CN101302497A - Method for cloning embryo by nuclear transfer of bovine somatic cells - Google Patents
Method for cloning embryo by nuclear transfer of bovine somatic cells Download PDFInfo
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Abstract
The invention belongs to the mammal reproduction technology field, particularly relates to a method for obtaining cloned embryos with cow somatic cells and discloses a method for producing cow cloned embryos simply and efficiently. According to the research results that pig somatic cells dissolves and disappears automatically in thirty minutes after being injected into ooplasm, whole cells with sound membrane are extracted from cows as donors and are injected into the whole cell cytoplast directly to construct cow cloned embryos, simplifying the operational process of somatic cell nuclear transfer. The method is characterized in that: (1) selecting and cultivating cow ovarian cumulus- oocytes complex; (2) enucleating of the occytes; (3) preparing donor cells; (4) preparing co-cultivation cell monolayer; (5) constructing reconstructed fetus; (6) activating the reconstructed fetus; and (7) cultivating the reconstructed fetus. The method has the best feather of easy operation, simple steps, stable results, providing a new idea and method for the expansion and the further study of cow somatic cells nuclear transplanting technology.
Description
Technical field
The present invention relates to the mammalian reproduction technical field, be specifically related to the method that a kind of utilization utilizes the nuclear transfer of bovine somatic cells clone embryos.
Background technology
Breakthrough has appearred in body-cell neucleus transplanting in latter stage in 20th century, after many jasmines sheep was born, increasing somatic cell clone animal was born, and the significance of body-cell neucleus transplanting progressively obtains people's approval.But the developmental rate of the reconstructed embryo of this technique construction is low, and in the technology of making reconstructed embryo, the pre-treatment of donor cell and micro-notes nuclear stoning operation are crucial conditioning steps.
People mainly contain serum starvation method (Campbell et al., 1996) and contact inhibition method (Kasinathan et al., 2001) to the pre-treatment of donor cell at present, in the hope of reaching the purpose of cell synchronization.The somebody thinks that passage number (Wang Yuge etc., 1999) is also influential to the effect of nuclear transplantation.The report that also has some other special processings (Sullivan etal., 2004) in addition.But whether the whole bag of tricks has significant effect still not have final conclusion.
On the pitting method of ovocyte, present main stream approach is blind suction method, and blind suction method is with reference to carrying out stoning with the first polar body, comprise again and attract to remove nuclear method (McGrath Jet al.Science, 1983,220:1300) remove two kinds of methods of nuclear method (Shiga etal., 1999) with extruding.Pitting method also has (Wang Zhenfei etc., 2006) and manual cloning (Peura et al., 1998).These three kinds of methods cut both ways, and blind suction method is to the chemical lesion of ovocyte, but the position of first polar body and nuclear can be offset along with the maturation of ovocyte, so the stoning rate is not too high, and the loss of ovocyte kytoplasm is more.Go the operation of zona pellucida nucleus transplantation comparatively loaded down with trivial details, and the process of dyeing and uv irradiating is also arranged in its experimental procedure, understanding pair cell equally has injury.On which kind of method the best of structure clone embryo, there is not unified viewpoint so far.
On the structure of reconstructed embryo, mainly contain two kinds of the interior injections (Wakayamaet al., 1998) of electro fusion method (Wilmut et al., 1997) and kytoplasm." many jasmines " just is to use electro fusion method to make up.Yet it is that inefficiency, process are loaded down with trivial details that aforesaid method is faced with a common difficult problem.The operating environment of microscopically is unfavorable for cells survival, long may being harmful to fetal development of operating time.Because it is high and technical strong that micrurgy requires plant and instrument, therefore operator's skill level also is restraining factors, cultivate a skilled experimenter and also need long time, so a current difficult problem that needs to be resolved hurrily invent exactly a kind of simply, nuclear transplantation method efficiently, be beneficial to popularization and the application of nuclear transfer technology in wider scope.
Direct injection is by direct injection in the sperm kytoplasm (ICSI:intracytopasmic sperminjection) development (Collas and Barnes, 1994) in the kytoplasm.The key of injection is to annotate nuclear rupture of membranes in the past to handle so that the plasmarrhexis of donorcells in the kytoplasm.Directly annotate the nuclear method and mainly be divided into dual mode: auxiliary direct notes nuclear of Piezo drive unit and the auxiliary direct notes of common micromanipulation system are examined (Wolf et al., 1998; Fulka et al., 1998; Heymanet al., 1998; Campbell et al., 2007).
