摘要
Embryonic stem cells (ES-5) were transfected with PSV 2-neo-EJ-Ras and the cell colonies were chosen in the selection medium containing Geneticin. Southern-blot analysis showed that all the transfected cells possessed the whole EJ-Ras DNA. Northern-blot analysis demonstrated that the selected clones express higher level of Ras mRNA than the control. When the transfected ES-5 cells were injected into the flanks of nude mice, they formed bigger tumors and more stem cell nests were found. The chimeric ability of the transfected ES-5 cells decreased when the cells were injected into the blastocysts which were then transferred back into the uterine of foster mother. We propose that the over-expression of EJ-Ras in ES-5 cells increase the proliferation ability but decreases the chimeric potential of cell.
转基因动物在研究基因的功能、医药、畜牧业等方面有重要的应用。利用胚胎干细胞（ＥＳ）制作转基因动物，比传统的显微注射法有无可比拟的优点。但是，目前、只在小鼠中建立起了ＥＳ细胞系，在其它哺乳动物（包括家畜）中尚未建成。近来，人们开始探索：改变胚胎细胞的遗传物质（如：把外源基因导入胚胎细胞）是否有利于获得ＥＳ细胞系．在各种外源基因中，癌基因是值得尝试的基因。因为：（１）ＥＳ细胞的建系是使胚胎的ＩＣＭ细胞永生化的过程；（２）在体外利用癌基因转染可使许多原代培养细胞永生化；（３）ＥＳ细胞高表达一些原癌基因。我们用癌基因ＥＪ－ｒａｓ转染小鼠ＥＳ细胞，观察其对ＥＳ－５细胞一些特性（增殖、嵌合能力）的影响，以便选择合适的癌基因用于家畜ＥＳ细胞的建系。ＥＳ－５细胞通常培养在丝裂霉素处理的小鼠胚胎成纤维细胞（ＭＥＦ）上（Ｆｉｇ．Ａ）。转染用ＥＳ－５细胞培养在无饲养层细胞的、含有大鼠肝细胞（ＢＲＬ）的条件培养液ＤＭＥＭ中。ＥＪ－ｒａｓ基因克隆在真核表达载体ＰＳＶ２ｎｅｏ的ＢａｍＨＩ位点上、使用磷酸钙法转染ＥＳ－５细胞，Ｇ－４１８法筛选阳性克隆，得到的抗性克隆仍以集落形式生长（Ｆｉｇ．Ｂ），选取５个较好的克隆（ＥＳ－ｒａｓ１，２，?
