CN103952368A - Culture solution for promoting in-vitro growth of porcine somatic cell cloned embryos - Google Patents
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Abstract
The invention provides a culture solution for promoting the in-vitro growth of porcine somatic cell cloned embryos. The culture solution contains an epigenetic modification agent EPZ00477. The EPZ00477 can specifically change the methylation modification level of histone H3K79 in the early stage growth process of cloned embryos to improve the deficiency of oocytes for somatic cell nuclear reprogramming. The culture solution can be used for carrying out in-vitro culture on the porcine somatic cell cloned embryos in order to promote the growth rate and the growth quality of blastulas of the embryos, so the production cost of somatic cell cloned pigs is reduced, the production efficiency of the somatic cell cloned pigs is improved, and it is helpful for further developing and applying a cloning technology.
Description
［ technical field ］
The present invention relates to animal embryo nutrient solution, particularly a kind of nutrient solution that promotes porcine somatic cell cloning embryos vitro Development of Embryos.
［ background technology ］
The somatic cell clone of animal, refer to and utilize certain equipment and technique means, it is body-cell neucleus transplanting that the somatocyte of animal is carried out to vitro recombination with the ovocyte of removing Mesoplast heredity material, then again by after extremely specific clone embryos vitro culture developmental stage, then be transplanted to the intrauterine of the replace-conceive parent that physiological status is identical, complete growth, produce and the process of somatocyte for homogeneity offspring in nucleome heredity.For over ten years, somatic cell clone technique binding molecule biology and cell culture technology have been produced a large amount of genetic modification offsprings, comprise the random integration of foreign gene and gene site-directed insertion, the animal knocking out.Produce transgenic animal by somatic cell clone, obtain the efficiency high more than procaryotic injection, this is in improvement of breed, rescue animals on the brink of extinction and even basic medical and clinical study aspect are with a wide range of applications, for example for transplanting, human organ opens up allos donor (L.X.Lai et al., Production of alpha-1, 3-galactosyltransferase knockout pigs by nuclear transfer coning, Science, 295 (2002) 1089-1092.), prepare human diseases medical faunae model and Basic of Biology research etc. a more satisfactory platform is provided.Also successfully set up mouse, rat, the human embryonic stem cell from clone embryos by somatic cell clone technique, for therapeutic cloning lays a solid foundation.
Somatic cell clone embryo's vitro culture is the very important link of cloned animal produced in vitro, and this step will be directly connected to efficiency that clone embryos produces and the problem such as optimization and body early embryo transplanting of quality, reconfiguration program.In order to ensure the effect of vitro culture, the composition of the nutrient solution using in laboratory and the environment of cultivation (comprising temperature, gas concentration and humidity etc.), be all the interior environment of as far as possible simulating parent reproductive tract.But owing to simulating after all, so the culture system in vitro of animal embryo still can not imitate the interior environment of parent reproductive tract completely, therefore in the culture effect outside embryoid body and body, the developmental capacity of embryo still has a certain distance.Liquid component in parent reproductive tract is comparatively complicated, contain a large amount of hormones and somatomedin, and these compositions are also non-constant, more or less embryo's later stage is grown and has regulating and controlling effect energetically, and the conventional nutrient solution composition in laboratory is relatively single, fixing, cannot fully simulate the liquid component in parent reproductive tract, why this also explained the cloning efficiency lower partly cause of animal.Therefore, in laboratory, conventional nutrient solution also needs by adding or the growth regulation factor, hormone etc., can reach best emulation mode, thereby make embryo's culture system in vitro further perfect, in laboratory, can obtain abundant embryo quality, the material providing for later experiments research of this animal.
