使用phiC31整合酶构建奶牛INSIG1基因乳腺恒定表达细胞系

Establishment of mammary cell line stably expressing bovine INSIG1 using phiC31 integrase

为了使用phiC31整合酶建立奶牛胰岛素诱导基因1(INSIG1)的稳定表达乳腺细胞系,首先从奶牛乳腺组织中克隆得到INSIG1基因的编码区,然后将克隆片段与phiC31载体进行BamHⅠ和KpnⅠ双酶切构建INSIG1-C31真核表达质粒;将其与phiC31整合酶质粒共同转染到小鼠乳腺细胞中,采用嘌呤霉素筛选细胞,荧光显微镜观察细胞绿色荧光蛋白的表达,PCR鉴定细胞基因组中INSIG1基因的表达,荧光定量PCR检测细胞中INSIG1基因的表达。结果表明,试验成功构建INSIG1-C31真核载体,与phiC31整合酶共转染小鼠乳腺细胞,经过8mg/L嘌呤霉素筛选后获得完全表达绿色荧光蛋白的乳腺细胞系;对细胞系鉴定表明INSIG1基因已经整合到细胞基因组中,与对照组相比,INSIG1基因的mRNA表达丰度增加了597.98倍(P<0.001)。本研究获得了稳定表达奶牛INSIG1基因的乳腺细胞系,为深入研究INSIG1基因功能打下基础。

phiC31 integrase can mediate integration of plasmids containing attB site into the eukaryotic genomes by a site-specific manner and resulting in the expression of integrated genes. To establish the mammary cell line stably expressing bovine INSIG1 gene using phiC31 integrase,the CDS sequences of INSIG1 was amplified from the bovine tissue by PCR. The eukaryotie expression plasmid INSIG1-C31 was constructed with cloned sequence and phiC31 vector digested By Kpn I and BamH I. The INSIG1-C31 vector was eotransfected into mouse mammary cells with phiC31 integrase vector. The cell line was selected by puromycin and the expression of GFP was observed using fluorescence microscope. The genomie DNA was extracted from mammary cell and detected the expression of INSIG1. The mRNA expression of INSIG1 was analysed by real-time PCR. Re- sults indicated that INSIG1-C31 vector was constructed successfully. After the INSIG1-C31 vector was cotransfected into the mammary ceils with phiC31 integrase,the cell line was selected by 8μg/L pu- romycin and expressing the GFP fluorescence was observed. The PCR test of genomic DNA showed that the bovine INSIG1 gene was recombinanted in the genomic DNA. The mRNA expression of INSIG1 was 597.98 folds （P〈0. 001） compared with control group. The mouse mammary cell line of expressing INSIG1 was successfully established, which will provide a foundation for further research on the role of INSIG1 gene.