CN101050458A - Method for once more cloning milch cow - Google Patents
Method for once more cloning milch cow Download PDFInfo
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- CN101050458A CN101050458A CN 200710064767 CN200710064767A CN101050458A CN 101050458 A CN101050458 A CN 101050458A CN 200710064767 CN200710064767 CN 200710064767 CN 200710064767 A CN200710064767 A CN 200710064767A CN 101050458 A CN101050458 A CN 101050458A
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Abstract
This invention relates to a method for re-cloning milk cow. The method comprises: culturing donor cells and receptor oocytes, transplanting, fusing, activating, culturing reconstructed embryo, and transplanting reconstructed embryo. The culture medium for the receptor oocytes contains: M199 + 10% FCS + 10 mu.g/mL FSH + 1 mu.g/mL LH + 1 mu.g/mL estradiol + 0.0625 g penicillium + 0.05 g streptomycin + 0.57 mmol/mL cysteine + 1 mmol/mL L-glutamine + 10 ng/mL EGF (epidermal growth factor). The culture medium for the donor cells contains: 1 L DMEM (dry powder) + 2.75 g NaHCO3 + 10% FCS + 1 mmol/L Na-pyruvate + 1 mmol/mL L-glutamine + 0.0625 g penicillium + 0.05 g streptomycin + 0.1 mmol/L beta-mercaptoethanol + 10 ng/mL EGF. The method can magnify the cloning effect, and can increase cloning efficiency.
Description
Technical field
The invention belongs to the method for embryo's bioengineering field, particularly a kind of once more cloning milch cow.
Background technology
From then on the birth of clone sheep Dolly, animal cloning enter the somatic cell clone epoch.Follow closely after the Dolly sheep, somatic cell clone research is carried out in many laboratories, various cloned animals come out one after another (Baguisi, 1999; Wakayama, 1998; Kao, 1998; Onishi, 2000; Shin, 2002; Chense, 2002; Galli, 2003; Woods, 2003).By somatic cell clone technique, people can obtain countless homogeneity individualities, have overcome embryonic cell and have cloned the shortcoming that can only reach limited individuality.The application of this technology except protect kind of procreation animal aspect at present, aspect transgenosis, the embryonic stem cell aspect obtained application, demonstrated tempting application prospect.
Somatic cell clone technique is that animal protects one of kind of important method.Protect kind by somatic cell clone technique, can become an animal to the somatocyte of an animal.If preserve species, just can draft out earlier the animal individual number of maintenance genetic stability, gather thousands of cells on one's body each animal then, carry out permanent preservation, if when needing to reappear an animal species or kind, only of the same race or interspecies cloning need be carried out, just this animal can be obtained.The somatocyte of an animal can be kept in the tubule, and a kind or species just can be kept in the capsule, and perhaps the animal species of China can be contained in the big room, so just can set up a genuine gene pool.Comizzoli etc. (2000) think that body-cell neucleus transplanting also opens up a new way for the rescue rare species.1998 (1999) such as Zelanian Wells utilize (2000) such as Lanza that this technology successfully cloned the local soil species ox U.S. in 2000 that is on the verge of to commit genocide method by inter-species nuclear transplantation to obtain the inter-species nuclear transplantation embryo with the ovocyte of tame ox, transplant to have obtained pregnant behind the ox of getting home and born the offspring; The old big units of 1999 China etc. have obtained the giant panda embryo of inter-species nuclear transplantation with rabbit oocyte; these results show that the quantity by body-cell neucleus transplanting method increase animals on the brink of extinction is feasible, may have breakthrough progress in the near future by inter-species nuclear transplantation method protection rare animal.
Somatic cell clone technique also is in conceptual phase, and also there are some problems in somatic cell clone:
1) unstable result, efficient is low at present, and clone sheep has only the reconstituted embryo about 3% to grow to be normal offspring.In the research process of clened cows, according to the result of kato etc., the high energy of the blastocyst rate of reconstituted embryo reaches 49%, have 80% transplanting embryo to grow approximately and be normal fetus, but the postnatal mortality ratio of fetus reaches 50%.Cibelli etc. are in the production process of clened cows, and the blastaea rate of reconstituted embryo is 17% only, have only 14% blastaea to grow normal offspring after the transplanting.The clone mouse is only succeedd in University of Hawaii medical college, have only the reconstructed embryo of 2%-3% can grow farrowing, the newborn mouse mortality ratio also shows that up to the above result of 40%o somatic cell clone technique only is in the preliminary study stage, also need further study on other animals, enhances productivity.