Direct injection is with behind the donor rupture of membranes in the kytoplasm, will examine cell space by Piezo driving injection device and directly be injected in the recipient cytoplasm, and the researchist adopts this kind method successfully to obtain clone mouse (Wakayama et al., 1998), clone pig.The latter does not then need this device, can realize direct injection in the complicated kytoplasm with common micromanipulation system.In order further to simplify the nuclear transplantation operating process, some researchists attempt utilizing the donorcells that does not carry out the rupture of membranes processing to carry out the nuclear transplantation of full cell direct injection, thereby the separation of nuclear and the process that electricity merges have been avoided, this kind method also successfully obtains clone Asia Mongolian gazelle offspring (Lee et al., 2005; Chen et al., 2007; Zhou et al., 2000).Full cell direct injection has been simplified the nuclear transplantation operating process greatly in the kytoplasm, has reduced SCNT is laid a good foundation for applying of this technology to the requirement of operator and testing installation.Injection progress of research situation sees Table 1 in the relevant kytoplasm.
Great milestone in the evolution of table 1 somatic cell clone
Up to now, Shang Weijian does donor about utilizing without the donorcells of serum starvation and rupture of membranes processing, adopts full cell direct injection to produce the research of ox clone embryos.
Summary of the invention
The objective of the invention is to overcome the defective that prior art exists, utilize the bovine oocyte stoning, utilize the nuclear transplantation method to obtain the method for ox embryo cloning.The present invention is simplifying unnecessary step as far as possible aspect the donorcells cultivation, improved the stoning of existing blind suction method in stoning operation, adopt the success of full cell direct injection acquisition the Niu Kelong blastaea.The present invention through a large amount of revision tests verified this method easy, quick, effective and and stablize, can obtain more satisfactory blastaea rate, for other mammiferous nuclear transplantation certain reference and reference are arranged.
The present invention is achieved in that
A kind of method that obtains the ox clone embryos, its step comprises: 1) get bovine oocyte and cultivate in maturation culture solution; 2) the ox donorcells is injected the acceptor ovocyte, obtain the reconstruct ovocyte; 3) with step 2) the reconstruct activation of oocytes, obtain reconstructed embryo, the invention is characterized in, the bovine oocyte that takes out is cultivated 22h in the maturation culture solution of improvement, 39 ℃ of culture temperature, make described bovine oocyte be in MII, remove the nucleus of mature oocyte, obtain the ox recipient cell, adopt full cell direct injection (Chen et al., 2007) the ox donorcells is injected described ox recipient cell, obtain the ox reconstructed embryo, place the droplet of the maturation culture solution of improvement to cultivate 3-4h described ox reconstructed embryo, then described ox embryo is exposed in the activator 1 and activates 5min, transfer to then in the activator 2 and activate 3-4h, activated ox reconstructed embryo is cultivated in embryo medium (do not added foetal calf serum in first day in 7 days in the cultivation of ox reconstructed embryo, from the foetal calf serum that began to add 10% volume in second day of vitro culture, to reach the effect that improves ox reconstructed embryo potentiality of development).
Nutrient solution that the present invention adopts or substratum moiety and proportioning are as follows:
Phosphoric acid buffer (DPBS): sodium-chlor 8.0g/L+ Repone K 0.2g/L+ sodium hydrogen phosphate 1.15g/L+ potassium primary phosphate 0.2g/L egg-sucking liquid: heparin 10ug/mL, 3mg/mL BSA+ Sodium.alpha.-ketopropionate 2.2mg/100mL+ penicillin 100IU/mL+ Streptomycin sulphate 100IU/mL+ phosphoric acid buffer (D-PBS), preserve two weeks for 4 ℃;
The maturation culture solution proportioning of improvement is as follows:
With TCM-199 commercial product culture medium (liquid nutrient medium, available from U.S. Gibco company product, article No.: 12340) be minimum medium, additional foetal calf serum 10%, ethyl piperazidine ethyl sulfonic acid 25mmol, Sodium.alpha.-ketopropionate 0.55mg/mL, penicillin 100IU/mL, Streptomycin sulphate 100IU/mL, follitropin 0.1IU/mL, lutropin 0.1IU/mL, oestrogenic hormon 1ug/mL, Urogastron 100ng/mL, pH 7.0-7.2;
It is as follows to activate substratum-1 proportioning:
Basal liquid for the improvement maturation culture solution+ionomycin (Ionopbore, available from U.S. Sigma company, article No.: I9657) 5 μ mol/L, pH 7.0-7.2;
It is as follows to activate substratum-2 proportioning:
Basal liquid for the improvement maturation culture solution+dimethylaminopurine (6-DMAP, available from U.S. Sigma company, article No.: P2629) 2mmol/L, pH 7.0-7.2;
The embryo medium basigamy is such as following:
The phenol red 10.0 μ g/mL+ matrimony vines acid of sodium-chlor 107.63mmol/L+ Repone K 7.16mmol/L+ potassium primary phosphate 1.19mmol/L+ sal epsom 1.51mmol/L+ benzylpenicillin sodium 0.012g/L+ Vetstrep 50 μ g/L+ Sodium.alpha.-hydroxypropionate 5.35mmol/L+ sodium bicarbonate 25.00mmol/L+ Sodium.alpha.-ketopropionate 7.27mmol/L+ calcium chloride 1.78mmol/L+ inositol 2.77mmol/L+ sodium 0.34mmol/L+ indispensable amino acid 30.0 μ l/ml+ non-essential amino acid 10 μ l/mL+L-glutamine 0.20mmol/L+BSA3mg/mL, pH 7.0-7.2.
