CN101880689B - Construction and ectogenesis method of Tibetan antelope-cattle interspecific somatic nucleus transferred embryo - Google Patents
Construction and ectogenesis method of Tibetan antelope-cattle interspecific somatic nucleus transferred embryo Download PDFInfo
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- CN101880689B CN101880689B CN201010200550XA CN201010200550A CN101880689B CN 101880689 B CN101880689 B CN 101880689B CN 201010200550X A CN201010200550X A CN 201010200550XA CN 201010200550 A CN201010200550 A CN 201010200550A CN 101880689 B CN101880689 B CN 101880689B
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Abstract
The invention relates to a construction and ectogenesis method of a Tibetan antelope-cattle interspecific somatic nucleus transferred embryo. In the method, a Tibetan antelope-cattle 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 yellow cattle, a cow or a yak as a recipient cytoplasm by a zona pellucida-free hand guide type clone technology, and a high-activity Tibetan antelope-cattle interspecific somatic nucleus transferred embryo 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 to a kind mesosome nuclear transplantation embryo structure and ectogenesis method, relate in particular to body-cell neucleus transplanting embryo's between Tibetan antelope-Niu kind structure and ectogenesis method.
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, and makes the caryoplasm recombinant chou the same with the normal fertilization ovum, through cell fission, differentiation and in parent, develop into a new individuality.It is individual that body-cell neucleus transplanting can produce unlimited clone in theory, might utilize a large amount of rare species of nuclear transfer technology clone.Up to the present, in the world interspecies cloning success gaur (Lanza et al., 2000), east argali (Loi et al. arranged; 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, in the rescue of the conservation of wildlife, rare animal and animals on the brink of extinction, can bring into play its vital role.
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 being difficult to Tibetan antelope is furtherd investigate; Research about Tibetan antelope is main with macroscopical zoological research mainly at present, 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-Niu kind.
Summary of the invention
Technical problem to be solved by this invention provides body-cell neucleus transplanting embryo's between a kind of Tibetan antelope-Niu kind that obtains high vigor embryo structure and ectogenesis method.
For addressing the above problem, body-cell neucleus transplanting embryo's structure and ectogenesis method between a kind of Tibetan antelope of the present invention-Niu kind may further comprise the steps:
(1) collection of ovary: behind the ovary clip of the ox that will just butcher, put into that saline water, temperature are housed is 20～25 ℃ vacuum flask, extract the ovarian follicle between diameter 2～8mm on the ox ovary surface then; The liquor folliculi that extracts with its 1～2 times of volume choose the dilution of ovum liquid after, 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 said 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 said 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 said 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 said step (4) gained, treat that a donorcells and two stoning acceptor ovocytes all merge after, balance 3h in embryo medium; Then clone embryos between said kind is placed the solution of 1mL calcium ions carrier (Ca ionophore), in the saturated humidity CO2gas incubator, cultivate 5min; Again clone embryos between said kind is transferred in the 6-dimethylaminopurine (6-DMAP) that droplet volume is 10 μ L, in the saturated humidity CO2gas incubator, accomplishes the activation of clone embryos between Tibetan antelope-Niu kind behind the cultivation 4.5h; Put into little cave of the 4 orifice plates bottom that the 0.5mL embryo medium is housed after clone embryos between Tibetan antelope-Niu kind activated, in the saturated humidity CO2gas incubator, cultivate and get final product.
Ox ovary in the said step (1) is meant any one the ovary in yak, ox, the milk cow.
The ovum liquid of choosing in the said step (1) is made up of 3% NBCS (NBS) that accounts for total liquor capacity and 97% Du Shi phosphoric acid buffer (D-PBS).
Ripe liquid in the said 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 HEPES (HEPES) of 238mg/100mL, the penicillium mould 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 20 μ g/mL, 17 β Theelin,dihydro-(the 17-β E of 1 μ g/mL
2) form.
Saturated humidity CO2gas incubator in the said step (2) is meant that temperature is 38.5 ℃, contains 5%CO
2The CO2gas incubator of saturated humidity air.
