CN100350039C - Method for cloning buffalo somatic cell - Google Patents
Method for cloning buffalo somatic cell Download PDFInfo
- Publication number
- CN100350039C CN100350039C CNB2005100190685A CN200510019068A CN100350039C CN 100350039 C CN100350039 C CN 100350039C CN B2005100190685 A CNB2005100190685 A CN B2005100190685A CN 200510019068 A CN200510019068 A CN 200510019068A CN 100350039 C CN100350039 C CN 100350039C
- Authority
- CN
- China
- Prior art keywords
- buffalo
- donorcells
- somatic cell
- culture
- cloning
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a buffalo somatic cell cloning method. The technology comprises the steps that a buffalo follicular fluid and oocytes of a culture receptor of growth factors of epithelial cells are added to a mature culture fluid, and enucleation is carried out in a spindle image system; donor cells are cultured by an inhibiting agent Aphidicolin which is synthesized by DNA, and hunger culture is then carried out; reconstructed embryos are activated by the combination of ionomycin and 6-dimethylaminopurine, and the embryos are cultured in vitro by improved TCM199 and serum of estrus buffaloes. Somatic cells of buffaloes are successfully cloned by the method of the present invention, and the first case of a buffalo cloned through adult somatic cells in the world is obtained.
Description
Technical field
The invention belongs to bionic Animal Biotechnology, particularly buffalo breeding and directed genetic improvement field.
Background technology
Cloning animal somatic cell is an epoch-making important breakthrough of biological technical field in latter stage in 20th century, it has proved that not only the adult animals somatocyte that has broken up can return to the initial state of growth under suitable condition, complete individuality of bud into, and the genetic improvement speed of accelerating animal, the efficient that improves transgenic animal had boundless application prospect.Though somatic cell clone is successively ox, mouse, rat, pig, goat, multiple animal such as horse and mule is achieved success on one's body, but buffalo is then owing to reasons such as areal distribution and ovum potentiality of development are low, and the clone of these species (buffalo that comprises the embryonic cell clone) does not achieve success in the world so far as yet.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of method with the cloning buffalo somatic cell buffalo.
The present invention solves the problems of the technologies described above with following technical scheme:
1. the maturation of acceptor ovocyte and stoning: in maturation culture solution, add ox liquor folliculi (BFF) and epithelial cell growth factor (EGF), in spindle body picture system (Spindle View) stoning down.
2. the cultivation of donorcells is handled: cultivate with DNA synthetic inhibitor Aphidincolin and handle donorcells, carry out hunger then and cultivate.
3. the activation of reconstituted embryo and vitro culture: adopt ionomycin (ionomycin) and 6-dimethylamino-purine (6-DMAP) to unite activation, add the external cultivation that the bovine serum of oestrusing (OCS) carries out the embryo with the TCM199 that improves.
Carry out cloning buffalo somatic cell with method of the present invention, obtained the first somatic cell clone buffalo in the world on November 19th, 2004, obtained the world the first adult body cell clone buffalo on March 17th, 2005, for cloning buffalo somatic cell and directed genetic improvement have stepped an important step.
Description of drawings
Fig. 1 is the situation of acceptor ovocyte of the present invention in the stoning of Spindle View microscopically.Left side figure is ovocyte not stoning as yet, and right figure examines the situation of having taken out; Its stoning success ratio reaches 100%.
Fig. 2 is the cloning buffalo somatic cell embryo that the present invention obtains.
Fig. 3 is the somatic cell clone buffalo (calf among the figure) that the present invention obtains.
