CN101880690A - Construction and in vitro culture method of Tibetan antelope-goat interspecific cloned embryo - Google Patents
Construction and in vitro culture method of Tibetan antelope-goat interspecific cloned embryo Download PDFInfo
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Abstract
The invention relates to a construction and in vitro culture method of a Tibetan antelope-goat interspecific cloned embryo. In the method, a Tibetan antelope-goat interspecific somatic nucleus transferred embryo is constructed by using an ear tissue fibroblast of a Tibetan antelope as a donor cell and using an enucleated oocyte of a goat as a recipient cytoplasm by a zona pellucida-free hand guide type clone technology, and a high-activity embryo which grows to a mulberry body can be obtained by using a chemical auxiliary fusion method and a micro hole type embryo culture system. The method provides the foundation for the application of animal clone technology in the protection of Tibetan antelopes.
Description
Technical field
The present invention relates between a kind activation and the extracorporeal culturing method of clone's embryo, relate in particular to clone embryos between Tibetan antelope-goat kind and make up method with vitro culture.
Background technology
The mammal body-cell neucleus transplanting technology is meant puts into the mature oocyte of stoning in advance with donorcells nuclear, form a new caryoplasm recombinant chou, move into nuclear under the recipient cytoplasm effect, the genesis and development program is rearranged, make the caryoplasm recombinant chou the same, through cell fission, differentiation and in parent, develop into a new individuality with the normal fertilization ovum.Body-cell neucleus transplanting can produce unlimited clone's individuality in theory, might utilize a large amount of rare species of nuclear transfer technology clone.Up to the present, the interspecies cloning success has a gaur (Lanza et al. in the world, 2000), east argali (Loi et al., 2001), African wildcat (G ó mez et al., 2004), grey wolf (Kim et al., 2007), Alps aegagrus (Folch et al., 2009).Their birth certificate inter-species nuclear transplantation is fully feasible in practice, can bring into play its vital role in the rescue of the conservation of wildlife, rare animal and animals on the brink of extinction.
Tibetan antelope (Pantholops hodgsoni) is under the jurisdiction of Artiodactyla Bovidae goat subfamily chiru and belongs to; the peculiar species in Qinghai-Tibet Platean; belong to animals under first-class state protection and " CITS " the active animals on the brink of extinction of forbidding to carry on trade in (CITES); mainly be distributed in high and cold desertification grassland, high-cold steppe and the high and cold grassy marshland area of within Chinese territory Qinghai, Tibet, Xinjiang San Sheng, on average live between height above sea level 4100～5500m in the rugged environment.Because environmental factors causes and is difficult to Tibetan antelope is furtherd investigate, at present about the research of Tibetan antelope mainly based on macroscopical zoological research, comprising: the physics of the diagnosis of animal behavior, population distribution, idiobiology, phylogenetics, parasite, disease and treatment, cashmere and chemical property etc.Research about Tibetan antelope fetology, developmental biology, molecular biology and cytobiology is less.Therefore, body-cell neucleus transplanting embryo's structure and the development that ectogenesis can promote Tibetan antelope cytobiology, molecular biology, fetology, conservation biology and developmental biology between research Tibetan antelope-goat kind.
Summary of the invention
Technical problem to be solved by this invention provides clone embryos between a kind of Tibetan antelope-goat kind that obtains high vigor embryo and makes up method with vitro culture.