2) the postnatal high mortality of the high fetus of offspring's mortality ratio also is one of problem of facing of somatic cell clone, and the reason that this phenomenon occurs is not clear.Can only start with to reduce fetal mortality from optimizing culture condition and schedule of operation at present.
3) it is present to do the exploration of donor with other somatocyte, and the donorcells kind that is used for somatic cell clone is very limited, and can other cell successfully remain further to be inquired into by nuclear transplantation.
4) the plasma inheritance problem is in clone's research of mouse and ox, it is found that cytoplasmic genetic material influence clone offspring's phenotype, as piebald, hair color etc., this is by the donorcells Mitochondrial DNA, still causes not clear by the genetic material in the ovum tenuigenin.Whether the genetic material in the tenuigenin exerts an influence to other proterties of cloning the offspring also needs further discussion. and along with the development of clone technology, the effect of genetic material can more and more cause people's attention in the tenuigenin.
5) one of sharpest edges that influence somatic cell clone of the preservation of donorcells and cell cycle are that donorcells can be cultivated and goes down to posterity and freezing preservation.But go down to posterity and refrigerating process in, the damage of genetic material and variation certainly will influence the growth of clone embryos in the nucleus.Simultaneously, the donorcells cycle directly influences cloning efficiency, but these problems are known little about it at present, is badly in need of being studied.
Somatocyte with cloned animal is cloned once more, is called clone again.In theory, by cloning and cloning again, can obtain unlimited many cloned animals.Clone technology is consistent with the clone technology ultimate principle again, but clone technology is further amplified clone's effect again, extends clone's intension, clones research again and will help to address the above problem.
Summary of the invention
The present invention is directed to the defective in the above-mentioned field, the cloning process again of a kind of milk cow is provided, clone's effect is amplified once more, is one of effective ways that improve cloning efficiency.
The cloning process again of a kind of milk cow, comprise the cultivation of donorcells and acceptor ovocyte, nuclear transplantation, fusion and activation, the cultivation of reconstructed embryo and reconstructed embryo are transplanted, and it is characterized in that: be added with in the nutrient solution of described recipient cell: M199+10%FCS+10 μ g/ml FSH+1 μ g/ml LH+1 μ g/ml Estradiol+0.0625g Penicillium+0.05gStreptomycin+0.57mmol/ml Cysteine+1mmol/ml L-Glutamine+10ng/ml EGF (Epidermal GrowthFactor); Be added with 1LDMEM (dry powder)+2.75g NaHCO in the nutrient solution of donorcells
3+ 10%FCS+1mmol/LNa-Pyruvate+1mmol/ml L-Glutamine+0.0625g Penicillium+0.05g Streptomycin+0.1mmol/L β-Mercaptoethanol+10ng/ml EGF.
The cultivation of described reconstructed embryo is adopted in three stages and is cultivated, the nutrient solution of described fs is: CR1+1%NEAA+1%EAA+1mmol/ml L-Glutamine+4mg/ml FAF-BSA+0.0625g Penicillium+0.05gStreptomycin, cultivation time: 0-2 days; The nutrient solution of described subordinate phase: fs nutrient solution and monolayer of particles cell, cultivation time: 2-5 days; The nutrient solution of phase III: the subordinate phase nutrient solution adds glucose, and its concentration is 1mg/ml, incubation time 5-9 days.
Described activation adopts A23187, CHX and CD to unite activation, and activationary time activated processing in 25 hours behind oocyte maturation.
Described activation is handled and was divided for three steps carried out: 1.CR1aa+5 μ molA23187+1mg/mlFAF-BSA, handle 4min at 38.5 ℃; 2. moving into CR1aa+10 μ g/ml CHX+3 μ g/ml CD handled 1 hour; 3. in the CR1-aa that contains 10 μ g/ml CHX, continue to cultivate 4 hours.
Described reconstructed embryo is transplanted to the intrauterine than its late one day recipient cattle of oestrusing.
The cloning process again of a kind of milk cow of the present invention, donor nuclei are the somatocyte of first-generation clened cows.The step of clone technology is identical with the step of clone technology again, the present invention is for improving cloning efficiency, at first optimized the culture condition of donorcells and recipient cell, some effective constituents have been added, as Cysteine, L-Glutamine, EGF (Epidermal Growth Factor) and β-Mercaptoethanol etc.These compositions can both promote that cell grows, and can improve the tenuigenin ripening degree, strengthen cell viability, and then strengthen the vitality of reconstructed embryo, help to improve the clone embryos pregnancy rate, reduce the birth mortality ratio of fetus.The concrete effect of these effective constituent pair cells is as follows:
Cysteine is the important component that constitutes GSH (Triptide), and GSH can make ovocyte avoid oxidational losses by oxidation-reduction, can remove the free radical in the ovocyte simultaneously, improves the whole developmental level of ovocyte.The pathways metabolism of GSH makes the important component part of kytoplasm ripening process, not only can promote oocyte maturation, and early embryonic development is also had promoter action.