Ovocyte stoning liquid: basal liquid is a maturation culture solution, add 5 μ g/mL cytochalasin Bs (Cytochalsin B, available from U.S. Sigma company, article No.: C6762)+10% polyvinylpyrrolidone (weight/volume)
Ovocyte is annotated karyolymph: the basal liquid of annotating karyolymph is a maturation culture solution, adds 10% polyvinylpyrrolidone (weight/volume) then in basal liquid.
As key operation step of the present invention, following technical characterictic is important:
Ox donorcells described in the above-mentioned steps is without serum starvation and vitro culture.Described donorcells is the ox cumulus cell, and this ox donorcells can be a granulosa cell.This ox donorcells can be handled without rupture of membranes.The cultivation of wherein said ox reconstructed embryo adopts the cumulus cell individual layer to cultivate altogether.Described ox cumulus cell individual layer and acceptor ovocyte homology.
The cultivation of described ox reconstructed embryo adopts ox cumulus cell individual layer to cultivate altogether.
The invention has the beneficial effects as follows:
1, the present invention is doing bold trial aspect the donorcells processing, the donorcells of nuclear transplantation is not done any processing, directly adopt from the cumulus cell that the ox cumulus cell-the ovocyte complex body is taken off, saved the shattering process of serum starvation and cytolemma, thereby simplified testing sequence, reduced requirement, saved test period operator's test operation technical ability, reduce the contaminated youngster of cell and led, be of value to the follow-up growth of ox reconstructed embryo.
2, the present invention annotates nuclear dimension in stoning and has used improved pitting method: promptly with the broken zona pellucida of kernel removing needle spine, in the other malleation of pushing with needle point in the zona pellucida dependence zona pellucida of thorn cut oocyte nuclei is extruded then, reduced the amount of losing ooecium matter, reduced damage simultaneously, thereby helped the follow-up growth of reconstructed embryo ovocyte.
More detailed technical scheme is seen " embodiment ".
Description of drawings
Fig. 1: be before cultivating bovine oocyte (opticmicroscope, 100x);
Fig. 2: be cultivate sophisticated bovine oocyte (opticmicroscope, 200x);
Fig. 3: be the bovine oocyte (150x) that removes cumulus cell;
Fig. 4: be through the painted ripe bovine oocyte of Hoechst (400x);
Fig. 5: be to be the 4-cell stage ox clone embryos (400x) that donor makes up with the cumulus cell;
Fig. 6: grow to 16-32-cell cell stage ox reconstructed embryo (400x);
Fig. 7: the ox reconstructed embryo (300x) that is expansion;
Fig. 8: be the ox reconstructed embryo (400x) in the hatching.