For addressing the above problem, the method for clone embryos structure and vitro culture between a kind of Tibetan antelope of the present invention-goat kind may further comprise the steps:
(1) collection of ovary: behind the ovary clip of the goat that will just butcher, put into that physiological saline, temperature are housed is 20～25 ℃ vacuum flask, then the goat ovary is placed on and fills in the culture dish of choosing ovum liquid, adopt the sectility method to choose and get ovarian cumulus in the liquor folliculi-ovocyte complex body;
(2) maturation in vitro of ovocyte is cultivated: after the ovarian cumulus-ovocyte complex body of described step (1) gained was cleaned with ripe liquid, ripe cultivation 20～24h obtained mature oocyte in the saturated humidity CO2gas incubator;
(3) the hand-guided stoning of mature oocyte: the mature oocyte of described step (2) gained after the ovarian cumulus granulosa cell is removed, the ovocyte chemistry shows nuclear, zona pellucida digestion, ovocyte stoning, is obtained stoning acceptor ovocyte;
(4) donorcells is prepared and cytogamy: the stoning acceptor ovocyte that will cultivate donorcells and described step (3) gained in 24 orifice plates adopts chemistry to assist fusion method to carry out cytogamy, clone embryos between obtaining planting;
(5) plant between clone embryos activate and vitro culture: with clone embryos balance 20～30min in the T20 droplet between the kind of described step (4) gained, treat that a donorcells and two stoning acceptor ovocytes all merge after, balance 3h in embryo medium; Then clone embryos between described kind is placed the solution of 1mL calcium ions carrier (Ca ionophore), in the saturated humidity CO2gas incubator, cultivate 5min; Again clone embryos between described kind is transferred in the 6-dimethylaminopurine (6-DMAP) that droplet volume is 10 μ L, in the saturated humidity CO2gas incubator, finishes the activation of clone embryos between Tibetan antelope-goat kind behind the cultivation 4.5h; Get final product putting into little cave of the 4 orifice plates bottom that the 0.5mL embryo medium is housed after the clone embryos activation between Tibetan antelope-goat kind, in the saturated humidity CO2gas incubator, cultivating.
The ovum liquid of choosing in the described step (1) is made up of 3% new-born calf serum (NBS) that accounts for total liquor capacity and 97% Du Shi phosphoric acid buffer (D-PBS).
Ripe liquid in the described step (2) is to contain 0.22g/100mL NaHCO by 10% the foetal calf serum (FBS) that accounts for total liquor capacity and 90%
3TCM199 basic culture solution and the Sodium.alpha.-ketopropionate of 0.22mg/100mL, the hydroxyethyl piperazine ethanesulfonic acid (HEPES) of 238mg/100mL, the penicillin of 75 μ g/mL, the Streptomycin sulphate of 100 μ g/mL, the follicular stimulating hormone (FSH) of 10 μ g/mL, the prolan B (LH) of 10 μ g/mL, 17 β estradiol (the 17-β E of 1 μ g/mL
2) form.
Saturated humidity CO2gas incubator in the described step (2) is meant that temperature is 38.5 ℃, contains 5%CO
2The CO2gas incubator of saturated humidity air.
Donorcells in the described step (4) is the inoblast of Tibetan antelope ear tissue.
Embryo medium in the described step (5) is by the KCl of NaCl, the 0.0534g/100mL of 0.6294g/100mL, the KH of 0.0162g/100mL
2PO
4, 0.0251g/100mL CaCl
22H
2The MgCl of the Sodium.alpha.-hydroxypropionate of O, 47 μ L/100mL (Sodium lactate), 0.01g/100mL
26H
2The NaHCO of O, 0.2106g/100mL
3, 0.0033g/100mL L-glutaminate (L-Glutamine), the indispensable amino acid of 2mL/100mL, the non-essential amino acid of 1mL/100mL, the bovine serum albumin (BSA) of 0.8g/100mL, the gentamicin of 0.005g/100mL and phenol red (Phenol-red) of 0.001g/100mL of Sodium.alpha.-ketopropionate (Sodium pyruvate), 0.0146g/100mL form.
T20 in the described step (5) is made up of 20% foetal calf serum that accounts for total liquor capacity and 80% the TCM199 basic culture solution that contains the 10mM hydroxyethyl piperazine ethanesulfonic acid, and adds two anti-ly after configuration, regulates the solution of pH value to 7.2～7.4 then with NaOH; Wherein two anti-the be meant penicillin of 75 μ g/mL and the Streptomycin sulphates of 100 μ g/mL.