But; except affecting the factor of various physiological conditions aspect of In vitro culture; the somatic cell clone embryo's that reconstruct obtains developmental potency itself is also well below natural fertilization embryo; because the epigenetic reprogrammed event comprising in the process of embryo's reconstruct is numerous; and nuclear reprogramming is difficult to complete reprogrammed occurs at short notice, its main matter comprises Chromatin Remodeling, DNA methylation, histone methylated and acetylize, genomic imprinting, x chromosome inactivation etc.Any one link in these programs goes wrong, and all likely affects the result of reprogrammed, finally has influence on the production efficiency of somatic cell clone animal.It seems from current experience; the imperfection of reprogramming of somatic cells level be cause that clone embryos is of poor quality, central factor (the L.Armstrong et al. of the bad phenomenon generation such as heteroplasia and dead miscarriage in body; Epigenetic modification is central to genome reprogramming in somatic cell nuclear transfer; Stem Cells, 24 (2006) 805-814; K.M.Whitworth et al., Somatic cell nuclear transfer efficiency:how can it be improved through nuclear remodeling and reprogramming, Mol Reprod Dev, 77 (2010) 1001-1015).In the process of somatic cell clone, donorcells is generally from the whole somatocyte of end differential period, and the genome of these cells has experienced the apparent modification of DNA methylation and the acetylation of histone etc. of high special.Therefore moving into after enucleation oocyte, donorcells must be through the apparent modification reprogrammed of reprogrammed factor mediation natural in ovocyte, first close and break up expression relevant, somatocyte specific gene, then activate the part gene that embryo is specific, early development is played an important role, clone embryos could obtain the totipotency of growing.But, ovocyte itself is reprogrammed Human Sperm Chromosome but not somatocyte, so the reprogrammed effect of nuclear transplantation guiding is not very desirable, can reprogrammed to grow embryonism be to be incomplete (X.C.Tian et al. slowly and comparatively speaking to well differentiated cell, Nuclear reprogramming by somatic cell nuclear transfer-the cattle story, Society of Reproduction and Fertility supplement, 64 (2007): 327-339), this may be because due to the not thorough and epigenetic aspect generation abnormal change of reprogramming of somatic cells, cause therefrom the efficiency of current cloning animal somatic cell still low.Therefore, investigator is so far still constantly exploring the method for intervening somatic cell clone embryo's early development from epigenetic aspect, to improving embryo's growth effect and quality.
Studies show that in recent years, epigenetic regulation is being played the part of more and more important effect at Growth of Cells and in growing, and histone methylated modification is one of epigenetics important regulating and controlling mechanism.Wherein, the modification of the 79th Methionin of histone H 3 (H3K79) is most important to setting up with the regulation and control that maintain euchromatin and heterochromatin two states, with nuclear reprogrammed close association.Several research groups such as Feng find, the histone-lysine methyltransferase DOT1 that one class is new can be by its catalysis (Q.Feng et al., Methylation of H3-lysine79is mediated by a new family of HMTases without a SET domain, Curr Biol, 12 (2012): 1052-1058).And according to the study, utilize micromolecular compound EPZ004777 (W.Y.Yu et al., Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors, Nat Commun3 (2012): 1288) dimethyl (H3K79me2) the methyltransgerase DOT1L of the 79th Methionin of selectively targeted inhibition of histone H3, at inductive pluripotent stem cells (Induced pluripotent stem cells, iPSCs) in work, can replace Klf4 and c-Myc gene is significantly accelerated reprogrammed speed, increase iPSCs positive colony quantity (T.T.Tian et al., Chromatin-modifying enzymes as modulators of reprogramming, Nature, 483 (2012) 598-602).Suppress in early days DOT1L in reprogrammed process relevant with two kinds of factor NANOG, LIN28 up-regulateds of bringing into play critical function effect in reprogrammed enhancement.Full genomic level is analyzed the distribution of H3K79me2 and is found, transforms relevant fibroblast-like cell specific gene lost H3K79me2 at the initial stage of reprogrammed to Epithelial and stromal.DOT1L suppresses to have promoted the loss of the gene H3K79me2 mark being suppressed in multipotency state.These results of study show that special chromatin modifying enzyme can serve as obstruction factor or promote the factor in reprogramming of somatic cells, how to have disclosed by regulating these chromatin modifying enzymes to reduce external source transcription factor etc., promote to generate more efficiently iPSCs.