Embodiment
Embodiment 1
1, sampling: the healthy ox ovary that will gather from red flag beef cattle slaughterhouse, Wuhan City, Hubei Province is stored in 39 ℃ DPBS, and prescription is seen " summary of the invention ", takes back the laboratory in 3 hours;
2, bovine oocyte is collected and is cultivated: with suction a small amount of egg-sucking liquid is arranged, prescription is seen " summary of the invention ", syringe (No. 18 syringe needles) is drawn ox cumulus cell-ovocyte complex body (COCs) from the 3-8mm ovarian follicle of ox ovary surface, with this COCs after DPBS washing 3 times, with maturation culture solution (prescription as mentioned above) washing 3 times, change over to and cultivate 22h in the 100ul maturation culture solution droplet (culture condition is: 39 ℃, relative humidity are 100%, 5%CO
2Incubator);
3, the ripening degree of bovine oocyte is judged: the bovine oocyte that will in maturation culture solution, cultivate 22h in 0.1% Unidasa through blowing and beating 3min repeatedly to purify peripheral cumulus cell.Then that kytoplasm is even, regular shape, fat are dripped content bovine oocyte abundant and the eliminating first polar body and are changed in the stoning droplet or stoning liquid (compound method is to add the 10ug/mL cytochalasin B in the maturation culture solution of above-mentioned improvement) of the 50ul that contains 10ug/mL cytochalasin B (Cytochalasin).Cover this droplet with paraffin oil, place 39 ℃, relative humidity is 100%, 5%CO
2Incubator in cultivate standby.In diameter is the culture dish lid of 60mm, makes stoning and annotate the nuclear droplet;
4, the preparation of donorcells: from come from the definite good female ox ovary of a genetic background, extract ox cumulus cell-ovocyte complex body (COCs) and carry out the maturation cultivation separately.After cultivating 22h, in cultivating droplet, use self-control suction pipe (william with 0.1% Unidasa, 2006) piping and druming gently, take off the peripheral cumulus cell of ox cumulus cell-ovocyte complex body, then, draw the above-mentioned maturation culture solution of 10ul in the stoning liquid of 90ul, prescription is seen " summary of the invention ", fully mixing after 3 10 times of dilutions, is drawn the stoning droplet that 10ul contains the cell suspension adding 50ul of cumulus cell, prescription is seen " summary of the invention ", in:
5, will be in the step 3 obtain sophisticated bovine oocyte stoning: 3 directions that sophisticated bovine oocyte first polar body are positioned at be similar to dial, locking pin is positioned at bovine oocyte first polar body right opposite, needle point with kernel removing needle punctures zona pellucida gently, push zona pellucida with needle point gently on thorn breakpoint next door then, the nucleus of bovine oocyte is pressed against outside the zona pellucida together with spindle body; Under fluorescent microscope, observe ovocyte and have or not fluorescent characteristics, be the stoning success as no blue-fluorescence person.At last the bovine oocyte of successful stoning is transferred to and annotated in the nuclear droplet, prescription " summary of the invention " is cultivated 10min.
6, the structure of ox reconstructed embryo: the ox cumulus cell adding that earlier step 3 is obtained is annotated the nuclear droplet and is done donor, again with annotating that the nuclear pin is inhaled as cytolemma is complete, the normal cell of form is as donor, fix this bovine oocyte with locking pin then, find first polar body and be located at 3 directions that are similar to dial.Thrust bovine oocyte with annotating the nuclear pin along the polar body direction, and will sting mouthful an amount of expansion.Extract notes nuclear pin out and push ovocyte up and down, so that polar body and nucleus are extruded in the lump at the thorn mouth.As extrude the group that material is shrunk to compactness, can be judged as the stoning success.Donorcells is injected in the ovocyte kytoplasm along above-mentioned thorn cut after extruding nucleus, the reorganization embryo that will make up then transfers to one side of droplet and discharges, and repeats above step till the micrurgy of all ovocytes is finished at once.
7, the reconstructed embryo of step 6 gained is forwarded in the droplet of maturation culture solution of improvement and cultivate (making its rest and reorganization) 3-4h.
8, reconstructed embryo that the cytolemma of step 7 gained is complete is transferred in the activation substratum-1 and is activated 5min, transfers to 3-4h in the activation substratum-2 then, activates to change in the embryo medium droplet after finishing and cultivates, and changes liquid every day once, the observed and recorded developmental state.
