CN101921808A - Method for improving pig nucleus transplantation efficiency - Google Patents
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- CN101921808A CN101921808A CN 201010256337 CN201010256337A CN101921808A CN 101921808 A CN101921808 A CN 101921808A CN 201010256337 CN201010256337 CN 201010256337 CN 201010256337 A CN201010256337 A CN 201010256337A CN 101921808 A CN101921808 A CN 101921808A
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Abstract
The invention discloses a method for improving pig nucleus transplantation efficiency, which belongs to the field of breeding or inseminating methods. The method comprises the following steps of: processing a donor cell by a small molecule medicament Calyculin A and inducing donor chromatin condensation, methylation rise and acetylation reduction, wherein a somatic nucleus expresses genetic modified zero resetting; then carrying out nucleus transplantation and constructing reconstructed embryos; and selecting optimal concentration of 10nM and the processing time of 30 minutes by an optimizing condition. The invention improves the ectogenesis blastocyst rate of a pig clone embryo by 9.7 percent, increases 23.78 blastomeres and lays the foundation for improving the production efficiency of clone animals.
Description
Technical field
The present invention relates to a kind of method that improves nucleus transplantation efficiency, especially a kind of by Calyculin A pre-treatment donorcells, improve the method for pig nucleus transplantation efficiency, belong to breeding or insemination method field.
Background technology
Since clone sheep " Dolly " was born, somatic cell nuclear transfer technique was successively achieved success on multiple Mammals, but cloning efficiency is still very low, on average between 0.1%-2.0%.For improving nuclear transplantation efficient, the researchist has carried out a large amount of research at aspects such as the screening of nuclear transfer technology program, oocyte maturation quality, external embryo culture condition, donor cell and pre-treatment, though obtained certain progress, but nuclear transfer embryo is grown the still very low (Li of rate that expires, J., Du, Y., et al.ChemicallyAssisted Handmade Enucleation of Porcine Oocytes, 2006 Cloning Stem Cells 8:241-50; Furnus, C.C., Matos, D.G., et al.Metabolic requirements associated with GSH synthesis during in vitromaturation of cattle oocytes, 2007 Anim.Reprod.Sci.doi:10.1016/j.anireprosci.2007.12.003.).
Think that at present the low main reason of cloning efficiency is exactly that the somatic cell nuclear reprogrammed is incomplete.The somatic cell nuclear reprogrammed is meant after donorcells nuclear moves into ovocyte and stops genetic expression program own, reverts to the necessary specific gene of fetal development and express program.This process mainly is that the donorcells epigenetic modification is write again, comprising karyomit(e) reinvent, dna methylation is rebuild, histone modification changes and imprinted gene expression regulation etc.In the body-cell neucleus transplanting process, ovocyte must carry out reprogrammed to donor nuclei, eliminate its tissue-specific epigenetic modification, the epigenetic pattern that can not eliminate tissue-specific epigenetic modification and reconstruction embryo effectively will influence embryo's normal development.Therefore solving the incomplete problem of somatic cell nuclear epigenetic reprogrammed will help to improve nuclear transplantation efficient.
It is generally acknowledged, in the nuclear transplantation process, the epigenetic modification of somatic cell nuclear normally is removed in very short time before the zygotic gene group activates, rebulid then, as seen 1-cell stage event pair cell is examined the particularly important (Zuccotti of reconstruction of epigenetic pattern, M., Garagna, S., Redi, C.A.Nuclear transfer, genome reprogramming andnovel opportunities in cell therapy, 2000 J Endocrinol Invest.23:623-9.).In general, embryo in vitro fertilization is higher than nuclear transfer embryo far away on omnidistance developmental potency, therefore can infer the key point of the incomplete problem of somatic cell nuclear epigenetic reprogrammed by comparing the difference of 1-cell stage ooecium confrontation donorcells nuclear and gametic nucleus reprogrammed process.In the normal fertilization embryo development procedure, the gametic nucleus of aggegation of after fertilization height and hyper-methylation has experienced the dynamic change of chromatin Structure, dna methylation, histone modification etc., demethylation takes place, deagglomeration forms female, male pronucleus, the epigenetic modification pattern of body early embryo is being set up in these variations, activates the required all-round genetic expression aspect of early embryonic development and plays a significant role.Yet, in the nuclear transplantation process, after donorcells is examined in just entering ooecium matter, can nuclear membrane take place under the effect of maturation promoting factor (MPF) breaks (NEBD) and premature chromosome condensation (PCC), but because nucleo-cytoplasmic interreaction is insufficient or MPF is active lower, cause PCC thoroughly or not take place, donor nuclei methylates not exclusively.After the activation, along with the active reduction of MPF, the donor nuclei deagglomeration forms the class protokaryon, and what accompany with it is the incomplete demethylation of donor nuclei.This shows that after donor nuclei entered in the ooecium matter, agglutinative thoroughly or did not take place, and methylated not exclusively be somatic cell nuclear inadequate cause of follow-up epigenetic reprogrammed and key.These may cause the continuous expression of somatocyte autologous tissue specific gene unusually, and the relevant versatility gene (as Oct4) of early embryonic development maybe can not activate and express too early, also may cause unusual epigenetic modification of imprinted gene and expression.In other words, if somatocyte nuclear energy as sperm nucleus and ovum nuclear, the state that was in height aggegation and hyper-methylation before reprogrammed has and is beneficial to raising nuclear transplantation efficient.
