畜牧兽医学报 ›› 2007, Vol. 38 ›› Issue (2): 149-154.doi:

• 遗传繁育 • 上一篇    下一篇

囊胚注射转基因ES细胞制作嵌合体的研究

付志新;刘宗平;梁建兰;李善刚;陈学进   

  1. 1河北科技师范学院,河北 066600;2 扬州大学,江苏 225009;3上海交通大学医学院附属新华医院上海发育生物学研究中心,上海 200025
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2007-02-25 发布日期:2007-02-25
  • 通讯作者: 付志新

Study on the production of chimeras derived from the transgenic embryonic stem cells by blastocysts injection


  

  • Received:1900-01-01 Revised:1900-01-01 Online:2007-02-25 Published:2007-02-25

摘要: 嵌合体小鼠的制作是判定胚胎干细胞系是否具有种系分化能力的唯一方法,其在胚胎干细胞系的建立、外源基因导入胚胎干细胞、种系嵌合体的获得中处于重要的地位,也是通过胚胎干细胞途径得到转基因小鼠的关键环节。本实验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。本实验用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17-19天的妊娠期后,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。本实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。

Abstract: cell system, introduction of foreign genes into ES cells and production of germ line chimeras, also plays as a key step to obtain transgenic mouse through embryonic stem(ES) cell. The experiment used ES cells line(S8 cells) transfected by LacZ gene, which was cultured on mouse mbryonic 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 p.c. 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 calls transfected LacZ gene.