The present invention compared with prior art has the following advantages:
1, the present invention selects that Qinghai-Tibet characteristic animal---Tibetan antelope is as research object; with the Tibetan antelope skin inoblast as donorcells; the goat ovocyte is as recipient cytoplasm; utilize the hand-guided clone technology of no zona pellucida to carry out the structure of body-cell neucleus transplanting embryo between the Tibetan antelope kind; set up little cave formula cultural method of clone embryos; explored with the goat ovocyte and carried out the feasibility that Tibetan antelope is cloned as recipient cytoplasm, the protection that is applied to Tibetan antelope for the animal cloning technology is laid a good foundation.
2, because the present invention on no pellucid zone ovum parent cell and somatocyte integration technology, has adopted chemical auxiliary fusion method, therefore, not only simplified operative technique, shortened the operating time, and effectively improved cytogamy efficient.
3, because the present invention has adopted the hand-guided clone technology of no zona pellucida, therefore, do not need valuable equipment, reduced the laboratory input, simplified operative technique, be suitable for the Northwest and carry out.
4,, therefore, help setting up the microenvironment that is fit to no zona pellucida embryo growth, thereby improved the developmental potency of clone embryos between kind because the present invention has adopted little cave formula embryo culture system.
5, after utilization the inventive method was carried out sectility to 536 goat ovaries gathering, obtaining ovum number was 1646, and every ovary on average gets several 3.07 of ovum, and its oocyte maturation rate is 66.61% ± 1.2%; The stoning ovum number of goat is 83, and fourth contact ovum number is 68, and the fourth contact rate is 82.27%.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is Tibetan antelope-goat morula that the present invention obtained.Shown in the figure is the reconstruct embryo who uses constructed Tibetan antelope somatocyte of the hand-guided cloning process of no zona pellucida and goat ovocyte, the morula behind vitro culture 4d, and magnification is 100 times.
Embodiment
Material: the goat ovary is collected in slaughterhouse, Le Jia gulf, Xining, Qinghai Province.
The method of clone embryos structure and vitro culture between a kind of Tibetan antelope-goat kind may further comprise the steps:
(1) collection of ovary: the ovary of the goat that will just butcher with sterilization scissors clip after, put into that physiological saline, temperature are housed is 20～25 ℃ vacuum flask, transport the laboratory in the 3h back.Wipe out reticular tissue, the fat on ovary surface and the uterine tube that adheres to, clean 3 times with physiological saline; The ovary of goat is adopted the sectility method---soon ovary is placed on and fills in the culture dish of choosing ovum liquid, with scalpel its surperficial ovarian follicle is cut, and extruding is as much as possible all flowed in choosing ovum liquid liquor folliculi gently, chooses under stereoscope then and gets ovarian cumulus in the liquor folliculi-ovocyte complex body.
Wherein: choose ovum liquid and form by 3% new-born calf serum (NBS) that accounts for total liquor capacity and 97% Du Shi phosphoric acid buffer (D-PBS).
(2) maturation in vitro of ovocyte is cultivated: ovarian cumulus-ovocyte complex body usefulness of step (1) gained is cleaned 2 times at the ripe liquid of saturated humidity CO2gas incubator inner equilibrium in advance, so that remove impurity; Then, the ripe 20～24h that cultivates obtains mature oocyte in the saturated humidity CO2gas incubator.
Wherein: ripe liquid is to contain 0.22g/100mL NaHCO by 10% the foetal calf serum (FBS) that accounts for total liquor capacity and 90%
3TCM199 basic culture solution and the Sodium.alpha.-ketopropionate of 0.22mg/100mL, the hydroxyethyl piperazine ethanesulfonic acid (HEPES) of 238mg/100mL, the penicillin of 75 μ g/mL, the Streptomycin sulphate of 100 μ g/mL, the follicular stimulating hormone (FSH) of 10 μ g/mL, the prolan B (LH) of 10 μ g/mL, 17 β estradiol (the 17-β E of 1 μ g/mL
2) form.
(3) the hand-guided stoning of mature oocyte: the mature oocyte of step (2) gained after the ovarian cumulus granulosa cell is removed, the ovocyte chemistry shows nuclear, zona pellucida digestion, ovocyte stoning, is obtained stoning acceptor ovocyte.
