CN1814750A - Mammal body-cell neucleus transplanting method - Google Patents
Mammal body-cell neucleus transplanting method Download PDFInfo
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- CN1814750A CN1814750A CNA2005100238720A CN200510023872A CN1814750A CN 1814750 A CN1814750 A CN 1814750A CN A2005100238720 A CNA2005100238720 A CN A2005100238720A CN 200510023872 A CN200510023872 A CN 200510023872A CN 1814750 A CN1814750 A CN 1814750A
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Abstract
This invention provides a nucleus transplantation method for mammal somatic cells characterizing in taking the oocyte of cross-fertilized F1 generation of the mammal as the recipient somatic cell for nucleus transplantation, which can increase the re-programming ability of recipient cytoplast of nucleus transplantation of mammals obviously, improve the growth of re-structured embryo to increase the clone efficiency of somatic cells.
Description
Technical field
The present invention relates to transgenic animal technology, embryo engineering and developmental biology field.Specifically, be about a kind of mammal body-cell neucleus transplanting method.
Background technology
The animal cloning technology is that the donor somatic cell nuclear is transplanted in the enucleation oocyte kytoplasm through the method for micrurgy and/or cytogamy, reformulates embryo's (reconstructed embryo).Reconstructed embryo is grown to blastaea through cultivation, again blastaea is transplanted to foster mother's intrauterine of synchronization of estrus, the animal of giving a birth out after waiting to reach maturity, this animal is called as " cloned animal ", this technology then be called as " somatic cell clone technique " or " somatic cell nuclear transfer technique " (Nuclear transfer, NT).
The mammalian somatic cell clone technology is through in a few years development, obtained remarkable progress already, the cloned animals such as sheep, goat, ox, pig, mouse, cat, rabbit that have been born in succession are for wide space has been opened up in the required donor organ of domestic animal stock breeding, animals on the brink of extinction rescue, production xenotransplant and the treatment of human diseases etc.But, present cloning efficiency is still very low, and cloned animal can occur many unusual in utero, as clone embryos developmental arrest, placental abnormality, abortion ratio height, the birth whose body weight is overweight and terateger rate is high (Han YM, Kang YK, Koo DB.Theriogenology, 2003,59 (1): 33～44).These problems have greatly limited the application and the development of clone technology.
We know that in the normal fertilization process, ovocyte is accepted sperm, and identification mutually starts fetal development; What move into the ovocyte kytoplasm in nuclear transfer technology then is the somatic cell nuclear that has broken up, its genome state and sperm have very big-difference, difficulty can take place to its " identification " in ovocyte, and then causes the failure of reprogrammed or not exclusively, clone embryos can't be finished growth.Therefore, as the ovocyte of reconstructed embryo recipient cytoplasm ability, be one of important factor that influences nuclear transplantation efficient to the reprogrammed of donor nuclei.
Yet the clened cows work that carry out in many laboratories has been subjected to the restriction in experiment material ovary source to a great extent.Because these ovaries slaughterhouse that mostly has drawn from, can't estimate situations such as the proterties of the ox that ovary is provided and physiological function, more have no way of understanding its genetic background.Simultaneously, from the recipient cell of different genetic backgrounds, its cytoplasmic structure composition also can be different.The difference of constituent mainly shows as the diversity and the interior environmental differences of tenuigenin of DNA type in the cell cytoplasm in the ovocyte kytoplasm.Different DNA form and in environmental differences can influence the division of reconstructed embryo of nuclear transplantation and follow-up growth thereof.