In embryo development procedure, gene presents the expression pattern of space-time in early days, and the variation that histone methylated modification also shows dynamics in this process, histone methylated modifying enzyme has participated in the network regulation of early embryonic development complexity.These also point out us, in the nutrient solution of different times, add epigenetic modification agent and regulate the generation of the nuclear reprogramming event of ovocyte mediation as EPZ004777 etc., the ectogenesis to somatic cell clone embryo and maybe will have positive effect for follow-up animal activity in production.
［ summary of the invention ］
An object of the present invention is to provide the application that epigenetic is repaiied reagent E PZ004777, for the preparation of the application of nutrient solution that promotes porcine somatic cell cloning embryos vitro Development of Embryos.
Another object of the present invention has been to provide a kind of nutrient solution that promotes porcine somatic cell cloning embryos vitro Development of Embryos, comprises that epigenetic repaiies reagent E PZ004777, and its content is 0.1～1nmol/L.Preferably, the concentration of described EPZ004777 is 0.5nmol/L.
According to the further feature of nutrient solution of the present invention, this nutrient solution also comprises the component of following content: sodium-chlor 6.305～6.315g/L; Sodium bicarbonate 2.115～2.125g/L; Repone K 0.740～0.750g/L; Potassium primary phosphate 0.045～0.055g/L; Magnesium sulfate 0.045～0.055g/L; Calcium lactate five water 0.600～0.610g/L; Sodium.alpha.-ketopropionate 0.015～0.025g/L; L-glutaminate 0.140～0.150g/L; Inositol 0.495～0.505g/L; Hypotaurine 0.540～0.550g/L; Penicillin G 0.060～0.070g/L; Streptomycin sulphate 0.045～0.055g/L; Bovine serum albumin 2.990～3.010g/L; Indispensable amino acid (50 ×) 2%; Non-essential amino acid (10 ×) 10%.
Preferably, described nutrient solution comprises the component of following content: sodium-chlor 6.312g/L; Sodium bicarbonate 2.119g/L; Repone K 0.746g/L; Potassium primary phosphate 0.048g/L; Magnesium sulfate 0.048g/L; Calcium lactate five water 0.606g/L; Sodium.alpha.-ketopropionate 0.022g/L; L-glutaminate 0.146g/L; Inositol 0.500g/L; Hypotaurine 0.546g/L; Penicillin G 0.066g/L; Streptomycin sulphate 0.050g/L; Bovine serum albumin 3.000g/L; Indispensable amino acid (50 ×) 2%; Non-essential amino acid (10 ×) 10%.
A further object of the present invention is to provide a kind of method that promotes porcine somatic cell cloning embryos vitro Development of Embryos, comprises the following steps:
A. the nutrient solution being just placed in as described in one of claim 2 to 5 through the porcine somatic cell cloning embryos embryo of reconstruct fusion is processed 6～30 hours;
B. by processing in steps A to enter not containing in the nutrient solution of EPZ004777, continue to be cultured to described vitro Development of Embryos and become blastaea.
Preferably, in steps A, it is 24 hours that described embryo cultivates the time of processing in nutrient solution.
The invention has the advantages that:
(1) the present invention promotes the nutrient solution of porcine somatic cell cloning embryos vitro Development of Embryos, somatic cell clone embryo is carried out after vitro culture processing, strengthen embryo's blastocyst rate and increased the total cell count in blastaea, thereby improve quantity, the quality of blastaea, therefore, can reduce the production cost of clone pig and improve its production efficiency, contributing to further developing and applying of clone technology;
(2) in the present invention, the contained epigenetic modification agent EPZ004777 of nutrient solution is the epigenetic modification agent of high specific, such as, it has been used at tumour medical field the propagation that selectivity suppresses MLL rearrangement property leukemia cell, compare commercially available similar most compounds, can't cause the intervention in the epigenetic site of wide spectrum, thereby there is higher biological safety;
(3) in the present invention, contained EPZ004777 transformation period of nutrient solution is very short, is handling after embryo's early development, can not grow and cause disadvantageous effect the later stage because of drug residue.
［ brief description of the drawings ］
Fig. 1 is the expression dynamic change figure of H3K79me2.
［ embodiment ］
For making the present invention easier to understand, will further set forth specific embodiments of the invention below.
