小鼠四倍体半克隆胚胎发育研究  

Development of Mouse Tetraploid Semi-cloned Embryos

作　　者：丁晨晖[1] 焦丽红[1] 陈欣洁[1] 王二耀[1] 于洋[1] 赵春丽[1] 侯晓军[1] 李雪梅[1] 季维智[2] 王柳[1] 

机构地区：[1]中国科学院动物研究所计划生育生殖生物学国家重点实验室,北京100080 [2]中国科学院昆明动物研究所动物生殖生物学实验室

出　　处：《科技导报》2005年第8期56-60,共5页Science & Technology Review

基　　金：国家自然科学基金面上项目(30300176);科学技术部国际科技合作重点项目;国家重点基础研究发展计划(973)项目(2001CB510103)

摘　　要：半克隆(Semi-Cloned)胚胎是通过注射体细胞核到未去核的卵母细胞中产生的。在半克隆胚胎中,体细胞被用来作为精子的替代物。然而,由于异常的染色体分离,构建的半克隆胚胎在激活后形成了非整倍体而导致胚胎发育受到严重影响,不能发育到期。本研究通过抑制小鼠半克隆胚胎在激活过程中染色体数目减半,避免非整倍体胚胎形成,研究四倍体半克隆(TetraploidSemi-cloned,TSC)胚胎的发育和体细胞核的掺入对胚胎发育的影响。结果显示,TSC胚胎的体外发育率显著高于二倍体半克隆胚胎,与正常受精卵及孤雌激活对照无显著性差异,但TSC胚胎的细胞数在桑椹胚和囊胚期比正常二倍体受精胚胎和孤雌激活胚胎少。通过Oct-4染色发现,TSC胚胎囊胚期内细胞团(InnerCellMass,ICM)细胞很少或者没有。移植63个四倍体半克隆胚胎到3只假孕母鼠体内,得到20个胎盘,但没有得到胎儿。组蛋白乙酰化和DNA甲基化检测显示,部分TSC胚胎在囊胚期没有形成正常受精胚胎在ICM和滋养外胚层(Trophectoderm,TE)之间的差异分布。TSC胚胎的基因表达不依赖于细胞分裂次数而依赖于发育时间。虽然TSC胚胎避免了二倍体半克隆胚胎形成非整倍体现象,但由于TSC胚胎没有ICM细胞或ICM细胞很少,所以只能形成胎盘而不能形成胎儿。本实验第一次较为全面地研究了TSC胚胎的发育,同时也为研究体细胞核再程序化、基因打靶技术提供了一种新的途径。Semi-cloned embryos could be produced by injecting cumulus cell nucleus into non-enucleated oocyte, which were considered a substitute for male gamete. However, most of the semi-cloned embryos were aneuploid caused by chromosomal segregation errors and impossible to implant. In this study, tetraploid semi-cloning （TSC） embryos were created in order to avoid the segregation errors and increase the development of TSC embryos. Results showed TSC embryos could develop to blastocyst in vitro at a high rate. There was no significant difference among the development of TSC, fertilized（Fer） and parthenogenetic（PA） embryos （2n）. We found that the cell number of TSC morula and blastocyst was less than normal Fer and PA embryos.There were less or no ICM cells in TSC blastocysts compared with Fer and PA blastocyst by OCT-4 immunostaining detection. Sixty-three TSC embryos were transferred into 3 pseudo-pregnant mice at 2-cell stage, 20 implantation sites were found in the uterus after 14 days, but only placentas were obtained. There was no different distribution of histone acetylation and DNA methylation between TE and ICM in some TSC blastocysts. Our results suggested that gene expression of TSC embryos depended on developmental time more than the number of cleavage. TSC embryos could not form fetus after embryos transfer, although TSC embryos avoided the segregation errors and formed placentas, which was caused by less ICM cells in TSC blastocysts.

关 键 词：四倍体 半克隆 胚胎发育 非整倍体 

分 类 号：Q813[生物学—生物工程]