The advantage of ovocyte in fetal development of first familiar generation that some experiments in the past are verified.In the production of transgenic animal, higher 8 times than the efficient of using inbred lines C57BL/6 mouse with hybridization system; At the female activated of orphan experimentally, be higher than with the lonely female activated embryo of the ovocyte of inbred lines C57BL/6 with lonely its blastaea hatching rate of female activated embryo of the ovocyte of (B6D2) F1.This may be mainly relevant with the heterosis, hybrid vigor of hybridization ovocyte.Any one hybridization is individual all might to have higher throughput than its father and mother.When two purebred hybridization, we will obtain to have the crossbred of higher hybridization heritability.An experiment has confirmed the existence of hybridization ovocyte heterosis, hybrid vigor from molecular level.Utilize the amphitropic protein gel electrophoresis relatively the ovocyte of DBA/2 and C57BL/6 find to have at least 17 kinds of albumen to exist difference on synthetic.And the synthetic ratio that these all albumen can both synthesize and have on the ovocyte in hybridization (B6D2) F1 generation of last two kinds of strains is also higher, wherein also having two kinds of albumen of synthetic also to be considered at DBA/2 very likely is ovocyte modifying factor (Latham KE et al.Development, 1994; 120 (12): 3419-26).Its mechanism may be with the different and imprinted genes of allelotrope expression level between two strains different relevant.As everyone knows, somatocyte be moved into behind the ovocyte must be as soon as possible by reprogrammed expressing the required gene of early embryo development, and the reprogrammed factor in the ovocyte kytoplasm has the ability of removing the original memory of cell just.Therefore, the genetic diversity of hybridization ovocyte heterosis, hybrid vigor makes it have higher reprogrammed ability, ovum form phase and ripening stage hybridization ovocyte will be not only on kind and also the quantitatively synthetic more reprogrammed factor improve the growth of reconstructed embryo, thereby improve somatic cell clone efficient.
Existing many researchs are attempted by selecting different acceptor ovocytes to improve growth (Betthauser J, et al.Nat.Biotechnol., 2000,18 (10): 1055-9 of clone's embryo; Bondioli K, et al.Mo1.Reprod.Dev., 2001,60 (2): 189-95; Miyoshi K, et al.Biol.Reprod., 2002,67 (2): 540-5), as the contrast of MII phase ovocyte and MI phase ovocyte; The contrast of maturation in vitro and cylinder mature ovocyte and derive from prepuberal animal and the contrast of the ovocyte of adult animal or the like.But do not see as yet that up to now, the ovocyte of useful first familiar generation carries out the report of cloning of mammalian animal as recipient cell.
Summary of the invention
Purpose of the present invention just is to utilize the advantage of first familiar generation ovocyte in fetal development, thereby a kind of mammal body-cell neucleus transplanting method is provided.
The present inventor is through discovering, utilize mammiferous first familiar generation ovocyte can improve the reprogrammed ability of recipient cytoplasm ovocyte effectively as recipient cell, improve the growth of reconstructed embryo, and then improve the early development rate of animal nuclear transfer embryo.
Therefore, the topmost characteristics of mammal body-cell neucleus transplanting method of the present invention are to carry out nuclear transplantation with the ovocyte of mammiferous first familiar generation as recipient cell, specifically may further comprise the steps:
A, get mammalian somatic cell and prepare the donor nuclei cell;
B, get the ovocyte of first familiar generation as recipient cell;
C, the ovocyte of first familiar generation is examined removal;
D, will donorcells nuclear merge after injecting non-nucleus egg mother cell.
Wherein, the described mammalian somatic cell of steps A can be taken from the tissue of ox, sheep, pig, dog, cat, rabbit, monkey or mouse, the cell of organ, the cell of vitro culture and the cell that genetic modification is crossed.Preferably, described donor nuclei cell is clearly inoblast or the cumulus cell of cow of genetic background.
The female parent of the first familiar generation ovocyte of step B and donor nuclei cell are same strain, and preferably, described ovocyte is that F1 passes through live egg-fetching (ovum pick up, the fresh ovocyte that method OPU) obtains for catalo;
Among the step D, use the interior injection of kytoplasm that the donor nuclei cell is injected ovocyte ovum week crack, incorporate ovocyte by fusion method then.
Mammal body-cell neucleus transplanting method of the present invention has following advantage:
1, utilizing the heterosis, hybrid vigor of first familiar generation, is recipient cytoplasm with F1 for the ovocyte of catalo, has improved the reprogrammed ability of recipient cytoplasm, thereby has improved the growth of reconstructed embryo, improves somatic cell clone efficient.With solving at present the difficult problem of somatic cell clone inefficiency in the world to a certain extent, set up somatic cell nuclear transfer technique platform efficiently;
2, utilize hybridization ovocyte tenuigenin nuclear transfer technology, not only in the quantity of blastaea, and all increasing significantly qualitatively at blastaea.