At present, mostly count investigators adopt some to methylate or deacetylation inhibitor (as TSA or 5-AZA-dC etc.) is handled donorcells or clone embryos, methylate and improve its acetylize level and improve cloning efficiency by reducing donorcells.Though this method obtains certain effect on the efficient of mouse body-cell neucleus transplanting, but on other species, do not obtain ideal results (Hochedlinger K, Jaenisch R.Nuclear transplantation:lessons from frogs and mice.2002 CurrOpin Cell Biol.14 (6): 741-748.; Enright BP, Kubota C, Yang X et al.Epigenetic characteristics anddevelopment of embryos cloned from donor cells treated by trichostatin A or 5-aza-2-deoxycytidine.2003 Biol.Reprod.69:896-901.; Enright BP, Sung LY, Chang CC et al.Methylation andacetylation characteristics of cloned bovine embryos from donor cells treated with5-aza-2-deoxycytidine.2005 Biol Reprod.72:944-948.).
Summary of the invention
Technical problem to be solved by this invention provides a kind of method that improves pig nucleus transplantation efficiency, to improve the production efficiency of cloned animal.
By with the comparison of zygote reprogrammed gametic nucleus process, we think, the first step of somatic cell nuclear reprogrammed mainly is that epigenetic modification to donor nuclei carries out " making zero " in the nuclear transplantation process." make zero " and instigate donor nuclei to form the gametic nucleus sample, have height agglutinative chromatin, hyper-methylation and low acetylizad epigenetic state.In view of the above, the present invention is by handling donorcells with small-molecule drug CalyculinA, lures the donor nuclei chromatin condensation into, methylating raises and acetylize reduces; make donor nuclei form the sperm nucleus sample; carry out nuclear transplantation then, make up reconstructed embryo, to improve the production efficiency of cloned animal.
For solving the problems of the technologies described above, technical scheme of the present invention is:
Use small-molecule drug Calyculin A to handle donorcells, lure the donor nuclei chromatin condensation into, methylating raises and acetylize reduces, and carries out nuclear transplantation then, makes up reconstructed embryo.
The concentration of described Calyculin A is below the 50nM, does not comprise 0nM, and optimum concn is 10nM.
The treatment time of described Calyculin A is below the 30min, does not comprise 0min, and the optimum handling time is 30min.
Described donorcells is a porcine fetus fibroblasts.
The present invention is by handling donorcells with small-molecule drug CalyculinA; lure the donor nuclei chromatin condensation into, methylating raises and acetylize reduces, and carries out nuclear transplantation then, makes up reconstructed embryo; improved and made pig nucleus transplantation efficiency, for the production efficiency that improves cloned animal is laid a good foundation.
By optimal conditions, select best Calyculin A 10nM, and treatment time 30min, make porcine clone embryos ectogenesis blastaea rate improve 9.7%, the blastomere number has increased by 23.78.
Figure of description
Fig. 1 somatic cell nuclear transfer technique route.
A: fixedly polar body was in 3 o'clock to 5 o'clock positions; B: remove polar body and ooecium matter on every side thereof; C: entry needle is drawn an individual cells; D: somatocyte is expelled under the ovocyte zona pellucida, contacts with the archiblast film.Scale is 50uM.
Fig. 2 different concns CalyculinA is to the influence of porcine fetus fibroblasts motility rate.
