奶牛胎儿细胞多位点基因打靶的研究

Research on gene targeting to multiple loci in cow fetal fibroblasts

利用奶牛rDNA基因间的ITS重复序列作为靶位点,对奶牛胎儿成纤维细胞进行多位点基因打靶,建立以重复序列为靶位点的多位点基因打靶技术,并为克隆定点转基因奶牛提供核供体。首先分离培养出奶牛胎儿成纤维细胞,并进行性别鉴定和核型分析。采用MTT比色法确定了G418和GCV正负筛选的最低有效浓度。然后通过多位点基因打靶载体转染、正负筛选获得7个表达绿色荧光的克隆细胞系,经PCR,RT-PCR和测序证实其中1个细胞系为定点整合的克隆细胞系,且GFP基因表达。

The repetitive ITS（internal transcribed spacers） between rDNA genes were used as the target loci for gene targeting to multiple loci in cow fetal fibroblasts. The techniques of gene targeting to multiple loci using rDNA repetitive sequences as target loci have been set up. The nuclei donor cells for cloning the transgenic cows have been obtained. Firstly, the cow fetal fibroblasts have been isolated, cultured, identified the sex and analysed the karyotypes. The lowest efficient concentrations of G418 and GCV for the positive-negtive selection have been affirmed by MTT method. Secondly, the 7 cell clones expressing green fluorescence have been got by transfect of the gene targeting vectors to multiple loci and positive-negtive selection. One of the 7 cell clones was confirmed as cell clone of gene targeting in site and GFP expression by PCR, RT-PCR and DNA sequencing.