Embodiment
For the ease of understanding the present invention, the spy enumerates following examples.Its effect should be understood that being is not restriction to any way of the present invention to annotation of the present invention.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) for example, or the condition of advising according to manufacturer.
Experiment material and method:
Employed experiment reagent in following examples unless otherwise indicated is U.S. Sigma company product.
Present embodiment is chosen the young holstein cow, ox and the F1 that set up the oestrus cycle and is divided into groups for catalo (holstein cows * yellow race bull), is respectively holstein cow group, ox group and F1 and organizes for catalo.The age is 12-13 month when beginning to test, and body weight is similar with body condition.
The cumulus cell that adopts holstein cow in the present embodiment is as the donor nuclei cell, and with F1 for the ovocyte of catalo (holstein cows * yellow race bull) as the acceptor ovocyte, and simultaneously in contrast with the ovocyte of holstein cow and ox.
The preparation of embodiment 1, donor nuclei cell
Get the cumulus cell in 1～5 generation of holstein cow or inoblast with the D-MEM/F-12 (Gibco that adds 10% foetal calf serum (FBS), Grand Island, NY) cultivate, at the bottom of cell grows to bottle 80～90% o'clock, nutrient solution is changed into the nutrient solution serum starvation 2～3 days that contains 0.5%FBS.Use about 3 minutes attached cells of tryptic digestion of 0.25% then, behind the cell washing that digests 2 times, add the suspension that an amount of TCM199+10%FBS blows and beats into individual cells repeatedly, it is stand-by promptly to obtain the donor nuclei cell.
The preparation of embodiment 2, acceptor ovocyte
The Niu Jinhang live egg-fetching of each experimental group is obtained ovocyte, the concrete grammar of live egg-fetching mainly as Petyim etc. (Petyim SR, et al.J.Vet.Med.A.Physio1.Pathol.Clin.Med.2000,47:627-640) described, specific as follows:
Make cow Baoding of standing, sterilization anesthesia.Whole process is carried out under the Type B ultrasonic surveillance.To have the light and slow vagina arched roof that is inserted into of the probe of adopting the ovum pin, operator draw ovary to the Ultrasonic-B probe end across rectal wall by rectum, the ovum pin is adopted in propelling, simultaneously, with push-switch control vacuum pump suction liquor folliculi, pressure is about 95mmHg, afterwards, adopt ovum pin and conduit with DPBS (containing 3%BSA and 2u/ml heparin) flushing, washing fluid is also together put into collection tube.Write down the OPU number of operations of catalo group, holstein cow group and ox group respectively, and the quantity of the ovocyte that obtains.
The ovocyte of collecting is moved into ripe droplet (TCM-199 (Gibco, Grand Island NY) add the short ovarian follicle of 10% foetal calf serum, 10 μ g/ml prolan Bs, 1 μ g/ml estradiol and 1 μ g/ml and generate plain).Culture condition is 5%CO
2, 38.5 ℃, saturated humidity is cultivated 20h.The ovocyte of ripe 20h is put into hyaluronic acid enzymic digestion 1～2min of 0.5%, with inhaling embryonic tube pressure-vaccum repeatedly, slough the cumulus cell that adheres to simultaneously.Then, wash 3 times through TCM199 liquid, under stereoscopic microscope, the ovocyte of selecting to discharge first polar body places the TCM199 droplet stand-by as the acceptor ovocyte.
Embodiment 3, nuclear transplantation and fusion
The ovocyte that donor nuclei cell that embodiment 1 is obtained and embodiment 2 obtain moves in the operation liquid (TCM199+10%FBS+5 μ g/ml cytochalasin B), according to (Park SH, et al.Mol Reprod Dev 2004 such as Park; 68:25-34) described method is removed the nuclear of ovocyte at microscopically, then the donor nuclei cell is moved into ovum week crack.
The concrete operations of stoning and notes nuclear are as follows: after in the ovocyte immigration operation liquid 20 minutes, together place donor cell and ovocyte on the slide, at microscopically, with holding fixedly ovocyte of ovum pin, the polar body that ovocyte is discharged is in 3 positions of clock and watch, kernel removing needle faces or departs from polar body slightly, removes polar body and a part of kytoplasm.Immediately 1 donor cell is moved into after the stoning in all cracks of ovum of enucleation oocyte, repeat the operation of next piece ovum, after all ovocytes operations are finished, place TCM199 liquid to cultivate 30min the ovocyte that moves into donor cell.