Given an example contriver's the standard laboratory practice of following implementation column, for example pattern of the present invention, and should not be interpreted as the present invention the scope that is defined in these embodiment.These embodiment, and those skilled in the art's open according to the present invention general level, technician will understand following only for example, can in scope of the present invention, carry out various variations, modification and transformation being no more than.Wherein related technology unless stated otherwise, is all normal experiment technology well known to those skilled in the art.
If do not specialize, the reagent using in following examples and material all can obtain from commercial channels.
The preparation of embodiment 1. porcine oocytes maturation culture solutions
In basic TCM-199 (Earle ' s salts), add 10% pig follicle liquid, 5% foetal calf serum, 10IU/mL pregnant mare serum gonadotrop(h)in (PMSG), 10IU/mL human chorionic gonadotrophin, 10ng/mL Urogastron, 100IU/mL penicillin and 100 μ g/mL Streptomycin sulphates formulated.
Obtaining and maturation in vitro of embodiment 2. porcine oocytes
By the pig ovary of just having won from slaughterhouse, put into 37 DEG C of physiological saline containing penicillin, Streptomycin sulphate, in 2 hours, transport laboratory back; The ovary of fetching with 75% alcohol spray disinfectant once, stroke-physiological saline solution washing 3 times, then extract the ovarian follicle of 2～6mm diameter on ovary with the 10mL plastic injector for temporary use of being furnished with No. 18 syringe needles, be placed in the 15mL centrifuge tube of 38.5 DEG C of thermostat water baths extracting liquid injection, after taking out ovum and finishing, centrifuge tube is placed on 38.5 DEG C of Thermostatic platforms and leaves standstill 15 minutes, allow ovarian cumulus-ovocyte complex body (Cumulus-Oocyte Complexes, COCs) natural subsidence; Supernatant liquor in reject centrifuge tube, and add egg-cleaning liquid (containing the DPBS damping fluid of 0.01%PVA) dilution precipitate and mix, under the body formula mirror with hot platform, pick rapidly kytoplasm even, have two-layer above cumulus cell and wrap up complete COCs.
After being washed to 3 times with oocyte maturation nutrient solution, the COCs of acquisition proceeds at 38.5 DEG C, 5%CO
2, 4 hours above paraffin oils of balance cover in 100% humidity incubator maturation cultivates 42 ± 2 hours in cultivating and dripping, every 50 μ L are ripe, and drops are cultivated 15 pieces of COCs.
Embodiment 3. acquisitions of pig MII phase ovocyte and the preparation of nuclear transplantation donorcells
The COCs that maturation was cultivated after 42 ± 2 hours moves into containing in the DPBS damping fluid of 1mg/mL Hya Unidasa with 38.5 DEG C of preheatings, blow and beat gently and repeatedly about 200 times or 3 minutes with pipettor again, check afterwards COCs under mirror around, whether cumulus cell has removed totally, the ovocyte that has taken off ovarian cumulus is moved into T2 operation liquid (containing the TCM199 of 2% foetal calf serum) drop washing 3 times, during the T2 that shift-in mineral oil covers drips; Select all gaps of ovum obvious, dotter haut is complete, and discharges the ovocyte of first polar body, is MII phase ovocyte.
Donor cell adopts the porcine fetus fibroblasts or the ear inoblast somatocyte that are cultured to 3rd～9 generations, selection degree of converging is at 70%～90% cell, starting to carry out micrurgy first 1 hour, after 37 DEG C of digestion are single cell suspension, be kept in 4 DEG C of refrigerators stand-by with 0.25% trypsinase-EDTA.