Donorcells in the said step (4) is the inoblast of Tibetan antelope ear tissue.
Embryo medium in the said step (5) 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 qingfengmeisu qiong 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 said 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 HEPES, and after configuration, adds two anti-ly, regulates the solution of pH value to 7.2～7.4 then with NaOH; Wherein two anti-the be meant penicillium mould 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; Bovine oocyte is as recipient cytoplasm; Utilize the hand-guided clone technology of no zona pellucida to carry out body-cell neucleus transplanting embryo's between the Tibetan antelope kind structure; Set up little cave formula cultural method of clone embryos, explored with bovine oocyte 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 running 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, utilization the inventive method is to after the ovary of 257 yaks, 808 oxes and 722 milk cows of collection aspirates respectively; Obtain ovum number and be respectively 1177,3066,3694; Every ovary on average gets the ovum number and is respectively 4.58,3.79,5.11, and its oocyte maturation rate is respectively 67.94% ± 4.1%, 85.07% ± 2.2%, 69.43% ± 1.8%; The stoning ovum number of yak, ox, milk cow is respectively 344,479,249, and fourth contact ovum number is respectively 302,321,211, and the fourth contact rate is respectively 87.79%, 67.01%, 84.73%.
Description of drawings
Do further detailed explanation below in conjunction with the accompanying drawing specific embodiments of the invention.
Fig. 1 is body-cell neucleus transplanting embryo between Tibetan antelope-yak kind that the present invention obtained.Figure is shown is yak ovocyte and Tibetan antelope somatocyte of two stonings after the auxiliary fusion of chemistry 10 minutes; Through the position of Hoechst 33342 dyeing showed cell nuclear under fluorescent microscope captured reconstructed embryo form, shinny points are donorcells nuclear in the middle of two ovocytes.Magnification is 100 times.
Fig. 2 is the developmental state of body-cell neucleus transplanting embryo vitro culture 72h in the culture systems of little cave between Tibetan antelope of the present invention-yak kind.Figure is shown be two stonings yak ovocyte and Tibetan antelope somatocyte through chemistry is auxiliary merge after, activate cultivation 16～32 cell stages that 72h produced in the culture systems of little cave with chemical process.Magnification is 40 times.
Embodiment
Material: the ox ovary is collected in slaughterhouse, Le Jia gulf, Xining, Qinghai Province.
Body-cell neucleus transplanting embryo's structure and ectogenesis method between a kind of Tibetan antelope-Niu kind may further comprise the steps:
(1) collection of ovary: the ovary of the ox that will just butcher with sterilization scissors clip after, put into that saline water, 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 saline water; Plastic injector for temporary use with 20mL extracts the ovarian follicle between diameter 2～8mm on the ox ovary surface then; The liquor folliculi that extracts with its 1～2 times of volume choose the dilution of ovum liquid after, under stereoscope, choose and get ovarian cumulus in the liquor folliculi-ovocyte complex body.
Wherein: choose ovum liquid and form by 3% NBCS (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 HEPES (HEPES) of 238mg/100mL, the penicillium mould 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 20 μ g/mL, 17 β Theelin,dihydro-(the 17-β E of 1 μ g/mL
2) form.
(3) the hand-guided stoning of mature oocyte: the mature oocyte of said 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 peripheral ovarian cumulus granulosa cell of ovocyte zona pellucida removes in the petridish 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, in the saturated humidity incubator, cultivates 2h then.After cultivation finishes ovocyte is taken out from nutrient solution, clean up with T20 (HEPES buffered TCM199 adds 20% foetal calf serum), subsequent use.
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, in the 35mm petridish, adds 2mLT20 solution then and serves as and cut ovum liquid.The ovocyte of no zona pellucida being transferred in the petridish, 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 (referring to Fig. 1) between obtaining planting.