Adding L-Glutamine can promote each seed amino acid to enter cytolemma, its contained amino is the source of purine and pyrimidine in the nucleic acid, also be the source of energy and carbon, visible ovocyte needs L-Glutamine rely nucleic acid and protein, promotes embryo growth and division.
EGF is the somatomedin of a peptide species.Can improve the cell development environment greatly, promote nuclear, the matter maturation of cell.
β-Mercaptoethanol also can promote the synthetic of the interior GSH of cell, improves cell development potentiality and tolerance, prevents the cellular oxidation apoptosis.
At reconstructed embryo in different developmental phases to nutritional need and own characteristic, adopt stage by stage cultural method to adjust its nutrient solution nutritive ingredient, so more help growing of reconstructed embryo, can improve the developmental rate of blastaea simultaneously.Adding granulosa cell in subordinate phase cultivates altogether.Cultivate altogether with granulosa cell and to help to overcome embryo's ectogenesis blocking-up, because granulosa cell energy secrete polypeptide and glycoprotein, play the effect of mitogenesis stimulating factor, promote embryo's separatist activities, overcome the retardance of ox embryo 8-16 cell effectively, so after cultivation, carried out common cultivation on the 2nd day.Along with fetal development, the later stage metabolism is more and more vigorous, needs more multipotency, and glucose is the easiest energy matter that is absorbed by the embryo, adds glucose and can in time replenish fetal development institute energy requirement.So on the nutrient solution of phase III is basis in subordinate phase, add glucose again.
The present invention adopts A23187, CHX and CD to unite activation, activationary time is 25 hours (it is 0 hour that ovocyte begins maturation) behind the oocyte maturation, clone activationary time and adopted 24 hours the first time of most literature record, the present invention activates and postpones 1 hour, increase the time that donor nuclei is exposed to the female kytoplasm of acceptor ovum like this, can make for nuclear and kytoplasm fully to interact, help to revise and clone the mistake that causes the nuclear programming first time, postpone 1 hour so activate ratio clone's first time.
Because the clone embryos growth in vitro is grown the inconsistent of kinetics and conceived naturally cow embryo again, often show as hypoevolutism, for endometrial growth of acceptor and donor clone embryos developmental stage are adapted, give simultaneously and be transplanted to intrauterine one period time of recovery of clone embryos again, select the recipient cattle of a little later oestrusing for use, the two normal development synchronously than donor embryo.More suitable through experiment showed, that the blastaea of cloning 7 days again moves into the recipient cattle intrauterine oestrused 6 days.
The present invention obtains 282 reconstructed embryos, grows to obtain 76 blastaeas, transplants 28 blastaeas; growth obtains 2 and clones milk cow again; the appearance of clened cows again and every physical signs of birth are all normal, show through the little satellite analytical results of DNA, and two is cloned the milk cow genotype again and cloned the identical of milk cow.
In a word, according to once more cloning milch cow method of the present invention, successfully obtain to clone individuality, the birth mortality ratio is zero again, and calving rate is cloned basically identical with the first time, and from the entire operation program, present method should be suitable for equally clones the first time of milk cow.
Embodiment
The present invention is described in further detail below by embodiment.
Embodiment 1
1. key instrument equipment, medicine
Micrurgy instrument, electricity melt instrument, draw pin and card grinding instrument, CO
2Incubator, constant temperature platform, view volume microscope, water-bath, thermostat container, super clean bench.
Related to medicine and reagent be the commercially available prod.
2. cows
Acceptor cows are set up in experimental requirement, and the clened cows of being born needs 20 recipient cattle approximately.
3. the preparation of donorcells
After cutting a fritter tissue from clone's holstein cow the ears of an ox or cow portion, the vacuum flask that usefulness is equipped with 0.9% physiological saline is taken back the laboratory.The ear tissue of cutting in super clean bench washs 3 times, shreds, goes cartilage, transfer to 4 ℃ of cold digestion in the culturing bottle after adding an amount of 0.25% pancreatin mixing, 37 ℃ of heat digestion 40 minutes, after digestion is good, add nutrient solution: 1L DMEM (dry powder)+2.75g NaHCO after 18-20 hour
3+ 10%FCS (Fetal Calf Serum)+1mmol/L Na-Pyruvate+1mmol/mlL-Glutamine+0.0625gPenicillium+0.05g Streptomycin+0.1mmol/L β-Mercaptoethanol+10ng/mlEGF (Epidermal Growth Factor) places 37 ℃, 5%CO
2Cultivate in the incubator.After cell attachment covers with, go down to posterity.Cell freezing before two generations is preserved.