Embodiment 4. pig nuclear transplantation are somatic cell clone embryo's preparation
With the somatocyte of preparing in T2 liquid washing refrigerator, the MII phase ovocyte of choosing is put into together with somatocyte to the nuclear transplantation operation liquid of micrurgy ware, this nuclear transplantation operation liquid is the TCM199 containing 7.5 μ g/mL cytochalasin Bs and 2% foetal calf serum of HEPES buffering, and operation ware is placed on the micrurgy platform of 38.5 DEG C of Thermostatic platforms; Fix after ovocyte with locking pin, draw first polar body and oocyte nuclei is contained at approximately 1/5～1/4 place kytoplasm thereof around with kernel removing needle, draw afterwards somatocyte (diameter 15～20 μ m, refractivity is strong, circular, smooth), enter ovocyte zona pellucida bet core from stoning otch, press zona pellucida, make somatocyte and cytoplasmic membrane close contact; After having operated, reconstruct ovum is transferred in the T2 drop that paraffin oil covers, in the thermostat container of 38.5 DEG C, recovered 30 minutes; The reconstruct ovum having recovered in T2 drop is moved in fusion/activation solution and washed 3 times, put into for 10 pieces every batch and be paved with the integration slot that merges liquid, that donorcells-acceptor oocyte membrane contact surface is vertical with direction of an electric field, with CF-150B type fusion instrument (BLS company, Hungary) the electric pulse induction that applies single 156V/mm, 100 μ s merges electricity simultaneously and activates, with embryo medium washing 3 times, proceed to immediately in the PZM-3 nutrient solution that covers paraffin oil 38.5 DEG C, 5%CO subsequently
2, 100% humidity cultivates after 30 minutes and takes out, decision fusion under Stereo microscope.
The preparation of embodiment 5. pig embryo mediums
Get respectively embryo culture water 50mL and dissolve 0.024g magnesium sulfate and 0.303g calcium lactate five water, after merging completely by the two mixing, and be sequentially added into 3.156g sodium-chlor, 1.053g sodium bicarbonate, 0.373g Repone K, 0.024g potassium primary phosphate, 0.011g Sodium.alpha.-ketopropionate, 0.073g L-glutaminate, 0.250g inositol, 0.273g Hypotaurine, 0.033g penicillin G, 0.025g Streptomycin sulphate, 10mL indispensable amino acid, 50mL non-essential amino acid, 0.5nmol/L EPZ004777(experimental group is added, control group does not add), fully mix and add embryo culture water with volumetric flask and be settled to 500mL afterwards, finally, add 1.5g bovine serum albumin, adjust pH is 7.2～7.4,4 DEG C of preservations.
Embodiment 6. porcine somatic cell cloning embryos embryos' vitro culture
By the clone embryos of decision fusion, move in chemical assisted activation liquid (containing embryo medium, 10 μ g/mL cycloheximides, 5 μ g/mL cytochalasin Bs) and activate and cultivate 4 hours, then, with embryo medium washing 3 times, proceed at 38.5 DEG C, 5%CO
2, 4 hours above paraffin oils of balance cover in 100% humidity incubator embryo culture drips middle cultivation and processes 24 hours, every 50 μ L embryo culture drops are cultivated approximately 15 pieces of embryos; Subsequently with the same but not containing the embryo medium washing of EPZ004777 3 times, then proceed to this embryo culture that balance crosses and continue to be cultured to ectogenesis to blastaea in dripping.
Production, cultivation and the observation of control group clone embryos, qualification, except omnidistance use contains the embryo medium of EPZ004777, all testing sequences are all consistent with above-mentioned.
Observation and the statistics of embodiment 7. porcine somatic cell cloning embryos vitro Development of Embryos situations
Embryo cultivates after 48 hours and 168 hours and (while starting chemical assisted activation cultivation, counts the 0th hour) in incubator, the formational situation of the difference observed and recorded spilting of an egg and blastaea under Stereo microscope; Blastaea total cell count is to adopt fluorescence dye Propidium Iodide to carry out core to embryonic cell to dye, and then under fluorescent microscope, adds up.Testing data adopts One-way ANOVA in SPSS13.0 software to carry out data statistic analysis, and in all analytical resultss, P < 0.05 represents significant difference.
By the test-results of the control group of the present embodiment and experimental group is compared (experiment repeats 6 times), its data statistics result is as shown in table 3.
Table 3.EPZ004777 processes the impact of porcine somatic cell cloning embryos embryo on early development
Note: 1. spilting of an egg rate=spilting of an egg number/cultivation number; 2. blastaea rate=blastaea number/cultivation number; 3. on same hurdle, mark different letter representation significant differences (P<0.05).