Wherein: the ovarian cumulus granulosa cell is removed and to be meant mature oocyte is moved in the centrifuge tube that the 0.5mL Unidasa is housed, and blows and beats 2min gently with liquid-transfering gun, whirlpool concussion 2min again after piping and druming finishes.The ovarian cumulus granulosa cell of ovocyte zona pellucida periphery removes in the culture dish at this moment.
The apparent nuclear of ovocyte chemistry is meant and places 400 μ L to contain in the oocyte maturation liquid of 20 μ L demecolcines (Sigma D 1925) ovocyte, cultivates 2h then in the saturated humidity CO2gas incubator.After cultivation finishes ovocyte is taken out from nutrient solution, clean up with T20 (HEPES buffered TCM199 adds 20% foetal calf serum), standby.
The digestion of zona pellucida is meant places ovocyte in the T20 droplet, moves in the 50 μ L pronase droplets again, and digestion zona pellucida 3～5min treats after the whole digestion of zona pellucida the no pellucid zone ovum parent cell to be placed in the T20 droplet, stablizes 15min.
The ovocyte stoning is meant that in T20 adding concentration is the cytochalasin of 5 μ g/mL, adds 2mL T20 solution then and serve as and cut ovum liquid in the 35mm culture dish.The ovocyte of no zona pellucida being transferred in the culture dish, forming a line, is sign with first polar body and no pellucid zone ovum parent cell rat then, with the stoning cutter first polar body and rat is cut away.Cut away part and be 1/3 size of whole ovocyte, a part of in addition seedless ovocyte matter is transferred among the T20, recovers 15min, treats to carry out cytogamy behind its complete fourth contact.
(4) donorcells is prepared and cytogamy: will cultivate the donorcells in 24 orifice plates---and the inoblast (Northwest Plateau-organisms Research Inst. of Chinese Academy of Sciences provides) that Tibetan antelope ear organizes adopts the auxiliary fusion method of chemistry to carry out cytogamy with the stoning acceptor ovocyte of step (3) gained, clone embryos between obtaining planting.
Wherein:
The inoblast of donorcells---Tibetan antelope ear tissue adopts following method to obtain:
1. sampling: during sampling, cut ear's hair of female adult Tibetan antelope individuality earlier with the scissors of sterilization, reject residual root of hair as much as possible with razor blade again.Clean last clip 1cm then successively with perfumed soap, distilled water, bromogeramine, physiological saline, 75% alcohol, D-PBS
2Skin histology, and put it in the 50mL centrifuge tube that stock solution is housed.
2. Tibetan antelope ear skin tissue in primary culture: Tibetan antelope ear skin tissue is embathed 5 times with containing two anti-D-PBS, soak 3min at every turn; With after containing two anti-D/F12 nutrient solutions flushing 3 times, the sample tissue block is put into the 35mm culture dish again, tissue block is shredded as far as possible with scissors; With suction pipe broken tissue block is moved in advance in the culturing bottle with the foetal calf serum coating then, put equably in the culturing bottle bottom; At last culturing bottle is slowly overturn, inversion is placed in the saturated humidity CO2gas incubator, and the culturing bottle that overturns gently behind the 3h adds 3～5mL D/F12 nutrient solution and continues to cultivate 10～18 days, obtains growing to 80%～90% primary cell that converges.
3. go down to posterity and purifying: the primary cell cultivation of going down to posterity; At first with the no Ca of primary cell
2+, Mg
2+PBS clean 2 times, the mixed solution that contains 0.05% trypsinase and 0.01% ethylenediamine tetraacetic acid (EDTA) (EDTA) with 1～2mL digests again, obtains Digestive system A (Digestive system A with the confluent culture bottle at the bottom of be advisable); Digestive system A is put into saturated humidity CO2gas incubator 2～3min, take out, examine under a microscope the digestion situation, beat the bottom gently, adding stops digestion with the D/F12 nutrient solution of Digestive system A equivalent then, obtains Digestive system B; Digestive system B is transferred in the centrifuge tube with 1000 rev/mins of centrifugal 5min, remove supernatant liquor after, obtain cell precipitation thing A; With the D/F12 nutrient solution with cell precipitation thing A mixing gently, import in another culturing bottle, continue to cultivate 3～6 days in the saturated humidity CO2gas incubator, obtain growing to 80%～90% skin flbroblast of converging, this skin flbroblast is the shuttle type.