Wherein:
The inoblast of donorcells---Tibetan antelope ear tissue adopts following method to obtain:
1. sampling: during sampling, cut the individual ear's hair of female adult Tibetan antelope with the scissors of sterilization earlier, reject residual root of hair as much as possible with razor blade again.Clean last clip 1cm then successively with perfumed soap, zero(ppm) water, SANIZOL C, saline water, 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-DMEM/F12 nutrient solutions flushing 3 times, the sample tissue piece is put into the 35mm petridish 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, be inverted and be placed in the saturated humidity CO2gas incubator, the culturing bottle that overturns gently behind the 3h adds 3～5mL DMEM/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 the PBS of primary cell with no Ca2+, Mg2+ cleaned 2 times, the mixed solution that contains 0.05% trypsinase and 0.01% YD 30 (EDTA) with 1～2mL again digests, and 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 DMEM/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 after, obtain cell precipitation thing A; With the DMEM/F12 nutrient solution with cell precipitation thing A mixing gently; Import in another culturing bottle; In the saturated humidity CO2gas incubator, continue to cultivate 3～6 days, and obtained growing to 80%～90% SF that converges, this SF is the shuttle type.
4. cell cryopreservation: SF is contained the mixed solution digestion of 0.05% trypsinase and 0.01% YD 30 (EDTA) with 1～2mL; Centrifugal back adds the cells frozen storing liquid mixing; Preserve half a 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 SF.
5. cell recovery: the frozen pipe that frozen SF 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 DMEM/F12 nutrient solution of 9 times of said 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 DMEM/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 DMEM/F12 nutrient solution is all by 10% the foetal calf serum that accounts for total nutrient solution volume, 90% DMEM/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 HEPES, 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 NaCl of 0.8g/100mL, the KCl of 0.0203g/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 penicillium mould 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 NaCl of 0.8g/100mL, the KCl of 0.02g/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% DMSO 99.8MIN., 70% DMEM/F12 and two anti-composition.
Above-mentioned two anti-the be meant penicillium mould 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, in the 60mm petridish, making volume by ordinary method is the fusion droplet of 25 μ L.
Successively the acceptor ovocyte is moved in T20, phytohaemagglutinin, T2B, the fusion liquid droplet then and operate, cover Yellow Protopet 2A on the droplet, avoid droplet evaporation and position to move.
Earlier the acceptor ovocyte is moved in the T20 droplet; Move again in acceptor ovocyte to the phytohaemagglutinin droplet; Transfer to then to glue in the T2B droplet and get a donorcells; Again be transferred to again in the phytohaemagglutinin droplet, from the T20 droplet, move again in acceptor ovocyte to the phytohaemagglutinin droplet, because phytohaemagglutinin can make cell surface have viscosity; So the position through adjustment donorcells and acceptor ovocyte complex body and another ovocyte can form the complex body that two ovocytes are mingled with a donorcells; 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 HEPES, and after configuration, adds two anti-ly, regulates the solution of pH value to 7.2～7.4 then with NaOH; Wherein two anti-the be meant penicillium mould 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 Z 150PH fully are dissolved in the 196mL zero(ppm) water, add the MgSO that 2mL prepares in advance respectively to it then
4And CaCl
22H
2O solution promptly gets after the filtration.
(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 petridish 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 Yellow Protopet 2A on the droplet, each droplet is put into a clone embryos.Then it is cultivated the activation of accomplishing clone embryos between Tibetan antelope-Niu kind behind the 4.5h in the saturated humidity CO2gas incubator; Little cave with putting into the 4 orifice plates bottom that the 0.5mL embryo medium is housed after the clone embryos activation between Tibetan antelope-Niu kind covers Yellow Protopet 2A, in the saturated humidity CO2gas incubator, cultivates and gets final product (referring to Fig. 2).
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 qingfengmeisu qiong 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 HEPES, and after configuration, adds two anti-ly, regulates the solution of pH value to 7.2～7.4 then with NaOH; Wherein two anti-the be meant penicillium mould of 75 μ g/mL and the Streptomycin sulphates of 100 μ g/mL.