Experimental design, with the cultivation of thawing in preceding 48 hours, 37 ℃ of had digestive transfer culture of the pancreatin with 0.25% or collection, collected cell is blown and beaten repeatedly with pipettor, makes cell with single suspension, is used for nuclear transplantation.
4. the maturation of ovocyte:
Collect ovary from the slaughterhouse.With being added with two anti-25 ℃ of physiological saline, within 3 hours, be transported to the laboratory.Extract the big ovarian follicle of 2-7mm on the ovary with syringe, under stereoscopic anatomical lens, select the ovocyte (COCs) that is surrounded by complete granulosa cell and be used for ripe the cultivation.Maturation culture solution is to be made of M199+10%FCS (Fetal Calf Serum)+10 μ g/mlFSH (Follicle-stimulating Hormone)+1 μ g/ml LH (Luteinizining Hormone)+1 μ g/ml Estradiol+0.0625g Penicillium+0.05g Streptomycin+0.57mmol/ml Cysteine+1mmol/ml L-Glutamine+10ng/mlEGF (Epidermal Growth Factor), at 38.5 ℃, 5%CO
2, the CO of saturated humidity
2Cultivated 18 hours in the incubator.Slough granulosa cell with 0.2%Hyaluronidase, selecting under stereoscopic microscope has ovocyte first polar body, the kytoplasm uniformity, places the Hepes-199 liquid that contains 10% serum stand-by.
5. nuclear transplantation
Blind suction method is adopted in the stoning of ovocyte.20 pieces of mature oocytes of sloughing granulosa cell are put into an end of operating drop, the operation drop is by Hepes-199,10% serum and 7.5ug/ml Cytochalasin B form, pre-treatment is after 5 minutes at room temperature, hold one piece of ovocyte with holding the ovum pin, adjust the position of polar body, make its first polar body be positioned at the position of 2 on clock.With first polar body and its underpart 1/4-1/3 material sucking-off, inhale the inoblast that diameter is 15-20 μ m with kernel removing needle with kernel removing needle then, and be injected under the zona pellucida, and the donorcells film is closely contacted with egg membrane from same otch.
6. merge and activation
(USA) type cell electricity fusion instrument merges for BTX, San Diego, and merging liquid is 0.28MMannitol+0.05mM CaCL with BTX ECM-2001
2.2H
2O+0.1mM MgCL
2.6H
2O+0.5mM Hepes+5mg/ml BSA (BovineSerum Albumin).Ovocyte and somatic complex body are merged balance 2min in the liquid at electricity, move into then and be full of between electricity fusion liquid two electrodes, stir with thin glass needle and to make somatocyte parallel with wire electrode with the contact surface of acceptor, induce fusion with twice 1.3kv/cm, 10 μ s electric pulses, 0.5h observes situation about merging behind the mixing operation.
All merge clone embryos again and are activating processing (clone is to activate processing in back 24 hours in maturation for the first time, and ovocyte begins ripe the cultivation and is 0h) after the maturation about 25h.Activating to handle divided for three steps carried out: 1.CR1aa+5 μ molA23187+1mg/mlFAF-BSA (Fatty Acid Free-Bovine Serum Albumin), handle 4min at 38.5 ℃; 2. moving into CR1aa+10 μ g/ml CHX (Cycloheximide)+3 μ g/ml CD (Cytochalasin D) handled 1 hour; 3. in the CR1-aa that contains 10 μ g/ml CHX (Cycloheximide), continue to cultivate 4 hours.
7. cultivate the preparation of granulosa cell individual layer altogether
With thin Glass tubing with the mature oocyte sucking-off in the ripe drop, the part granulosa cell is stayed, change to fetal development liquid [CR1+1%NEAA (Non-essential Amino Acids)+1%EAA (Essential Amino Acids)+1mmol/mlGlutamine+4mg/ml FAF-BSA (Fatty Acid Free-Bovine Serum Albumin)] then, soon just form granular cell layer, changed liquid once in per 2 days, this is the common cultivation drop of the subordinate phase of reconstructed embryo.
