and transport go back to laboratory with the physiological saline of 37 DEG C, the antral follicles collection cumulus oocyte complex of extraction 3-5mm diameter, select under stereomicroscope there are more than three layers complete cumulus cells ovocyte for maturation culture; The culture condition of maturation culture is 38.5 DEG C, the atmosphere surrounding of 5% carbonic acid gas, 95% air, saturated humidity; Maturation culture adopts 0.5% Unidasa to remove cumulus cell after 42 hours, the MII phase ovocyte obtained, as nuclear transplantation acceptor.
2. the acquisition of porcine fetus fibroblasts and cultivation
Aseptic technique Qu35Tian Zhu fetal tissue, adopt conventional 0.25% trypsin Gibco) digestion method chorista cell, DMEM (Gibco) adds 20% foetal calf serum original cuiture, DMEM adds 10% foetal calf serum Secondary Culture, culture condition is 38.5 DEG C, the atmosphere surrounding of 5% carbonic acid gas, 95% air, saturated appropriateness; The digestion of the fetal fibroblast of the contact inhibition in 3-5 generation is adopted to carry out nuclear transplantation for single suspension cell.
3. body-cell neucleus transplanting
Pig nucleus transplantation adopts fusion method to carry out, process is as follows: blind suction method removes spindle body and the first polar body of M II ovum, somatocyte is noted under zona pellucida, and make it and oocyte plasma membrane close contact, direct current shock (1.2kv/cm, 30 μ s, 2 subpulses) inducing somatic and stoning ovum cytomixis form reconstructed embryo and make it to be activated, and be incubated in PZM-3 nutrient solution, culture condition is: 38.5 DEG C, 5%CO2, saturated humidity.Be cultured to 48h and calculate cleavage rates, 146h calculates blastocyst rate.5mg/LHoechst 33342 pairs of blastaeas carry out dyeing 5min, viewed under fluoroscopy note blastomere number; Used tool, liquid, instrument are as follows: fixed tube internal diameter is 30 μm, syringe inner diameter 25 μm; Ovocyte operation liquid is the modified version TCM199 (Gibco) containing 5mg/ml BSA, ovocyte micrurgy liquid is the operation liquid of interpolation 7.5 μ g/ml cytochalasin B (CB), and merging liquid is 3% mannitol solution containing 1.0mM calcium ion and 0.1mM magnesium ion; Micromanipulation system is Japanese Narishige company NT-88NE system; Fusion instrument is U.S. BTX company BTX2001 type electricity cell fusion apparatus.
4.iPS induction and alkaline phosphatase staining
Adopt mouse source 6 factor method induction iPS (Wang, J., Gu, Q., Hao, J., Jia, Y., Xue, B., Jin, H., Ma, J., Wei, R., Hai, T., Kong, Q., Bou, G., Xia, P., Zhou, Q., Wang, L., and Liu, Z. (2013) .Tbx3and Nr5alpha2play important roles in pig pluripotent stem cells.Stem Cell Rev.9,700 – 708.).Retrovirus and feeder layer cells is prepared according to method before.The PEF in P3 or P4 generation is for inducing iPSCs.Day-1: inoculation 1 × 10
5pEF is in the hole of each 6 orifice plates; Day0: by MOI be 5 virus quantity the virus of 6 factors is added to simultaneously in the PEF that inoculated in advance, add 8ug/mL polybrene simultaneously; Day1: change liquid, can add the nutrient solution of penicillin streptomycin from today; Day3: change liquid, observation of cell, and prepare feeder layer; Day4: when cells infected grows to the degree of converging of more than 90%, passes to PEF on feeder layer, cultivates with the DMEM nutrient solution of 10%FBS; Day5: the DMEM nutrient solution discarding 10%FBS, cultivate with X nutrient solution, later every day changes liquid; X nutrient solution formula is that (50mL): 19mL KO-DMEM, 12.25mLDMEM/F12,12.25mL Neurabasal, 5mL KOSR, 250uL B27,125uL N2,250uL glutamine (2mM), 25uL beta-mercaptoethanol, 500uL are dual anti-, 250uL non-essential amino acid, 4uL bFGF, 5uL hLif, 31.25uL 2%BSA.Day9: do alkaline phosphatase staining.First wash three times with PBS, 90s is fixed again by 4% paraformaldehyde room temperature, PBS washes three times, each 5 minutes (or placing room temperature 15 minutes), with 100mmol/L Tris-HCl (pH 9.5), 100mmol/L NaCl, 50mmol/L MgCl2 cocktail buffer washes 5 minutes, the bromo-4-of tetrazole indigo plant (NBT) 4.5 μ l and 5-chloro-3-indolyl phosphate (BCIP) 3.5 μ l is added in 1ml PBS, lucifuge develops the color 20 minutes ~ 1 hour, observes staining conditions at any time.Wash 3 times with PBS, be directly observable under inverted microscope, take a picture.