Ox ovary among the present invention is meant any one the ovary in yak, ox, the milk cow.The saturated humidity CO2gas incubator is meant that temperature is 38.5 ℃, contains 5%CO
2The CO2gas incubator of saturated humidity air.
Claims (3)
1. body-cell neucleus transplanting embryo's structure and ectogenesis method between Tibetan antelope-Niu kind may further comprise the steps:
(1) collection of ovary: behind the ovary clip of the ox that will just butcher, put into that saline water, temperature are housed is 20～25 ℃ vacuum flask, extract the ovarian follicle between diameter 2～8mm on the ox ovary surface then; The liquor folliculi that extracts with its 1～2 times of volume choose the dilution of ovum liquid after, choose and get ovarian cumulus in the liquor folliculi-ovocyte complex body; Choosing ovum liquid is made up of 3% NBCS that accounts for total liquor capacity and 97% Du Shi phosphoric acid buffer;
(2) maturation in vitro of ovocyte is cultivated: after the ovarian cumulus-ovocyte complex body of said step (1) gained was cleaned with ripe liquid, ripe cultivation 20～24h obtained mature oocyte in the saturated humidity CO2gas incubator; Ripe liquid is to contain 0.22g/100mL NaHCO by 10% the foetal calf serum that accounts for total liquor capacity and 90%
3The Sodium.alpha.-ketopropionate, the HEPES of 238mg/100mL, the penicillium mould 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 20 μ g/mL, the 17 β Theelin,dihydro-of 1 μ g/mL of TCM199 basic culture solution and 0.22mg/100mL form;
(3) the hand-guided stoning of mature oocyte: the mature oocyte of said 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 peripheral ovarian cumulus granulosa cell of ovocyte zona pellucida removes in the petridish 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 ovocyte, in the saturated humidity incubator, cultivates 2h then; After cultivation finishes ovocyte is taken out from nutrient solution, clean up with T20, subsequent use;
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, in the 35mm petridish, adds 2mLT20 solution then and serves as and cut ovum liquid; The ovocyte of no zona pellucida being transferred in the petridish, 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;
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 HEPES, and after configuration, adds two anti-ly, regulates the solution of pH value to 7.2～7.4 then with NaOH; Wherein two anti-the be meant penicillium mould of 75 μ g/mL and the Streptomycin sulphates of 100 μ g/mL;
(4) donorcells is prepared and cytogamy: the stoning acceptor ovocyte that will cultivate donorcells and said step (3) gained in 24 orifice plates adopts chemistry to assist fusion method to carry out cytogamy, clone embryos between obtaining planting;
Wherein:
The inoblast of donorcells---Tibetan antelope ear tissue adopts following method to obtain:
1. 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-DMEM/F12 nutrient solutions flushing 3 times, the sample tissue piece is put into the 35mm petridish 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, be inverted and be placed in the saturated humidity CO2gas incubator, the culturing bottle that overturns gently behind the 3h adds 3～5mL DMEM/F12 nutrient solution and continues to cultivate 10～18 days, obtains growing to 80%～90% primary cell that converges; The DMEM/F12 nutrient solution is all by 10% the foetal calf serum that accounts for total nutrient solution volume, 90% DMEM/F12 and two anti-composition;
2. go down to posterity and purifying: the primary cell cultivation of going down to posterity; At first primary cell is used no Ca
2+, Mg
2+PBS clean 2 times, the mixed solution that contains 0.05% trypsinase and 0.01% YD 30 with 1～2mL again digests, and obtains Digestive system A; 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 DMEM/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 after, obtain cell precipitation thing A; With the DMEM/F12 nutrient solution with cell precipitation thing A mixing gently; Import in another culturing bottle; In the saturated humidity CO2gas incubator, continue to cultivate 3～6 days, and obtained growing to 80%～90% SF that converges, this SF is the shuttle type;
3. cell cryopreservation: SF is contained the mixed liquor digestion of 0.05% trypsase and 0.01% ethylenediamine tetra-acetic acid 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 SF;
