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Study of cloning embryos in bovine using parthenogenetic cells

利用孤雌生殖细胞克隆牛胚胎的研究

基本信息

批准号：
05806041
负责人：
SUZUKI Tatsuyuki
金额：
$ 1.02万
依托单位：
United Graduate School of Veterinary Science, Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05806041/
关键词：
Aggragation Parthenogenetic IVF Chimera Cloning 牛移植一卵性双子 1卵性双子

项目摘要

Oocytes were aspirated from the ovaries collected from abatoir and matured for 30-32h at 38.5C under 5% CO2 in air. To induce parthenogenetic activation, these matured oocytes were suspended in culture media containing 7% ethanol for 10mins followed by treatment using cytochalasin D (5h) to suppress the second polar body extrusion, so producing diploid parthenogenes. The oocytes were then washed and cultured in vitro on feeder layrs of bovine cumulus cells for further development. The normal fertilized embryos were obtained by in virtro maturation, fertilization and culture procedures. The follicular oocytes were matured for 20-24h at 38.5C under 5%CO2 in air. These matured oocytes were then fertilized by in vitro capacitated sperm and followed by cultured in vitro. Aggregation chimeras were produced by methods : (1) injection of 2 blastomeres obtained from 16 cell stage fertilized embryo into 4-cell parthenogenetic cell ; 2/16F->4/4P, (2) injection of 4 balstomeres obtained from 16 cell stage fertilized embryo into 4-cell parthenogenetic cell ; 4/16->4/4P.(3) injection of fertilized demi-embryo (8-cell stage) into parthenogenetic demi-embryo (8cell-stage) ; 4/8F->4/8P, (4) aggregation of whole fertilized embryo and parthenogenetic cell ; 8/8F<->8/8P.The developmental rate of aggregated embryos produced by aggragation of whole embryos to morulae and balstocysts were significantly higher (P<.05) than injection of 2/16F-> 4/4p, 4/16F-> 4/4P and 4/8F -> 4/8P.By method (2), the identical twin male calves were delivered on day 267 from 1 recipient in which XY chromosome plates were detected in each calf. From method (4), single male calf which XX and XY chromosome plates were detected was delivered on day 261.

卵母细胞从屠宰场采集的卵巢中抽离，在38.5℃、5% CO2的空气中成熟30-32h。为了诱导孤雌生殖激活，将这些成熟卵母细胞悬浮在含有7%乙醇的培养基中10min，然后用细胞松弛素D （5h）抑制第二极体挤压，产生二倍体孤雌生殖。卵母细胞洗涤后，体外培养于牛积云细胞饲养层上进行进一步发育。经体外成熟、受精和培养获得正常受精卵。卵泡卵母细胞在38.5℃、5%CO2空气中成熟20-24h。这些成熟的卵母细胞然后被体外有能力的精子受精，然后在体外培养。聚集嵌合体的制备方法：(1)将16细胞期受精卵的2个卵裂球注入4细胞孤雌生殖细胞；(2)将16细胞期受精卵获得的4个balstomer注入4细胞孤雌生殖细胞；4/16——> 4/4P。(3)将受精卵半胚胎（8细胞期）注入孤雌生殖半胚胎（8细胞期）；4/ 8f ->4/ 8p,(4)整个受精卵和孤雌生殖细胞聚集；8/8F < - > 8/8P。与注射2/16F-> 4/4p、4/16F-> 4/4p和4/8F -> 4/8P相比，全胚向胚胚和囊胚聚集产生的聚集胚发育率显著提高（P< 0.05）。通过方法(2)，在第267天从1个受体中取出同卵双胞胎雄性小牛，每只小牛都检测到XY染色体板。采用方法(4)，于第261天分娩单头雄犊，检测到XX和XY染色体板。