The dissimilar ox donorcellses of table 3 are to the influence of SCNT ox reconstructed embryo potentiality of development
Annotate: the different subscript person's significant differences (P＜0.05) of same column; Blastaea rate I: blastaea number/spilting of an egg embryo number;
Blastaea rate II: blastaea number/mature oocyte number
The ox donorcells of table 4 different treatment is to the influence of SCNT ox reconstructed embryo potentiality of development
Annotate: the different subscript person's significant differences (P＜0.05) of same column; Blastaea rate I: blastaea number/spilting of an egg embryo number; In this table: ovarian cumulus (being cell) is the ox cumulus cell.Blastaea rate II: blastaea number/mature oocyte number
Reference
1.Collas?P，Barnes?F?L.Nuclear?transplantation?by?microinjection?of?inner?cellmass?and?granulosa?cell?nuclei.Mol?Reprod?Dev，1994，38：264-267
2.Wolf?E，Zakhartchenko?V，Brem?G.Nuclear?transfer?in?mammals：recent?developmentsand?future?perspectives.J?Biotechnol，1998，65：99-110
3.Fulka?J，Jr.，First?N?L，Loi?P，Moor?R?M.Cloning?by?somatic?cell?nuclear?transfer.Bioessays，1998，20：847-851
4.Heyman?Y，Vignon?X，Chesne?P，Le?Bourhis?D，Marchal?J，Renard?J?P.Cloning?incattle：from?embryo?splitting?to?somatic?nuclear?transfer.Reprod?Nutr?Dev，1998，38：595-603
5.Campbell?K?H，Fisher?P，Chen?W?C，Choi?I，Kelly?R?D，Lee?J?H，Xhu?J.Somaticcell?nuclear?transfer：Past，present?and?future?perspectives.Theriogenology，?2007，68?Suppl?1：S214-231
6.Wakayama?T，Perry?A?C，Zuccotti?M，Johnson?K?R，Yanagimachi?R.Full-termdevelopment?of?mice?from?enucleated?oocytes?injected?with?cumulus?cell?nuclei.Nature，1998，394：369-374
7.Lee?B?C，Kim?M?K，Jang?G，Oh?H?J，Yuda?F，Kim?H?J，Hossein?M?S，Kim?J?J，KangS?K，Schatten?G，Hwang?W?S.Dogs?cloned?from?adult?somatic?cells.Nature，2005，436：641
8.Chen?D?Y，Jiang?M?X，Zhao?Z?J，Wang?H?L，Sun?Q?Y，Zhang?L?S，Li?R?C，Cao?H?H，Zhang?Q?J，Ma?D?L.Cloning?of?Asian?yellow?goat(C.hircus)by?somatic?cell?nucleartransfer：telophase?enucleation?combined?with?whole?cell?intracytoplasmic?injection.Mol?Reprod?Dev，2007，74：28-34
9.Zhou?Q，Boulanger?L，Renard?J?P.A?simplified?method?for?the?reconstruction?offully?competent?mouse?zygotes?from?adult?somatic?donor?nuclei.Cloning，2000，2：35-44。
Claims (8)
1, a kind of method that obtains the ox clone embryos, its step comprises: 1) get bovine oocyte and cultivate in maturation culture solution; 2) the ox donorcells is injected the acceptor ovocyte, obtain the reconstruct ovocyte; 3) with step 2) the reconstruct activation of oocytes, obtain reconstructed embryo, it is characterized in that, the bovine oocyte that takes out is cultivated 22h in the maturation culture solution of improvement, 39 ℃ of culture temperature, make described bovine oocyte be in MII, remove the nucleus of mature oocyte, obtain the ox recipient cell, adopt full cell direct injection that the ox donorcells is injected described ox recipient cell, obtain the ox reconstructed embryo, place the droplet of the maturation culture solution of improvement to cultivate 3-4h described ox reconstructed embryo, described ox embryo is exposed in the activation substratum-1 activates 5min then, transfer to then in the activation substratum-2 and activate 3-4h, activated ox reconstructed embryo was cultivated in embryo medium 7 days
Wherein:
The maturation culture solution proportioning of improvement is as follows:
With TCM-199 commodity liquid nutrient medium is minimum medium, additional foetal calf serum 10%, ethyl piperazidine ethyl sulfonic acid 25mM, Sodium.alpha.-ketopropionate 0.55mg/mL, penicillin 100IU/mL, Streptomycin sulphate 100IU/mL, follitropin 0.1IU/mL, lutropin 0.1IU/mL, oestrogenic hormon 1ug/mL, Urogastron 100ng/mL, pH 7.0-7.2;
It is as follows to activate substratum-1 proportioning:
Basal liquid is maturation culture solution+ionomycin 5 μ mol/L of improvement, pH 7.0-7.2;
It is as follows to activate substratum-2 proportioning:
Basal liquid is the maturation culture solution+dimethylaminopurine 2mmol/L of improvement, pH 7.0-7.2;
The embryo medium proportioning is as follows:
The phenol red 10.0 μ g/mL+ matrimony vines acid of sodium-chlor 107.63mmol/L+ Repone K 7.16mmol/L+ potassium primary phosphate 1.19mmol/L+ sal epsom 1.51mmol/L+ benzylpenicillin sodium 0.012g/L+ Vetstrep 50 μ g/L+ Sodium.alpha.-hydroxypropionate 5.35mmol/L+ sodium bicarbonate 25.00mmol/L+ Sodium.alpha.-ketopropionate 7.27mmol/L+ calcium chloride 1.78mmol/L+ inositol 2.77mmol/L+ sodium 0.34mmol/L+ indispensable amino acid 30.0 μ l/ml+ non-essential amino acid 10 μ l/mL+L-glutamine 0.20mmol/L+BSA 3mg/mL, pH 7.0-7.2.