4. cell recovery: the frozen pipe that frozen SF 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 DMEM/F12 nutrient solution of 9 times of said 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 DMEM/F12 nutrient solution, and be inoculated in the culturing bottle, place the saturated humidity CO2gas incubator to cultivate and get final product;
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 then; T2B 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;
Secondly, in the 60mm petridish, making volume by ordinary method is the fusion droplet of 25 μ L;
Successively the acceptor ovocyte is moved in T20, phytohaemagglutinin, T2B, the fusion liquid droplet then and operate, cover Yellow Protopet 2A on the droplet, avoid droplet evaporation and position to move;
Earlier the acceptor ovocyte is moved in the T20 droplet; Move again in acceptor ovocyte to the phytohaemagglutinin droplet; Transfer to then to glue in the T2B droplet and get a donorcells; Again be transferred to again in the phytohaemagglutinin droplet, from the T20 droplet, move again in acceptor ovocyte to the phytohaemagglutinin droplet, because phytohaemagglutinin can make cell surface have viscosity; So the position through adjustment donorcells and acceptor ovocyte complex body and another ovocyte can form the complex body that two ovocytes are mingled with a donorcells; 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;
(5) plant between clone embryos activate and vitro culture: with clone embryos balance 20～30min in the T20 droplet between the kind of said step (4) gained, treat that a donorcells and two stoning acceptor ovocytes all merge after, balance 3h in embryo medium; Then clone embryos between said kind is placed the solution of 1mL calcium ions carrier, in the saturated humidity CO2gas incubator, cultivate 5min; Again clone embryos between said kind is transferred in the 6-dimethylaminopurine that droplet volume is 10 μ L, in the saturated humidity CO2gas incubator, accomplishes the activation of clone embryos between Tibetan antelope-Niu kind behind the cultivation 4.5h; Put into little cave of the 4 orifice plates bottom that the 0.5mL embryo medium is housed after clone embryos between Tibetan antelope-Niu kind activated, in the saturated humidity CO2gas incubator, cultivate and get final product; 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 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 qingfengmeisu qiong of 0.005g/100mL and the phenol red composition of 0.001g/100mL; 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 HEPES, and after configuration, adds two anti-ly, regulates the solution of pH value to 7.2～7.4 then with NaOH; Wherein two anti-the be meant penicillium mould of 75 μ g/mL and the Streptomycin sulphates of 100 μ g/mL.
2. body-cell neucleus transplanting embryo's structure and ectogenesis method between Tibetan antelope as claimed in claim 1-Niu kind is characterized in that: the ox ovary in the said step (1) is meant any one the ovary in yak, ox, the milk cow.
3. body-cell neucleus transplanting embryo's structure and ectogenesis method between Tibetan antelope as claimed in claim 1-Niu kind is characterized in that: the saturated humidity CO2gas incubator in the said step (2) is meant that temperature is 38.5 ℃, contains 5%CO
2The CO2gas incubator of saturated humidity air.
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| Title |
|---|
| Interspecies nuclear transfer of tibetan antelope using caprine oocyte as recipient;Zhen-Jun Zhao et al;《Moleclular reproduction and development》;20071231;第74卷;第412-419页 * |
| Rabbit oocyte cytoplasm supports development of nuclear transfer embryos derived from the somatic cells of the camel and tibetan antelope;Zhen-Jun Zhao et al;《Journal of reproduction and development》;20061231;第52卷(第3期);全文 * |
| Zhen-Jun Zhao et al.Interspecies nuclear transfer of tibetan antelope using caprine oocyte as recipient.《Moleclular reproduction and development》.2007,第74卷第412-419页. |
| Zhen-Jun Zhao et al.Rabbit oocyte cytoplasm supports development of nuclear transfer embryos derived from the somatic cells of the camel and tibetan antelope.《Journal of reproduction and development》.2006,第52卷(第3期),全文. |
| 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》.2009,(第8期),全文. |
| 盘羊-绵羊异种核移植技术程序的优化研究;潘晓燕;《中国博士学位论文全文数据库》;20090815(第8期);全文 * |
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