4. cell cryopreservation: skin flbroblast is contained the mixed solution digestion of 0.05% trypsinase and 0.01% ethylenediamine tetraacetic acid (EDTA) (EDTA) with 1～2mL, centrifugal back adds the cells frozen storing liquid mixing, preserve half an hour at 4 ℃ earlier, then the cell cryopreservation box is directly put into-70 ℃ of refrigerator overnight, the last liquid nitrogen that directly drops into is preserved, and obtains frozen skin flbroblast.
5. cell recovery: the frozen pipe that frozen skin flbroblast will be housed takes out from liquid nitrogen, putting into 37 ℃ of water-baths rapidly melts, the sucking-off cell suspension is put in the 15mL centrifuge tube, after washing with the D/F12 nutrient solution of 9 times of described cell suspension volumes, with 1000 rev/mins of centrifugal 5min, after removing supernatant, obtain cell precipitation thing B; With the abundant suspension cell sediment B of D/F12 nutrient solution, and be inoculated in the culturing bottle, place the saturated humidity CO2gas incubator to cultivate and get final product.
Above-mentioned steps 2., 3. and the saturated humidity CO2gas incubator 5. be meant that temperature is 37.5 ℃, contains 5%CO
2The CO2gas incubator of saturated humidity air; The D/F12 nutrient solution is all by 10% the foetal calf serum that accounts for total nutrient solution volume, 90% D/F12 and two anti-composition.
The stock solution of above-mentioned steps in 1. is for adding two anti-T20; Add two anti-T20 and form by 20% foetal calf serum that accounts for total liquor capacity and 80% the TCM199 basic culture solution that contains the 10mM hydroxyethyl piperazine ethanesulfonic acid, and after configuration, add two anti-, then with the solution of NaOH adjusting pH value to 7.2～7.4.
The Du Shi phosphoric acid buffer of above-mentioned steps in 2. is by the KCl of NaCl, the 0.0203g/100mL of 0.8g/100mL, the Na of 0.1152g/100mL
2HPO
4, 0.0203g/100mL KH
2PO
4, 0.0134g/100mL CaCl
22H
2The MgCl of O, 0.01g/100mL
26H
2The Streptomycin sulphate of the Sodium.alpha.-ketopropionate of O, 0.0036g/100mL, the penicillin of 0.0075g/100mL, 0.005g/100mL and the glucose of 0.1g/100mL are formed.
The phosphate buffered saline buffer of above-mentioned steps in 3. is by the KCl of NaCl, the 0.02g/100mL of 0.8g/100mL, the Na of 0.115g/100mL
2HPO
4KH with 0.02g/100mL
2PO
4Form.
The frozen storing liquid of above-mentioned steps in 4. is by 20% the foetal calf serum that accounts for total liquor capacity, 10% dimethyl sulfoxide (DMSO), 70% D/F12 and two anti-composition.
Above-mentioned two anti-the be meant penicillin of 75 μ g/mL and the Streptomycin sulphate of 100 μ g/mL.
The auxiliary fusion method of chemistry is meant:
At first, donorcells is cultivated in 24 orifice plates; The cell that selection culture hole inner bottom part all covers with during nuclear transplantation is as donorcells.Each digestion 1 porocyte is moved into small amounts of cells in the droplet of T2B (HEPES buffered TCM199 liquid adds 2% foetal calf serum, and other adds 8mg/mL BSA) then.
Secondly, making volume in the 60mm culture dish according to a conventional method is the fusion droplet of 25 μ L.
The acceptor ovocyte is moved in T20, phytohaemagglutinin, T2B, the fusion liquid droplet successively then and operate, cover paraffin oil on the droplet, avoid droplet evaporation and position to move.
