小鼠Dazl基因真核表达载体的构建及表达检测

Construction of Mouse Dazl Eukaryotic Expression Vector and Expression

为研究小鼠Dazl基因的功能及探究Dazl基因过表达在干细胞向生殖细胞分化过程中的作用,采用RTPCR方法,从13.5 dpc雌性胎鼠卵巢中克隆出Dazl基因的全部编码区,测序正确后利用限制性内切酶将其连接到真核表达载体pc DNA3.1上。线性化后对小鼠成纤维细胞进行转染,qRT-PCR及Western Blot技术显示外源基因Dazl已经成功转染并表达。为研究Dazl基因的功能及干细胞向生殖细胞的诱导分化奠定了基础。

To study the function of Dazl gene and to confirm its promotion role during the differentiation of stem cells to germ cells,the full coding sequence of Dazl gene was cloned from the ovaries of female mice of 13.5 days post coitum（dpc）.Then it was ligated into eukaryotic expression vector pc DNA3.1 after digested by the restriction endonuclease Hind Ⅲ and Kpn Ⅰ.The mouse fibroblasts were transfected using the linearized pc DNA3.1-Dazl.The results of qRT-PCR and Western Blot showed the foreign gene Dazl had transfected into eukaryotic cells.The study would provide the foundations for the induction and differentiation of stem cells to germ cells.