Embodiment
For improving the potentiality of development of ovocyte, ovocyte is cultivated 20～22h in the ripe liquid of TCM199 (being No. 199, tissue culture medium)+5%OCS (bovine serum of promptly oestrusing)+2%BFF (being the ox liquor folliculi)+10ng/ml EGF (being epithelial cell growth factor); For improving the stoning rate, guarantee the normal development of reconstituted embryo, after the Unidasa with 0.15～0.25% removes cumulus cell, in the stoning of Spindel View microscopically.After donorcells is cultivated through going down to posterity, for making donorcells same period, cultivate processing 24h, in the nutrient solution that contains 0.5% foetal calf serum (FCS), cultivate 2～5d then with the Aphidicolin (a kind of DNA synthetic inhibitor) of 0.1～0.2 μ g/ml in the G0/G1 phase.Donorcells through cultivate handling is with tryptic digestion 1～2min of 0.2%, and the acceptor ovocyte ovum week that is then injected into stoning is merged the fusion of inducing donorcells and acceptor ovocyte plasma membrane by electricity, the formation reconstituted embryo in the crack.For fully activating reconstituted embryo, guarantee the external normal development of reconstituted embryo, reconstituted embryo activates processing 5～10min with the ionomycin of 5 μ mol/L, in 2mmol/L 6-DMAP (a kind of inhibitor of protein phosphorylation), cultivate again and handle 2～4h, and be placed in the granulosa cell monolayer cell droplet that contains 30 μ l nutrient solutions (TCM199+5%OCS of improvement), containing 5%CO
238.5 ℃ of incubators in cultivate 6～7d, make it grow the blastaea stage.The blastaea that obtains is grafted directly in the acceptor water cow horn of uterus, or is transplanted to after freezing preservation in the acceptor water cow horn of uterus again.Adopt method of the present invention, the division rate of reconstituted embryo reaches 70～80%, and blastocyst rate reaches 30%.Till the applying date, oneself obtains the achievement of 7 water cows of becoming pregnant applicant of the present invention, wherein two miscarriage, and two childbirth is waited to produce for three.
Embodiment 1
On December 5th, 2003, we handle 24h to the female buffalo fetal fibroblast through the cultivation of going down to posterity (3 generation) with the Aphidicolin cultivation of 0.1 μ g/ml with method of the present invention, then with the hungry 5d that cultivates of the nutrient solution that contains 0.5%FCS.On December 11st, 2003 these are cultivated the cell of handling and be transplanted in the local buffalo ovocyte of stoning, be built into reconstituted embryo.Reconstituted embryo was cultivated through 6 days and is formed blastaea, was transplanted to acceptor buffalo intrauterine on December 17th, 2003.Through after 343 days gestation, obtain a female sex clone buffalo in c-section on November 19th, 2004.The body weight of this clened cows is 29kg, and sex is consistent with donorcells, identifies through microsatellite DNA, and this clone buffalo and donorcells are in full accord.
Embodiment 2:
On March 31st, 2004, we cultivate processing 24h with the Aphidicolin of 0.1 μ g/ml to the adult buffalo gonad granulocyte of the cultivation of going down to posterity (5 generation), then with the hungry 3d that cultivates of the nutrient solution that contains 0.5%FCS.On April 4th, 2004, as donorcells, the local buffalo ovocyte reorganization formation reconstructed embryo with stoning forms clone's blastaea through external cultivation in 6 days with these buffalo gonad granulocytes of cultivating processing.On April 10th, 2004, will clone the non-operation transplantation of blastaea to acceptor buffalo intrauterine, through after 348 days gestation, in female sex clone buffalo of natural labor on March 17 in 2005.The body weight of this clone buffalo is 23kg, and body is long to be 86cm, height 62.5cm, and sex is consistent with donorcells.Identify through microsatellite DNA, confirm that this clone buffalo is really from donorcells.
Claims (1)
1. the method for a cloning buffalo somatic cell is characterized in that processing step is:
(1) maturation of acceptor ovocyte and stoning: in bovine serum+2% maturation culture solution that N liquor folliculi+the 10ng/ml epithelial cell growth factor is formed of oestrusing by tissue culture medium No. 199+5%, add ox liquor folliculi and epithelial cell growth factor, stoning under the spindle body picture system;
(2) cultivation of donorcells is handled: cultivate with DNA synthetic inhibitor Aphidicolin and handle donorcells, hungry then the cultivation;