Fig. 3 10nM CalyculinA handles the influence (40X) of 30min to the porcine fetus fibroblasts motility rate.
Fig. 4 cultivates the clone embryos (100X) behind the 146h.
Fig. 5 pig clone blastomere number (200X).
Embodiment
Come further to illustrate the present invention by the following examples, the following example is used for illustration purpose but not is used to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is all operated according to the described condition of common somatic cell clone technique basically.
The maturation of embodiment 1 porcine oocytes is cultivated
Collect ovary and transport go back to the laboratory by the slaughterhouse, extract the chamber ovarian follicle that has of 3-5mm diameter and collect ovarian cumulus ovocyte complex body, select under the stereomicroscope and have the complete ovocyte of cumulus cell more than three layers and be used for the maturation cultivation with 37 ℃ physiological saline; The ripe culture condition of cultivating is 38.5 ℃, the atmosphere surrounding of 5% carbonic acid gas, 95% air, saturated humidity; The ripe cultivation adopts 0.5% Unidasa to remove cumulus cell after 42 hours, the MII phase ovocyte that obtains is as the nuclear transplantation acceptor.
The acquisition and the cultivation of embodiment 2 porcine fetus fibroblastses
35 days pig fetus ear tissues are got in aseptic technique, adopt conventional 0.25% trypsin Gibco) digestion method chorista cell, DMEM (Gibco) add 20% foetal calf serum former be commissioned to train foster, DMEM adds the cultivation of going down to posterity of 10% foetal calf serum, culture condition is 38.5 ℃, the atmosphere surrounding of 5% carbonic acid gas, 95% air, saturated appropriateness; Adopt the fetal fibroblast of the contact inhibition in 3-5 generation to be used for nuclear transplantation, adopt 50,30 before the nuclear transplantation respectively, 10nM CalyculinA (sigma) pre-treatment fetal fibroblast 30,20,10min, adopting 0.25% tryptic digestion then is single suspension cell, as the nuclear transplantation donor.
Embodiment 3 body-cell neucleus transplantings
Pig nucleus transplantation adopts fusion method to carry out (Fig. 1), process is as follows: blind suction method is removed the spindle body and the first polar body of MII ovum, somatocyte is annotated under zona pellucida, and make it closely to contact with the ovocyte plasma membrane, direct current shock (1.2kv/cm, 30 μ s, 2 subpulses) inductor cell and stoning ooecium matter merge the formation reconstructed embryo and make it to be activated, and are incubated in the PZM-3 nutrient solution, and culture condition is: 38.5 ℃, 5%CO2, saturated humidity.Be cultured to 48h and calculate spilting of an egg rate, 146h calculates the blastaea rate.33342 pairs of blastaeas of 10mg/LHoechst 5min that dyes, fluoroscope observe note blastomere number down; Used tool, liquid, instrument are as follows: the fixed tube internal diameter is 30 μ m, syringe inner diameter 25 μ m; Ovocyte operation liquid is the modified version TCM199 (Gibco) that contains 5mg/mlBSA, and ovocyte micrurgy liquid is for adding the operation liquid of 7.5 μ g/ml cytochalasin Bs (CB), and merging liquid is 3% mannitol solution that contains 1.0mM calcium ion and 0.1mM magnesium ion; Micromanipulation system is a NT-88NE system of Japanese Narishige company; Fusion instrument is the U.S. BTX2001 of a BTX company type electricity cytogamy instrument.
Embodiment 4 cell motility rates detect
Collect the porcine fetus fibroblasts handled under each condition adopt the computer-assisted cell analysis system (CASY) I cell-analyzing unit (
Systems, Reutlingen, Germany) cytoanalyze detects the cell motility rate.Select suitable concentration and time to handle porcine fetus fibroblasts, with 0.4% trypan blue dyeing 3min, microscopically is observed and is counted, and the result of CASYI cell motility rate analyser is verified.The result shows that 10nM treatment group and control group motility rate do not have significant difference (93.8%vs95.4%), and remarkable decline of 50nM and 30nM treatment group cell motility rate is respectively 71.2% and 88.4% (Fig. 2).10nM CalyculinA handles the influence minimum of 30min to the porcine fetus fibroblasts motility rate, and motility rate reaches 96.4%, and blue cell shown in the arrow is dead cell (Fig. 3) among the figure.