In addition, if EPZ004777 processing porcine somatic cell cloning embryos embryo's time is too short or long, cannot produce promoter action to embryo's early development, the treatment time is long, even can suppress embryo's growth.
The histone methylated immunofluorescence dyeing of embodiment 8. porcine somatic cell cloning embryos embryo
After DPBS damping fluid washing cultivation each stage clone embryos of 0 to 24 hour, use 4% paraformaldehyde to fix 15 minutes; DPBS damping fluid washs after 3 times (5 minutes/time), uses penetrating 30 minutes of 0.5%Triton X-100; After DPBS damping fluid washing 3 times (5 minutes/time), embryo is processed and within 15 minutes, makes DNA sex change in 2N HCl, then move in Tris-HCL, process 20 minutes with in and HCl; Using 4% paraformaldehyde to carry out secondary fixes; 3 times (5 minutes/time) of DPBS damping fluid washing, then with the DPBS damping fluid sealing treatment containing 1%BSA; After repetitive scrubbing, add successively primary antibodie (antibody of anti-H3K79me2 is purchased from Abcam company), two anti-(Alexa Fluor594 is purchased from Molecular Probes companies) to hatch during this time; After finally the Embryo Cell Squash Technique dyeing being processed, use conventional fluorescence microscope.With " ImageJ " software collection and add up fluorescent signal value, result shows as Fig. 1, compare control group, can there is H3K79me2 demethylation in the initial stage that the porcine somatic cell cloning embryos embryo that EPZ004777 processed cultivates growth in vitro, make it the embryo close to natural fertilization more quickly.
The production of embodiment 9. somatic cell clone pigs
In multiparity Gilt Uterus by the clone embryos of preparing by such scheme with Nonoperative method immigration spontaneous estrus, carry out gestation.The step of Nonoperative method embryo transfer, for after slightly anaesthetizing with Sodital, is inserted the thick conduit of external diameter 10mm from acceptor sow vagina to cervical canal, then the fine duct of diameter 5mm is inserted to thick conduit inner side, extend into body of uterus or a side horn of uterus.Embryo is preserved to liquid together with 2mL and transplant into by fine duct, the carbonic acid gas specifically generating with dry ice is blown into intrauterine 1～3 minute through conduit by embryo.After embryo transfer, whether 30 days B ultrasounds detect gestation.
Result shows: compared with control group, adding the porcine somatic cell cloning embryos embryo medium of 0.5nmol/L EPZ004777 processes after embryo 24h, embryo's spilting of an egg rate does not significantly improve (73.60%vs67.29%, P > 0.05), (the 28.97%vs17.13% but the developmental rate of blastaea and total cell count are significantly increased, 45vs31, P < 0.05); The commitment of the reprogrammed being mediated by ovocyte at somatic cell nuclear, can there is quickly demethylation in H3K79me2, and this is conducive to cell and reprogrammed occurs thoroughly and chromatinicly normally reinvent; And the clone embryos obtaining is for embryo transfer, become pregnant can normal development gestation in sow body.Therefore, by using EPZ004777 to carry out vitro culture processing to porcine somatic cell cloning embryos embryo, can improve the deficiency of ovocyte to somatic cell nuclear reprogrammed, and promote embryo's cell proliferation, finally improve the efficiency of somatic cell clone.
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although the present invention is explained in detail with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not depart from essence and the scope of technical solution of the present invention.
Claims (7)
1. epigenetic is repaiied the application of reagent E PZ004777 for the preparation of the nutrient solution of promotion porcine somatic cell cloning embryos vitro Development of Embryos.
2. a nutrient solution that promotes porcine somatic cell cloning embryos vitro Development of Embryos, is characterized in that: comprise that epigenetic repaiies reagent E PZ004777, its content is 0.1～1nmol/L.
3. nutrient solution according to claim 2, is characterized in that: the concentration of described EPZ004777 is 0.5nmol/L.