2, method according to claim 1 is characterized in that, described ox donorcells is without serum starvation and vitro culture.
3, method according to claim 1 is characterized in that, described ox donorcells is the ox cumulus cell.
4, method according to claim 1 is characterized in that, described ox donorcells is the ox granulosa cell.
5, method according to claim 1 is characterized in that, described ox donorcells is handled without rupture of membranes.
6, method according to claim 1 is characterized in that the cultivation of ox reconstructed embryo adopts ox cumulus cell individual layer to cultivate altogether.
7, method according to claim 1 is characterized in that, described ox cumulus cell individual layer and recipient cattle ovocyte homology.
8, method according to claim 1 is characterized in that, cultivates at the ox reconstructed embryo and does not add foetal calf serum in first day, from the foetal calf serum that began to add 10% volume in second day of vitro culture.
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Cited By (8)
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| CN101492669B (en) * | 2009-01-15 | 2011-04-13 | 华中农业大学 | Method for cloning embryo with cattle spermatozoon |
| CN102250831A (en) * | 2011-07-12 | 2011-11-23 | 中国农业大学 | Culture medium for enhancing ectogenesis of ox preantral follicle and application thereof |
| CN102344941A (en) * | 2010-07-29 | 2012-02-08 | 内蒙古大学 | Method for breeding cloned animal or transgenic cloned animal |
| CN102344907A (en) * | 2010-07-29 | 2012-02-08 | 内蒙古大学 | Method for activating reconstructed embryo |
| CN102559585A (en) * | 2012-02-03 | 2012-07-11 | 兰诺生物技术无锡有限公司 | Culture medium for in vitro culture of bovine embryo |
| CN104206375A (en) * | 2013-05-29 | 2014-12-17 | 深圳华大方舟生物技术有限公司 | Device and method for encapsulating embryos |
| CN105018419A (en) * | 2015-07-27 | 2015-11-04 | 广西壮族自治区水牛研究所 | Buffalo oocyte enucleation method |
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| CN100350039C (en) * | 2005-07-06 | 2007-11-21 | 广西大学 | Method for cloning buffalo somatic cell |
| CN101050458A (en) * | 2007-03-26 | 2007-10-10 | 北京锦绣大地农业股份有限公司 | Method for once more cloning milch cow |
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| CN101492669B (en) * | 2009-01-15 | 2011-04-13 | 华中农业大学 | Method for cloning embryo with cattle spermatozoon |
| CN102344941A (en) * | 2010-07-29 | 2012-02-08 | 内蒙古大学 | Method for breeding cloned animal or transgenic cloned animal |
| CN102344907A (en) * | 2010-07-29 | 2012-02-08 | 内蒙古大学 | Method for activating reconstructed embryo |
| CN102250831A (en) * | 2011-07-12 | 2011-11-23 | 中国农业大学 | Culture medium for enhancing ectogenesis of ox preantral follicle and application thereof |
| CN102559585A (en) * | 2012-02-03 | 2012-07-11 | 兰诺生物技术无锡有限公司 | Culture medium for in vitro culture of bovine embryo |
| CN104206375A (en) * | 2013-05-29 | 2014-12-17 | 深圳华大方舟生物技术有限公司 | Device and method for encapsulating embryos |
| CN104206375B (en) * | 2013-05-29 | 2016-04-20 | 深圳华大方舟生物技术有限公司 | The device and method of encapsulation embryo |
| CN105018419A (en) * | 2015-07-27 | 2015-11-04 | 广西壮族自治区水牛研究所 | Buffalo oocyte enucleation method |
| CN114075542A (en) * | 2021-11-02 | 2022-02-22 | 上海交通大学医学院附属第九人民医院 | Culture solution for cytoplasm mechanical separation and use method thereof |
| CN114075542B (en) * | 2021-11-02 | 2024-02-20 | 上海交通大学医学院附属第九人民医院 | Culture solution for cytoplasmatic mechanical separation and application method thereof |
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