【摘要】 为了获取转bFat-1基因牛胎儿成纤维细胞系,试验采用酶切连接方法构建pIRES2-eGFP-bFat-1载体,采用脂质体转染、G418筛选、荧光显微镜观察、基因组PCR和RT-PCR鉴定方法构建重组转bFat-1基因牛胚胎成纤维细胞。结果表明:SacⅠ和ApaⅠ双酶切和测序验证pIRES2-eGFP-bFat-1载体构建正确;经脂质体转染,G418筛选去除未转染细胞,最后通过荧光显微镜观察,基因组PCR和RT-PCR鉴定获得了4株既高表达绿色荧光蛋白又高表达bFat-1基因的牛胚胎成纤维转基因细胞株。说明已成功构建了转bFat-1基因载体并构建出转基因细胞系。更多还原

【Abstract】 To obtain a transferred bFat- 1 gene cell line from bovine fetal fibroblast,the transgenic vector pIRES2- eGFP- bFat- 1 was constructed by enzyme digesting. The recombinant bFat- 1 transgenic bovine embryo fibroblast cells were constructed using liposome transfection, G418 screening,observation by fluorescence microscope,identifications by PCR and RT- PCR tests. The results showed that the transgenic vector pIRES2- eGFP- bFat- 1 was constructed and identified correctly using double digestion with Sac Ⅰ and Apa Ⅰ restriction enzymes and DNA sequencing. Four transgenic cell strains of bovine fetal fibroblasts were obtained through liposome transfection,G418 screening to remove non- transfected cells,using observation by fluorescence microscope and identifications by PCR and RT- PCR tests,in which both of bFat- 1 gene and green fluorescent protein( eGFP) gene were highly expressed. The results indicate that the transgenic vector of bFat- 1 gene and the transgenic cell line from bovine fetal fibroblast cells were constructed successfully.更多还原