Earlier the acceptor ovocyte is moved in the T20 droplet, move an acceptor ovocyte again to the phytohaemagglutinin droplet, transfer to then to glue in the T2B droplet and get a donorcells, again be transferred in the phytohaemagglutinin droplet again, from the T20 droplet, move an acceptor ovocyte again to the phytohaemagglutinin droplet, because phytohaemagglutinin can make cell surface have viscosity, so, can form the complex body that two ovocytes are mingled with a donorcells by adjusting the position of donorcells and acceptor ovocyte complex body and another ovocyte; With complex body move to merge liquid inner equilibrium 2～3min after, again to the integration slot with alternating-electric field 7V, pulsed electrical field 70V, time length 20 μ s continues twice, each 0.1s at interval carries out electricity fusion and gets final product.
T20 in present method is made up of 20% foetal calf serum that accounts for total liquor capacity and 80% the TCM199 basic culture solution that contains the 10mM hydroxyethyl piperazine ethanesulfonic acid, and adds two anti-ly after configuration, regulates the solution of pH value to 7.2～7.4 then with NaOH; Wherein two anti-the be meant penicillin of 75 μ g/mL and the Streptomycin sulphates of 100 μ g/mL.
Phytohaemagglutinin in present method is the solution that is dissolved in gained in the 5mL TCM199 basic culture solution by the 5mg phytohaemagglutinin.
T2B in present method is meant in the TCM199 basic culture solution 2% the foetal calf serum that adds total liquor capacity and the solution of 8mg/mL bovine serum albumin gained.
Fusion liquid in present method is meant earlier with 20mL dissolved in distilled water 0.025g MgSO
4, filter and preserve; Use 20mL dissolved in distilled water 0.0147g CaCl again
22H
2O filters and preserves; 10.93g N.F,USP MANNITOL and 0.2g polyvinyl alcohol fully are dissolved in the 196mL distilled water, add the MgSO that 2mL prepares in advance respectively to it then
4And CaCl
22H
2O solution, after the filtration promptly.
(5) plant between clone embryos activate and vitro culture: with clone embryos balance 20～30min in the T20 droplet between the kind of step (4) gained, treat that a donorcells and two stoning acceptor ovocytes all merge after, balance 3h in embryo medium; Begin then to activate.To plant a clone embryos earlier and place the 35mm culture dish of the solution of 1mL calcium ions carrier (Ca ionophore), in the saturated humidity CO2gas incubator, cultivate 5min; To plant a clone embryos again and be transferred in the 6-dimethylaminopurine (6-DMAP) that droplet volume is 10 μ L, and cover paraffin oil on the droplet, each droplet is put into clone's embryo.Then it is cultivated the activation of finishing clone embryos between Tibetan antelope-goat kind behind the 4.5h in the saturated humidity CO2gas incubator; With putting into little cave of the 4 orifice plates bottom that the 0.5mL embryo medium is housed after the clone embryos activation between Tibetan antelope-goat kind, cover paraffin oil then, in the saturated humidity CO2gas incubator, cultivate and get final product (referring to Fig. 1).
Wherein: embryo medium is by the KCl of the NaCl of 0.6294g/100mL, 0.0534g/100mL, the KH of 0.0162g/100mL
2PO
4, 0.0251g/100mL CaCl
22H
2The MgCl of the Sodium.alpha.-hydroxypropionate of O, 47 μ L/100mL (Sodium lactate), 0.01g/100mL
26H
2The NaHCO of O, 0.2106g/100mL
3, 0.0033g/100mL L-glutaminate (L-Glutamine), the indispensable amino acid of 2mL/100mL, the non-essential amino acid of 1mL/100mL, the bovine serum albumin (BSA) of 0.8g/100mL, the gentamicin of 0.005g/100mL and phenol red (Phenol-red) of 0.001g/100mL of Sodium.alpha.-ketopropionate (Sodium pyruvate), 0.0146g/100mL form.