The ovocyte of annotating after examining merges liquid (0.3M N.F,USP MANNITOL, 0.15mM Ca at the electricity that contains N.F,USP MANNITOL of 38.5 ℃ of preheatings
2+, 0.15mM Mg
2+) middle balance 1～2min, move in the electric integration slot, between electrode, pass to two DC pulse (2.5kV/cm, 10 μ s/ time, 1s at interval), induce fusion.Behind 30～60min, under stereoscopic microscope, check the fusion situation, merge ovum and be reconstructed embryo.Write down the quantity that catalo group, holstein cow group and ox are organized the fusion reconstructed embryo that obtains respectively.
The activation of embodiment 4, reconstructed embryo and vitro culture
After reconstructed embryo is washed 3 times with activation solution TCM199 (containing 10%FBS, 5 μ g/ml cytochalasin Bs, 10 μ g/ml cycloheximides), move in 38.5 ℃ of pre-warm good activation solution droplets and act on 5h.
Reconstructed embryo after the activation is put into the B2 nutrient solution, and (LABORATOIRE C.C.D, France) co-culture system with individual layer Vero cell (is that the Vero cell suspends into density 1 * 10 with the B2 nutrient solution
5Individual/ml, make to cultivate with this suspension then and drip, behind about 48h cultivate drip the bottom surface will layer overlay Vero cell, can put into the embryo at this moment), at 5%CO
2, 38.5 ℃, cultivate in the incubator of saturated humidity.Change the B2 nutrient solution every 48h half amount.The spilting of an egg situation that activation back 48h observes reconstructed embryo is calculated spilting of an egg rate (spilting of an egg embryo number/cultivation embryo number); Cultivate the quantity of the 8th day record blastaea, calculate blastaea rate (embryo number of blastaea number/cultivation).
In addition, also carry out respectively cell counting blastula stage (Petyim SR, et al.J.Vet.Med.A.Physiol.Pathol.Clin.Med.2000,47:627-640), specific as follows:
The blastaea of cultivating the 8th day crushes the embryo with 5 μ g/ml Hoechst33342 solution-dyed 5min with cover glass, and fluorescent microscope is counting down, each blastaea statistics three times, computation of mean values.
More than the statistic data of each step operation and calculation result as shown in the following Table 1:
Table 1, the isoacceptor ovocyte is not to the fusion of clone embryos and the influence of growth (Mean ± SE)
| Parameter | The catalo group | The He Sitanniu group | The ox group | Amount to |
| Ovocyte | 353 | 847 | 327 | 1527 |
| Sophisticated ovocyte (%) | 256(73) | 614(72) | 238(73) | 1108(73) |
| The reconstructed embryo (%) that merges | 110(43) a | 216(35) b | 120(50) a | 446(40) |
| Spilting of an egg rate (%) | 73(66) | 142(66) | 90(75) | 305(68) |
| The blastaea rate D(％) | 37(51) a | 52(37) b | 24(27) b | 113(37) |
| Cell count in the blastaea | 135±4.1 a | 116±3.6 b | 101±4.2 c | 118±3.1 |
| A level blastaea E(％) | 20(54) | 22(42) | 7(29) | 49(43) |
Every line number word has different subscript letter persons that significant difference (P＜0.05) is arranged;
D: the blastaea rate is a radix with spilting of an egg number;
E: A level blastaea color and luster becomes clear and has fine and close inner cell mass and the trophoderm that is evenly distributed; It is average or poor that the quality of the B level of comparing and C level then is respectively;
By the result of table 1 as seen, from the quantity angle of blastaea, all be the highest in catalo group aspect reconstructed embryo that merges and the blastaea rate; From the quality angle of blastaea, the cell count catalo group in the blastaea also is maximum, and the ratio of A level blastaea also is the highest.
Therefore, of the present invention is the body-cell neucleus transplanting method of recipient cytoplasm with F1 for the ovocyte of catalo, can significantly improve the reprogrammed ability of recipient cytoplasm, improves the growth of reconstructed embryo, thereby improves somatic cell clone efficient.It is relevant that its reason may make it have higher reprogrammed ability with the genetic diversity of hybridization ovocyte heterosis, hybrid vigor.
