无标记转Fat-1基因真核表达载体的构建及转基因绵羊细胞系的建立

Construction of Fat-1 eukaryotic expression vector of excision markers and the establishment of transgenic sheep cell lines

摘要:

为了建立无筛选标记基因的转Fat-1基因绵羊细胞系，本研究将PCR克隆得到的Fat-1基因，合成的attB、Loxp序列并克隆入pN1-EGFP框架载体，得到可删除筛选标记基因的pEGFP-N1-Fat-1真核表达载体。体外转录合成phiC31整合酶mRNA并与线性化的pEGFP-N1-Fat-1载体共转染绵羊胎儿皮肤成纤维细胞，G418筛选得到表达绿色荧光的单克隆，再利用pET-28a-His-NLS-TAT-Cre质粒诱导Cre重组蛋白表达，将纯化后的Cre穿膜肽转导表达绿色荧光的单克隆细胞，将荧光淬灭的细胞系扩繁，提取基因组DNA，进行PCR及测序鉴定，得到无标记转Fat-1基因绵羊胎儿皮肤成纤维细胞系，为生产无筛选标记基因的转基因绵羊奠定基础。

Abstract:

In order to establish marker-free transgenic cell lines, we cloned Fat-1 gene, attB and Loxp sequences by PCR. Then we inserted these sequences to pN1-EGFP vector and got pEGFP-N1-Fat-1 expression vector. PhiC31 integrase mRNA which was generated by in vitro transcription and a pEGFP-N1-Fat-1 expression vector co-electroporated into sheep fetal fibroblasts, and then we got transgenic cell lines expressing green fluorescence. Prokaryotic expression and purification of Cre recombinant protein was performed. Cre recombinant protein was transducted into stably-transfected cell colonies. We identified cell colonies by sequencing and established marker-free transgenic cell lines and eventually established marker-free transgenic cell lines which were building more safely basic for producing Fat-1 transgenic animals.