8. reconstructed embryo is grown
Fs (0-2 days): reconstructed embryo is cultivated in growing liquid CR1+1%NEAA+1%EAA+1mmol/ml L-Glutamine+4mg/ml FAF-BSA+0.0625g Penicillium+0.05g Streptomycin and is grown.Subordinate phase (2-5 days): the reconstructed embryo that will grow 2 days moves in the drop of cultivating altogether (CR1+1%NEAA+1%EAA+1mmol/ml L-Glutamine+4mg/ml FAF-BSA+0.0625g Penicillium+0.05g Streptomycin and granular cell layer).Phase III (5-9 days): add glucose in the 5th day embryo's nutrient solution, its concentration is 1mg/ml.Changed liquid once in per 2 days, the quantity of record morula and blastaea after the 6th day, and carry out statistical study.
9. embryo transfer
The blastaea that 1 piece of form is good move into than its late one day recipient cattle intrauterine of oestrusing (such as, the 7th day blastaea is transplanted to the 6th day recipient cattle intrauterine of oestrusing) after 2 months, surpass and definite conceived situation by B-.If there is not the suitable recipient cattle of oestrusing,, transplanting later on the freezing preservation of reconstructed embryo.
10. clone the calf birth again
At pregnancy duration, with the super fetation situation of observing of B-, the excessive or malpresentation of discovery fetus is carried out c-section just before giving birth before, if fetation normally allows its spontaneous labor.
The present invention obtains 282 reconstructed embryos, grows to obtain 76 blastaeas, and the blastaea rate is 26.9% (76/282); Transplant 28 blastaeas, obtain 2 and clone milk cow again, calving rate is 7.1% (2/28), and the external form of clened cows again and every physical signs of birth are all normal.The blastaea rate and the natality of the inventive method all are improved, so that cloning efficiency obtains large increase.
The little satellite analytical results of DNA shows that two is cloned the milk cow genotype again and cloned the identical of milk cow, shows that the cloning process again of milk cow of the present invention is feasible.
Claims (5)
1. the cloning process again of a milk cow, comprise that donorcells is cultivated, the acceptor ovocyte is cultivated, nuclear transplantation, fusion and activation, the cultivation of reconstructed embryo and reconstructed embryo are transplanted, and it is characterized in that: be added with in the nutrient solution of described acceptor ovocyte: M199+10%FCS+10 μ g/ml FSH+1 μ g/ml LH+1 μ g/ml Estradiol+0.0625g Penicillium+0.05gStreptomycin+0.57mmol/ml Cysteine+1mmol/ml L-Glutamine+10ng/ml EGF; Be added with 1LDMEM dry powder+2.75g NaHCO in the nutrient solution of donorcells
3+ 10%FCS+1mmol/L Na-Pyruvate+1mmol/mlL-Glutamine+0.0625g Penicillium+0.05g Streptomycin+0.1mmol/L β-Mercaptoethanol+10ng/ml EGF.
2. the cloning process again of milk cow according to claim 1, the cultivation of described reconstructed embryo is adopted in three stages and is cultivated, the nutrient solution of described fs is: CR1+1%NEAA+1%EAA+1mmol/ml L-Glutamine+4mg/mlFAF-BSA+0.0625g Penicillium+0.05g Streptomycin, cultivation time: 0-2 days; The nutrient solution of described subordinate phase: fs nutrient solution and monolayer of particles cell, cultivation time: 2-5 days; The nutrient solution of phase III: the subordinate phase nutrient solution adds glucose, and glucose concn is 1mg/ml, incubation time 5-9 days.
3. the cloning process again of milk cow according to claim 1, described activation adopt A23187, CHX and CD to unite activation, and activationary time activated processing in 25 hours behind oocyte maturation.
4. the cloning process again of milk cow according to claim 3, described activation are handled and were divided for three steps carried out: 1.CR1aa+5 μ molA23187+1mg/mlFAF-BSA, handle 4min at 38.5 ℃; 2. moving into CR1aa+10 μ g/ml CHX+3 μ g/ml CD handled 1 hour; 3. in the CR1-aa that contains 10 μ g/ml CHX, continue to cultivate 4 hours.
5. the cloning process again of milk cow according to claim 1, described reconstructed embryo is transplanted to the intrauterine than its late one day recipient cattle of oestrusing.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101302497B (en) * | 2008-06-27 | 2010-07-21 | 华中农业大学 | Method for cloning embryo by nuclear transfer of bovine somatic cells |
| CN102344941A (en) * | 2010-07-29 | 2012-02-08 | 内蒙古大学 | Method for breeding cloned animal or transgenic cloned animal |
-
2007
- 2007-03-26 CN CN 200710064767 patent/CN101050458A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101302497B (en) * | 2008-06-27 | 2010-07-21 | 华中农业大学 | Method for cloning embryo by nuclear transfer of bovine somatic cells |
| CN102344941A (en) * | 2010-07-29 | 2012-02-08 | 内蒙古大学 | Method for breeding cloned animal or transgenic cloned animal |
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