Though be that example is described body-cell neucleus transplanting method of the present invention just more than with the ox, but method of the present invention is equally applicable to other Mammals, as ox, sheep, pig, dog, cat, rabbit, monkey or mouse etc., this is conspicuous for a person skilled in the art, do not need performing creative labour fully, therefore, these are used and should belong to this too
Scope of invention.
Claims (8)
1, a kind of mammal body-cell neucleus transplanting method is characterized in that, described nuclear transplantation method with the ovocyte of mammiferous first familiar generation as recipient cell.
2, the method for claim 1 is characterized in that, may further comprise the steps:
A, get mammalian somatic cell and prepare the donor nuclei cell;
B, get the ovocyte of first familiar generation as recipient cell;
C, the ovocyte of first familiar generation is examined removal;
D, will donorcells nuclear merge after injecting non-nucleus egg mother cell.
3, method as claimed in claim 1 or 2 is characterized in that, described Mammals is an ox.
4, method as claimed in claim 2 is characterized in that, mammalian somatic cell described in the described steps A is cumulus cell or the inoblast of ox.
5, method as claimed in claim 2 is characterized in that, the female parent of described first familiar generation ovocyte and donor nuclei cell are same strain.
6, method as claimed in claim 5 is characterized in that, described F1 is the bovine oocyte that obtains by the live egg-fetching operation for ovocyte.
7, method as claimed in claim 2 is characterized in that, among the described step D, the donor nuclei cell is injected ovocyte ovum week crack, incorporates ovocyte by fusion method then.
8, method as claimed in claim 6 is characterized in that, the actual conditions of described fusion method is: balance is 1～2 minute in the electricity fusion liquid that contains N.F,USP MANNITOL of preheating, moves in the electric integration slot, passes to two DC pulse between electrode, induces fusion.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102899353A (en) * | 2011-07-30 | 2013-01-30 | 华中农业大学 | Method for screening bovine nuclear transplantation donor cells by using antioxidant |
| CN103184189A (en) * | 2011-12-29 | 2013-07-03 | 北京富通华生物科技有限公司 | Cultivation method of cross-bred wagy |
| CN108715869A (en) * | 2018-06-01 | 2018-10-30 | 华南农业大学 | A method of improving cloning of mammalian animal efficiency based on specific donorcells are obtained |
| WO2019141052A1 (en) * | 2018-01-17 | 2019-07-25 | 中国科学院上海生命科学研究院 | Method for preparing non-human primate somatic cell cloned animal |
| CN111690684A (en) * | 2020-04-30 | 2020-09-22 | 广州再生医学与健康广东省实验室 | Composition and application thereof |
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- 2005-02-04 CN CNA2005100238720A patent/CN1814750A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102899353A (en) * | 2011-07-30 | 2013-01-30 | 华中农业大学 | Method for screening bovine nuclear transplantation donor cells by using antioxidant |
| CN102899353B (en) * | 2011-07-30 | 2015-04-29 | 华中农业大学 | Method for screening bovine nuclear transplantation donor cells by using antioxidant |
| CN103184189A (en) * | 2011-12-29 | 2013-07-03 | 北京富通华生物科技有限公司 | Cultivation method of cross-bred wagy |
| WO2019141052A1 (en) * | 2018-01-17 | 2019-07-25 | 中国科学院上海生命科学研究院 | Method for preparing non-human primate somatic cell cloned animal |
| US12091676B2 (en) | 2018-01-17 | 2024-09-17 | Center For Excellence In Brain Science And Intelligence Technology, Chinese Academy Of Sciences | Method for preparing non-human primate somatic cell cloned animal |
| CN108715869A (en) * | 2018-06-01 | 2018-10-30 | 华南农业大学 | A method of improving cloning of mammalian animal efficiency based on specific donorcells are obtained |
| CN111690684A (en) * | 2020-04-30 | 2020-09-22 | 广州再生医学与健康广东省实验室 | Composition and application thereof |
| CN111690684B (en) * | 2020-04-30 | 2022-07-29 | 广州明迅生物科技有限责任公司 | Composition and application thereof |
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