小鼠白血病抑制因子基因的克隆、表达及其对维持鸡原始生殖细胞未分化状态的影响

Cloning and expression of mLIF and the effects of mLIF maintaining chicken primordial germ cells in the undifferentiated state

为了探索鸡原始生殖细胞(Primordial germ cells,PGCs)适合的培养体系,我们在已构建的分泌型真核表达载体pSecTag-mlif(sp-)的基础上,通过脂质体介导将mlif转染到鸡PGCs和鸡胚胎成纤维(Chicken embryonic fibroblast,CEF)细胞中,48h后收集细胞上清液,蛋白印迹均检测到小鼠白血病抑制因子(mLIF)的表达。以CEF细胞作饲养层,分七组来培养鸡PGCs,结果发现四组和五组培养的PGCs生长状态最好,三组传代后开始2-3天生长状况较好,3天后克隆周围有明显的分化现象。本实验将已构建了含mLIF基因的分泌型真核表达载体成功地瞬时转染到PGCs和CEF细胞中,且表达的mLIF具有维持鸡PGCs未分化状态的功能。

In order to establish the best cultivation system, we constructed eukaryotic expression vector pSecTag- mlif （ sp - ）, transfected chicken PGCs （Primordial germ cells） and CEF （Chicken embryonic fibroblast） cells with pSecTag-mlif （sp-） by the method of lipofectamine mediation, and collected the supernatant after centrifugating the cellular medium. We surveyed mLIF （Mice leukemia inhibitory factor） expression in cellular medium by means of of western-blotting. We cultured PGCs using seven different methods including three control groups, which all utilized CEF cells as feeder. The PGC clones cultured in the forth group and the fifth group proliferated perfectly. The second passage CEG clones cultured in the third group proliferated very well at first, but the CEG clones began differentiating three days later. We sucsesfully transfected chicken PGCs and CEF cells with pSecTag- mlif （sp - ）, and the mLIF expressed by pSecTag-mlif （sp） can maintain PGCs cells in an umdifferentiated state