4. nutrient solution according to claim 2, is characterized in that, also comprises the component of following content:
5. nutrient solution according to claim 4, is characterized in that, comprises the component of following content:
6. a method that promotes porcine somatic cell cloning embryos vitro Development of Embryos, is characterized in that, comprises the following steps:
A. the nutrient solution being just placed in as described in one of claim 2 to 5 through the porcine somatic cell cloning embryos embryo of reconstruct fusion is processed 6～30 hours;
B. embryo after treatment is moved into not containing in the nutrient solution of EPZ004777, continue to be cultured to described vitro Development of Embryos and become blastaea.
7. application according to claim 6, is characterized in that: in described steps A, it is 24 hours that described embryo cultivates the time of processing in nutrient solution.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834216A (en) * | 2017-02-20 | 2017-06-13 | 上海市农业科学院 | A kind of in vitro culture liquid and cultural method for the lonely female activation embryo of pig |
| CN107201343A (en) * | 2017-07-14 | 2017-09-26 | 阜阳师范学院 | A kind of goat cells clone embryos nutrient solution and cultural method |
| CN108060117A (en) * | 2017-12-01 | 2018-05-22 | 广东温氏食品集团股份有限公司 | A kind of method for improving porcine clone embryos development efficiency |
| CN108374028A (en) * | 2017-02-27 | 2018-08-07 | 扬州大学 | A method of improving azaphilone class compound productions in marine fungi using apparent stoichiometries genetic modification |
| CN113755428A (en) * | 2020-06-02 | 2021-12-07 | 江苏省农业科学院 | Culture method for improving livestock embryo in vitro development quality |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827812A (en) * | 2011-06-17 | 2012-12-19 | 中国科学院动物研究所 | Preparation method and application of induction type neural stem cells |
| CN103074294A (en) * | 2013-01-22 | 2013-05-01 | 安徽农业大学 | Pig embryo culture solution capable of improving ectogenetic efficiency of pig embryo and preparation method thereof |
-
2014
- 2014-03-28 CN CN201410122806.8A patent/CN103952368B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827812A (en) * | 2011-06-17 | 2012-12-19 | 中国科学院动物研究所 | Preparation method and application of induction type neural stem cells |
| CN103074294A (en) * | 2013-01-22 | 2013-05-01 | 安徽农业大学 | Pig embryo culture solution capable of improving ectogenetic efficiency of pig embryo and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| TAMER T.ONDER 等: "Chromatin-modifying enzymes as modulators of reprogramming", 《NATURE》 * |
| 张玲 等: "表观遗传学药物的研究进展", 《中国药理学通报》 * |
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| CN106834216A (en) * | 2017-02-20 | 2017-06-13 | 上海市农业科学院 | A kind of in vitro culture liquid and cultural method for the lonely female activation embryo of pig |
| CN106834216B (en) * | 2017-02-20 | 2020-06-16 | 上海市农业科学院 | In-vitro culture solution and culture method for swine parthenogenetic activation embryos |
| CN108374028A (en) * | 2017-02-27 | 2018-08-07 | 扬州大学 | A method of improving azaphilone class compound productions in marine fungi using apparent stoichiometries genetic modification |
| CN108374028B (en) * | 2017-02-27 | 2021-05-11 | 扬州大学 | Method for improving yield of azaphilone compounds in marine fungi by utilizing chemical epigenetic modification |
| CN107201343A (en) * | 2017-07-14 | 2017-09-26 | 阜阳师范学院 | A kind of goat cells clone embryos nutrient solution and cultural method |
| CN107201343B (en) * | 2017-07-14 | 2021-05-11 | 阜阳师范学院 | Goat cell clone embryo culture solution and culture method |
| CN108060117A (en) * | 2017-12-01 | 2018-05-22 | 广东温氏食品集团股份有限公司 | A kind of method for improving porcine clone embryos development efficiency |
| CN108060117B (en) * | 2017-12-01 | 2021-08-20 | 温氏食品集团股份有限公司 | Method for improving pig cloned embryo development efficiency |
| CN113755428A (en) * | 2020-06-02 | 2021-12-07 | 江苏省农业科学院 | Culture method for improving livestock embryo in vitro development quality |
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