Embodiment 2
1. the maturation culture (with embodiment 1) of porcine oocytes
2. the acquisition of porcine fetus fibroblasts and cultivation
PEF before nuclear transplantation respectively with 0.5 μM, 0.75 μM and 3 μMs of GSK126 process 48h.All the other are with embodiment 1.
3. body-cell neucleus transplanting (with embodiment 1)
Embodiment 3
1. the maturation culture (with embodiment 1) of porcine oocytes
2. the acquisition of porcine fetus fibroblasts and cultivation (with embodiment 1)
3. body-cell neucleus transplanting
Embryo after fusion is with 0.05 μM, 0.1 μM and 0.15 μM of GSK126 process 24h.Afterwards embryo is placed in normal PZM-3 to continue to cultivate.All the other are with embodiment 1.
Embodiment 4
1. the acquisition of porcine fetus fibroblasts and cultivation (with embodiment 1)
2.iPS induction and alkaline phosphatase staining
For iPS induction PEF before 6 orifice plates inoculations with 0.5 μM, 0.75 μM and 3 μMs of GSK126 process 48h.All the other are with embodiment 1.
Embodiment 5
1. different concns GSK126 on cell count and H3K27me3 level change impact
In order to reduce the H3K27me3 level of donorcells, we use the GSK126 process PEF 48h of different concns.Fig. 1 is PEF cell count (A) and H3K27me3 level (B) change after different concns GSK126 process 48.The cell state respectively organized when can find 0-3 μM from Fig. 1 is good, and the cell count of each group does not have difference (A) yet.The H3K27me3 level of each group of cell is detected, finds that GSK126 can reduce the H3K27me3 level of PEF, particularly effect comparatively significantly (P<0.05) (B) during concentration >0.5 μM.
The impact that 2.GSK126 process PEF grows clone embryos
Table 1 is through the clone embryos developmental rate of the PEF of GSK126 process
a,b indicates P<0.05.
Table 1 is the clone embryos developmental rate of the PEF through GSK126 process.Nuclear transplantation is carried out as can be seen from Table 1 with the cell through 0.5 μM, 0.75 μM and 3 μMs GSK126 process, the blastocyst rate of two groups of clone embryos is all significantly higher than control group (Con.v.s.0.5 μM of v.s.0.75 μM of v.s.3 μM, 21.99%v.s.31.82%v.s.27.96%v.s.30.23%, P<0.05).
Fig. 2 is the H3K27me3 level of 1-cell, 2-cell clone embryos after the PEF nuclear transplantation of GSK126 process.As can be seen from Figure 2, the H3K27me3 level of 1-cell and 2-cell of 0.75 μM of GSK126 treatment group clone embryos is all lower than control group.This illustrates that GSK126 can reduce the H3K27me3 level of PEF thus be conducive to the reprogrammed of clone embryos H3K27me3, and can significantly improve clone embryos and grow.
The impact that 3.GSK126 process clone embryos is grown clone embryos
The clone embryos developmental rate of table 2 after GSK126 process embryo 24h
Different superscript indicates P<0.05.
Table 2 is the clone embryos developmental rate after GSK126 process embryo 24h.The blastocyst rate of the clone embryos of 0.05 μM, 0.1 μM and 0.15 μM GSK126 process is significantly higher than control group (Con.v.s.0.05 μM of v.s.0.1 μM of v.s.0.15 μM as can be seen from Table 2,21.99%v.s.28.97%v.s.29.86%v.s.29.37%, P<0.05).
Fig. 3 is the H3K27me3 level of clone embryos after GSK126 process 24h.As can be seen from Figure 3, the H3K27me3 for the treatment of group 2-cell is starkly lower than control group level.This illustrates that GSK126 process clone embryos can promote H3K27me3 reprogrammed, and significantly improves porcine clone embryos growth.
