转肌细胞特异表达MSTN基因RNA干涉序列的小鼠成纤维细胞株的筛选

Screening of transgenic mouse fibroblast cell line in the RNAi sequence of myostatin gene with muscle cell-specific expression

为了筛选肌细胞特异表达MSTN基因RNA干涉序列的小鼠成纤维细胞株,研究利用人工合成的小鼠肌肉启动子SP,连接前期工作中获得带有GFP基因的小鼠MSTN基因RNA干涉载体pR-NAT-M1,构建真核表达载体pRNAT-SP-M1,将pRNAT-SP-M1线性化后转染小鼠成纤维细胞并通过300μg/mL G418筛选获得转基因阳性细胞。结果表明:获得的转基因阳性细胞呈现正常的小鼠成纤维细胞的梭形,且在荧光显微镜下呈现出表达绿色荧光蛋白的绿色;转基因阳性细胞的生长曲线呈"S"形;转基因阳性细胞经冷冻复苏后,仍具有正常的细胞形态和增殖特性;PCR鉴定结果显示转基因阳性细胞基因组DNA中整合有pRNAT-SP-M1序列。说明成功获得了转肌细胞特异表达的MSTN基因RNA干涉序列小鼠成纤维细胞株。

To establish a transgenic mouse fibroblast cell line, a synthetic SP （muscle - specific promoter） was linked with pRNAT- MI （ MSTN knockdown vector with GFP） by a series of molecular method. The eukaryotic expression vector pRNAT - SP - M1 was constructed. Mouse fibroblasts were transfected with the plasmid by cationic liposome method and filtered using 300 μg/mL G418 to obtain the transgenic positive cells. The results showed that the obtained cells had normal spindle morphology of mouse fibroblast, the cells growth curve was S - shaped, and GFP could be observed in microscope. After freezing and thawing, the transgenic positive cells still had normal cell morphology and cell proliferation characteristics. The PCR result showed that the constructed vector was integrated into the mouse genome. It indicated that the transgenic mouse fibroblast cell line with pRNAT - SP - M1 vector was obtained successfully.