囊胚注射转基因ES细胞制作嵌合体的研究

在小鼠胚胎成纤维细胞制作的饲养层上培养并成功的维持了携带LacZ基因的胚胎干细胞系（S8），在此基础上，以S8为供体细胞，以远交系昆明白小鼠3．5d胚胎为受体，通过显微注射法将供体细胞转移到受体的囊胚腔内，经过恢复培养，移植到代孕鼠昆明白雌鼠的子宫中；后代在嵌合体出生一周后进行判定。本试验用8～13代的S8细胞共注射胚胎597枚，经1～3h恢复培养，有585枚胚胎重新具有膨大的囊胚腔，细胞轮廓分明，滋养层细胞间连接也清晰可见，胚胎成活率为97％；胚胎移植后，代孕母鼠共移植胚胎228枚，经17～19d的妊娠期后，产仔37只（2只死胎），产仔率为16％；有35只仔鼠（雄鼠18只，雌鼠17只）存活到可以判断毛色，共获得8只S8细胞毛色嵌合体小鼠，嵌合体的产生率为21．6％。结果表明用S8细胞经囊胚注射后能够获得嵌合体，并且嵌合体明显发生了性偏离现象。本试验为国内利用囊胚注射携带LacZ基因的胚胎干细胞获得嵌合体小鼠的首例报道。

Study on the Production of Chimeras Derived from the Transgenic Embryonic Stem Cells by Blastocysts Injection

Using ES cells line(S8 cells) transfected by LacZ gene,which was cultured on mouse embryonic feeder layers made by fibroblast of mouse embryo,and through blastocyst injection,the S8 cells that had been digested into single cell was injected into the blastocyst of the outbreed mouse straining Kunming albino females as the provider for recipient-embryo which had grown for 3.5 days after coitum(p.c),then transplanted the manipulated embryos into the uterus of 2.5 days after coitum pseudopregant Kunming albino female recipients to allow the embryos to develop in vivo after recovering culture,which offsprings were identified one week after the chimera mouse being born according to the contribution of ES cells to tissues of chimeric mice by coat color.The experiment treated total 597 embryo with S8 cells of 8-13 generation through blastocyst injection,there were 585 embryo which got blastocyst cavity again after recovery culture of 1-3 hours,not only the figure of cells were clear,but also the juncture between the cells of feeder layers could be seen clearly,the surviving rate of embryo achieved 97%.All 37 liver-born offsprings were obtained after 17-19 days of gestation by using embryonic transfer(the fetiferous rate was 16%),among which 8 are chimeras(the generative rate of chimera was 21.6%)with coat color.The result showed that the chimeras mouse can be generated by blastocyst injection using the ES(S8) cells,and all of them occurred the sex-converted.This experiment was firstly shown that chimeras could be obtained in China at present by using S8 cells transfected LacZ gene.