4.GSK126 process is on the impact of AP positive colony number during iPS
Fig. 4 is the AP positive colony number when the PEF of GSK126 process induces iPS.As can be seen from Figure 4, carry out iPS induction after 0.5 μM, 0.75 μM and 3 μMs of GSK126 process PEF 48h, compared with control group, AP positive colony digital display work promotes, and increase rate reaches 111.3%.This illustrates that GSK126 can significantly improve iPS induced efficiency.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; can do various change and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (9)
1. one kind is improved the method for pig cell reprogrammed ability, to obtain and after cultivating porcine oocytes and porcine fetus fibroblasts, carry out body-cell neucleus transplanting, finally carry out iPS induction and carry out alkaline phosphatase staining, it is characterized in that, use small-molecule drug GSK126 in any one mode following:
1) porcine fetus fibroblasts utilizes small-molecule drug GSK126 process before nuclear transplantation;
2) after porcine fetus fibroblasts body-cell neucleus transplanting, small-molecule drug GSK126 process is utilized to merge embryo;
3) for the porcine fetus fibroblasts of iPS induction, after cultivation is ripe, small-molecule drug GSK126 process is utilized before inoculation.
2. utilizing small-molecule drug GSK126 process method described in claim 1, is characterized in that, 1), is utilize the GSK126 process 40-50h of 0.5-3.0 μM.
3. method described in claim 2, is characterized in that, describedly utilizes small-molecule drug GSK126 process, is to utilize the GSK126 process 48h of 0.5 μM.
4. utilizing small-molecule drug GSK126 process method described in claim 1, is characterized in that, 2), is utilize the GSK126 process 18-30h of 0.05-0.15 μM.
5. method described in claim 4, is characterized in that, describedly utilizes small-molecule drug GSK126 process, is to utilize the GSK126 process 24h of 0.1 μM.
6. method described in claim 1-5, is characterized in that, for improving the developmental rate of clone embryos.
7. utilizing small-molecule drug GSK126 process method described in claim 1, is characterized in that, 3), is utilize the GSK126 process 40-50h of 0.5-3.0 μM.
8. method described in claim 7, is characterized in that, describedly utilizes small-molecule drug GSK126 process, is to utilize the GSK126 process 48h of 0.75 μM.
9. method described in claim 1,7 and 8, is characterized in that, for improving the induced efficiency of inducing pluripotent stem cells.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913653A (en) * | 2018-06-04 | 2018-11-30 | 温氏食品集团股份有限公司 | A method of improving pig nuclear transfer efficiency |
| CN111269878A (en) * | 2020-01-19 | 2020-06-12 | 武汉大学 | Special culture medium for converting human pluripotent stem cells into expanded pluripotent stem cells and application of special culture medium |
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Non-Patent Citations (4)
| Title |
|---|
| ALEXANDER J. FEDERATION ET AL.: "The use of small molecules in somatic-cell reprogramming", 《TRENDS CELL BIOL.》 * |
| DANWEI HUANGFU ET AL.: "Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds", 《NATURE BIOTECHNOLOGY》 * |
| GIULIA FRAGOLA ET AL.: "Cell Reprogramming Requires Silencing of a Core Subset of Polycomb Targets", 《PLOS GENETICS》 * |
| H HIRAL, N KIKYO: "Inhibitors of suppressive histone modification promote direct reprogramming of fibroblasts to cardiomyocytelike cells", 《CARDIOVASCULAR RESEARCH》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913653A (en) * | 2018-06-04 | 2018-11-30 | 温氏食品集团股份有限公司 | A method of improving pig nuclear transfer efficiency |
| CN108913653B (en) * | 2018-06-04 | 2022-06-07 | 温氏食品集团股份有限公司 | Method for improving nuclear transplantation efficiency of pigs |
| CN111269878A (en) * | 2020-01-19 | 2020-06-12 | 武汉大学 | Special culture medium for converting human pluripotent stem cells into expanded pluripotent stem cells and application of special culture medium |
| CN111269878B (en) * | 2020-01-19 | 2022-02-11 | 深圳市北科生物科技有限公司 | Special culture medium for converting human pluripotent stem cells into expanded pluripotent stem cells and application of special culture medium |
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