T20 is made up of 20% foetal calf serum that accounts for total liquor capacity and 80% the TCM199 basic culture solution that contains the 10mM hydroxyethyl piperazine ethanesulfonic acid, and adds two anti-ly after configuration, regulates the solution of pH value to 7.2～7.4 then with NaOH; Wherein two anti-the be meant penicillin of 75 μ g/mL and the Streptomycin sulphates of 100 μ g/mL.
Saturated humidity CO2gas incubator among the present invention is meant that temperature is 38.5 ℃, contains 5%CO
2The CO2gas incubator of saturated humidity air.
Claims (7)
1. clone embryos makes up method with vitro culture between Tibetan antelope-goat kind, may further comprise the steps:
(1) collection of ovary: behind the ovary clip of the goat that will just butcher, put into that physiological saline, temperature are housed is 20～25 ℃ vacuum flask, then the goat ovary is placed on and fills in the culture dish of choosing ovum liquid, adopt the sectility method to choose and get ovarian cumulus in the liquor folliculi-ovocyte complex body;
(2) maturation in vitro of ovocyte is cultivated: after the ovarian cumulus-ovocyte complex body of described step (1) gained was cleaned with ripe liquid, ripe cultivation 20～24h obtained mature oocyte in the saturated humidity CO2gas incubator;
(3) the hand-guided stoning of mature oocyte: the mature oocyte of described step (2) gained after the ovarian cumulus granulosa cell is removed, the ovocyte chemistry shows nuclear, zona pellucida digestion, ovocyte stoning, is obtained stoning acceptor ovocyte;
(4) donorcells is prepared and cytogamy: the stoning acceptor ovocyte that will cultivate donorcells and described step (3) gained in 24 orifice plates adopts chemistry to assist fusion method to carry out cytogamy, clone embryos between obtaining planting;
(5) plant between clone embryos activate and vitro culture: with clone embryos balance 20～30min in the T20 droplet between the kind of described step (4) gained, treat that a donorcells and two stoning acceptor ovocytes all merge after, balance 3h in embryo medium; Then clone embryos between described kind is placed the solution of 1mL calcium ions carrier, in the saturated humidity CO2gas incubator, cultivate 5min; Again clone embryos between described kind is transferred in the 6-dimethylaminopurine that droplet volume is 10 μ L, in the saturated humidity CO2gas incubator, finishes the activation of clone embryos between Tibetan antelope-goat kind behind the cultivation 4.5h; Get final product putting into little cave of the 4 orifice plates bottom that the 0.5mL embryo medium is housed after the clone embryos activation between Tibetan antelope-goat kind, in the saturated humidity CO2gas incubator, cultivating.
2. clone embryos makes up the method with vitro culture between Tibetan antelope as claimed in claim 1-goat kind, it is characterized in that: the ovum liquid of choosing in the described step (1) is made up of 3% new-born calf serum that accounts for total liquor capacity and 97% Du Shi phosphoric acid buffer.
3. clone embryos makes up the method with vitro culture between Tibetan antelope as claimed in claim 1-goat kind, it is characterized in that: the ripe liquid in the described step (2) is to contain 0.22g/100mL NaHCO by 10% the foetal calf serum that accounts for total liquor capacity and 90%
3TCM199 basic culture solution and the Sodium.alpha.-ketopropionate of 0.22mg/100mL, the hydroxyethyl piperazine ethanesulfonic acid of 238mg/100mL, the penicillin of 75 μ g/mL, the Streptomycin sulphate of 100 μ g/mL, the follicular stimulating hormone of 10 μ g/mL, the prolan B of 10 μ g/mL, the 17 β estradiol of 1 μ g/mL form.
4. clone embryos makes up the method with vitro culture between Tibetan antelope as claimed in claim 1-goat kind, it is characterized in that: the saturated humidity CO2gas incubator in the described step (2) is meant that temperature is 38.5 ℃, contains 5%CO
2The CO2gas incubator of saturated humidity air.
5. the method for clone embryos structure and vitro culture between Tibetan antelope as claimed in claim 1-goat kind is characterized in that: the donorcells in the described step (4) is the inoblast of Tibetan antelope ear tissue.