The monitoring of embodiment 5 clone embryos
The clone embryos that builds, vitro culture 146h observes the blastaea rate, and the arrow indication is blastaea (Fig. 4).Clone blastaea after Hoechst 33342 dyeing, the photo under fluoroscope, blue is blastomere nuclear (Fig. 5).Monitoring result shows that clone embryos physically well develops, and adopts Calyculin A to handle donorcells, carries out nuclear transplantation then, makes porcine clone embryos ectogenesis blastaea rate improve 9.7%, and the blastomere number has increased by 23.78 (table 1).
Table 1Calyculin A handles donorcells to the ectogenetic influence of porcine clone embryos
Mark different letter representation significant differences (P＜0.05) in the same row.
Should be understood that in addition read of the present invention above-mentioned tell about content after, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (6)
1. method that improves body pig cell nuclear transplantation efficient is characterized in that using small-molecule drug Calyculin A to handle donorcells, lures the donor nuclei chromatin condensation into, methylating raises and acetylize reduces, and carries out nuclear transplantation then, makes up reconstructed embryo.
2. method according to claim 1, the concentration that it is characterized in that described Calyculin A is below the 50nM, does not comprise 0nM.
3. method according to claim 2, the optimum concn that it is characterized in that described Calyculin A is 10nM.
4. method according to claim 1, the treatment time that it is characterized in that described Calyculin A is below the 30min, does not comprise 0min.
5. method according to claim 4, the optimum handling time that it is characterized in that described CalyculinA is 30min.
6. according to the described method of claim 1-5, it is characterized in that described donorcells is a porcine fetus fibroblasts.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063091A (en) * | 2015-08-06 | 2015-11-18 | 中国农业科学院深圳农业基因组研究所 | Method for improving efficiency of mammalian somatic cell cloning technology |
| CN108588008A (en) * | 2018-04-18 | 2018-09-28 | 广西壮族自治区畜牧研究所 | A kind of black pig handmade cloning in Debao reconstructs drug and the application of vitro Development of Embryos |
| 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 |
| CN108913653A (en) * | 2018-06-04 | 2018-11-30 | 温氏食品集团股份有限公司 | A method of improving pig nuclear transfer efficiency |
Citations (2)
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| US6635802B1 (en) * | 2000-01-10 | 2003-10-21 | The Texas A&M University System | Nuclear transfer using cells cultured in serum starvation media containing apoptosis inhibitors |
| CN101679942A (en) * | 2007-02-23 | 2010-03-24 | 先进细胞技术公司 | Highly efficient methods for reprogramming differentiated cells and for generating animals and embryonic stem cells from reprogrammed cells |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US6635802B1 (en) * | 2000-01-10 | 2003-10-21 | The Texas A&M University System | Nuclear transfer using cells cultured in serum starvation media containing apoptosis inhibitors |
| CN101679942A (en) * | 2007-02-23 | 2010-03-24 | 先进细胞技术公司 | Highly efficient methods for reprogramming differentiated cells and for generating animals and embryonic stem cells from reprogrammed cells |
Non-Patent Citations (3)
| Title |
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| 《STEM CELLS》 20061231 ROBERT BLELLOCH et al Reprogramming Efficiency Following Somatic Cell Nuclear Transfer Is Influenced by the Differentiation and Methylation State of the Donor Nucleus 第24卷, 2 * |
| 《东北农业大学学报》 20080315 宋军等 利用体细胞核移植技术生产表达绿色荧光蛋白转基因猪胚胎的研究 , 第03期 2 * |
| 《中国科学(C辑:生命科学)》 20071215 刘忠华等 体细胞核移植克隆民猪:培养液对卵母细胞成熟及胚胎发育的影响 , 第06期 2 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063091A (en) * | 2015-08-06 | 2015-11-18 | 中国农业科学院深圳农业基因组研究所 | Method for improving efficiency of mammalian somatic cell cloning technology |
| CN108588008A (en) * | 2018-04-18 | 2018-09-28 | 广西壮族自治区畜牧研究所 | A kind of black pig handmade cloning in Debao reconstructs drug and the application of vitro Development of Embryos |
| 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 |
| CN108913653A (en) * | 2018-06-04 | 2018-11-30 | 温氏食品集团股份有限公司 | A method of improving pig nuclear transfer efficiency |
| CN108913653B (en) * | 2018-06-04 | 2022-06-07 | 温氏食品集团股份有限公司 | Method for improving nuclear transplantation efficiency of pigs |
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