(3) activation of reconstituted embryo and vitro culture: adopt ionomycin and 6-dimethylamino-purine to unite activation, add the vitro culture that the bovine serum of oestrusing carries out being merged by donorcells and acceptor ovocyte the reconstituted embryo that obtains for No. 199 with the tissue culture medium that improves.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100190685A CN100350039C (en) | 2005-07-06 | 2005-07-06 | Method for cloning buffalo somatic cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100190685A CN100350039C (en) | 2005-07-06 | 2005-07-06 | Method for cloning buffalo somatic cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1733907A CN1733907A (en) | 2006-02-15 |
| CN100350039C true CN100350039C (en) | 2007-11-21 |
Family
ID=36076525
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100190685A Expired - Fee Related CN100350039C (en) | 2005-07-06 | 2005-07-06 | Method for cloning buffalo somatic cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100350039C (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100432219C (en) * | 2006-07-28 | 2008-11-12 | 浙江大学 | Cow ovum cell in vitro ripening culturing liquid containing tea polyphenol and its culturing method |
| CN100419074C (en) * | 2006-07-28 | 2008-09-17 | 浙江大学 | Ox embryo in vitro culturing liquid containing tea polyphenol and its culturing method |
| CN101302497B (en) * | 2008-06-27 | 2010-07-21 | 华中农业大学 | Method for cloning embryo by nuclear transfer of bovine somatic cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1304443A (en) * | 1999-06-30 | 2001-07-18 | 黄禹锡 | Method for producing clened cows |
| CN1503623A (en) * | 2001-04-10 | 2004-06-09 | 长沙惠霖干细胞工程有限公司 | Method for construction of clone embryo of mammal |
| WO2004053090A2 (en) * | 2002-12-10 | 2004-06-24 | Gtc Biotherapeutics, Inc. | Method and system for utilizing somatic cell nuclear transfer embryos as cell donors for additional nuclear transfer |
-
2005
- 2005-07-06 CN CNB2005100190685A patent/CN100350039C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1304443A (en) * | 1999-06-30 | 2001-07-18 | 黄禹锡 | Method for producing clened cows |
| CN1503623A (en) * | 2001-04-10 | 2004-06-09 | 长沙惠霖干细胞工程有限公司 | Method for construction of clone embryo of mammal |
| WO2004053090A2 (en) * | 2002-12-10 | 2004-06-24 | Gtc Biotherapeutics, Inc. | Method and system for utilizing somatic cell nuclear transfer embryos as cell donors for additional nuclear transfer |
Non-Patent Citations (2)
| Title |
|---|
| 不同来源的供体细胞对水牛体细胞核移植效果的影响 陆凤花等.广西农业生物科学，第24卷第2期 2005 * |
| 重组牛生长激素对山羊卵泡卵母细胞体外成熟的影响 辛晓玲等.河南农业科学，第10期 1997 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1733907A (en) | 2006-02-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Grupen | The evolution of porcine embryo in vitro production | |
| US6107543A (en) | Culture of totipotent embryonic inner cells mass cells and production of bovine animals | |
| US9994868B2 (en) | Method and culture medium for preparing mammalian ovum or embryo in which zona pellucida has been thinned or eliminated, and method for fertilization using mammalian ovum prepared by same method | |
| WO2019061560A1 (en) | In vitro maturation culture medium of immature oocytes and use thereof | |
| CN101302497B (en) | Method for cloning embryo by nuclear transfer of bovine somatic cells | |
| CN106520838A (en) | New method for gene injection for somatic cell nuclear transfer reconstructed embryo | |
| Kim et al. | Improved preimplantation development of porcine somatic cell nuclear transfer embryos by caffeine treatment | |
| CN100350039C (en) | Method for cloning buffalo somatic cell | |
| CN105838668B (en) | In-vitro maturation culture solution for small follicle oocytes and application thereof | |
| CN101195834A (en) | Early pickling transgene method for cultivated silkworm diapause breed variety | |
| CN101962626B (en) | Calf in vitro embryo culture solution | |
| CN114410573B (en) | Oocyte in-vitro maturation culture solution additive and application thereof | |
| CN100404675C (en) | Production process of somatic cell clone pig | |
| CN105349485B (en) | Improved method for cutting buffalo blastocyst by bare hand and cutting fluid | |
| CN101260383A (en) | Single-semen injection activation method | |
| CN102344941A (en) | Method for breeding cloned animal or transgenic cloned animal | |
| CN102344907A (en) | Method for activating reconstructed embryo | |
| Wakayama et al. | Production of cloned mice from somatic cells, ES cells, and frozen bodies | |
| Nandedkar et al. | Parthenogenesis and somatic cell nuclear transfer in sheep oocytes using polscope | |
| Kim et al. | In vitro development of canine somatic cell nuclear transfer embryos in different culture media | |
| Chen et al. | Somatic cell bovine cloning: Effect of donor cell and recipients | |
| JP2003503045A (en) | Clonal tiger production method using xenogeneic nuclear transfer technology | |
| KR102552211B1 (en) | Method for producing in vitro rat 0vary | |
| CN112226404B (en) | Culture medium composition and culture method for promoting animal embryo in-vitro development | |
| CN100526456C (en) | Method for transplanting consubstantial cell nucleus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20071121 Termination date: 20120706 |