6. the method for clone embryos structure and vitro culture between Tibetan antelope as claimed in claim 1-goat kind, it is characterized in that: the embryo medium in the described step (5) is by the KCl of NaCl, the 0.0534g/100mL of 0.6294g/100mL, the KH of 0.0162g/100mL
2PO
4, 0.0251g/100mL CaCl
22H
2The Sodium.alpha.-hydroxypropionate of O, 47 μ L/100mL, the MgCl of 0.01g/100mL
26H
2The NaHCO of O, 0.2106g/100mL
3, the Sodium.alpha.-ketopropionate of 0.0033g/100mL, the L-glutaminate of 0.0146g/100mL, the indispensable amino acid of 2mL/100mL, the non-essential amino acid of 1mL/100mL, the bovine serum albumin of 0.8g/100mL, the gentamicin of 0.005g/100mL and the phenol red composition of 0.001g/100mL.
7. the method for clone embryos structure and vitro culture between Tibetan antelope as claimed in claim 1-goat kind, it is characterized in that: the T20 in the described step (5) is made up of 20% foetal calf serum that accounts for total liquor capacity and 80% the TCM199 basic culture solution that contains the 10mM hydroxyethyl piperazine ethanesulfonic acid, and after configuration, add two resisting, regulate the solution of pH value to 7.2～7.4 then with NaOH; Wherein two anti-the be meant penicillin of 75 μ g/mL and the Streptomycin sulphates of 100 μ g/mL.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132363A (en) * | 2015-09-09 | 2015-12-09 | 南京农业大学 | First cleavage time- and blastocyst gene expression level-based method for screening related genes for detecting goat cloned embryo quality |
| CN105670988A (en) * | 2016-02-02 | 2016-06-15 | 中国农业科学院北京畜牧兽医研究所 | Mammal embryo flushing solution as well as preparation method and application thereof |
| CN110055212A (en) * | 2019-02-14 | 2019-07-26 | 中国科学院西北高原生物研究所 | A method of embryo is produced using Ovisammon Linnaeus tissue sperm in vitro |
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| CN1524121A (en) * | 2001-04-20 | 2004-08-25 | ����ϣ��ѧ | Method of nuclear transfer |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1524121A (en) * | 2001-04-20 | 2004-08-25 | ����ϣ��ѧ | Method of nuclear transfer |
Non-Patent Citations (3)
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| ZHEN-JUN ZHAO ET AL: "Interspecies nuclear transfer of tibetan antelope using caprine oocyte as recipient", 《MOLECULAR REPRODUTION AND DEVELOPMENT》 * |
| ZHEN-JUN ZHAO ET AL: "Rabbit ooctye cytoplasm supports development of nuclear transfer embryos derived from the somatic cells of the camel and tibetan antelope", 《JOURNAL OF REPRODUCTION AND DEVELOPMENT》 * |
| 潘晓燕: "盘羊-绵羊异种核移植技术程序的优化研究", 《中国博士学位论文全文数据库》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132363A (en) * | 2015-09-09 | 2015-12-09 | 南京农业大学 | First cleavage time- and blastocyst gene expression level-based method for screening related genes for detecting goat cloned embryo quality |
| CN105670988A (en) * | 2016-02-02 | 2016-06-15 | 中国农业科学院北京畜牧兽医研究所 | Mammal embryo flushing solution as well as preparation method and application thereof |
| CN105670988B (en) * | 2016-02-02 | 2020-06-12 | 中国农业科学院北京畜牧兽医研究所 | Mammal egg-washing liquid and its preparation method and use |
| CN110055212A (en) * | 2019-02-14 | 2019-07-26 | 中国科学院西北高原生物研究所 | A method of embryo is produced using Ovisammon Linnaeus tissue sperm in vitro |
| CN110055212B (en) * | 2019-02-14 | 2022-12-23 | 中国科学院西北高原生物研究所 | Method for producing embryo in vitro by using sperm of pannage testis tissue |
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|---|---|
| CN101880690B (en) | 2012-12-19 |
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