摘要
为了优化建立转染绵羊成纤维细胞系的外源基因转染体系,本研究利用带有绿色荧光蛋白（GFP）的外源基因,采用阳离子脂质体法,探讨了DNA用量、脂质体用量和转染时间等因素对转染绵羊成纤维细胞的影响。结果表明,在500μL转染培养基中成纤维细胞达到85%汇合时,添加由50μL DMEM中含有0.2μg DNA和50μL DMEM中含有2.0μL Lipo-fectamineTM2000的两种溶液混合构成的转染液并转染12 h,可获得最高的转染效率。当转染12 h后,用基础培养液培养至第24小时,细胞核周区出现强绿色荧光,而当转染后48～64 h观察时,细胞核周区的荧光强度明显弱,而远离核周区的胞质中的荧光强度增加。
In order to establish highly efficient system of transfected sheep fibroblast, cationic liposome method was used to transfect subcultured 3 to 5 passage ovine fibroblasts by plasmids with green fluorescence protein （GFP） report gene, with the view of confirming the best transfection method and its optimization. Results showed that transfection efficiency could get max imal with 0.2 μg DNA and 2.0 μl. LipofectamineTM 2000 in 12 hours under the circumstance of using plasmid DNA taken by Ptasmid Purification Mini Kit spin column. Green fluorescence near the nucleus became dimmer and dimmer with the lapse of time after the initial strong expression, on the contrary, it enhanced gradually in the cytoplasm.
出处
《中国畜牧兽医》
CAS
北大核心
2009年第12期67-70,共4页
China Animal Husbandry & Veterinary Medicine
基金
东北林业大学大学生创新性实验项目(2008008)
关键词
质粒DNA
绵羊成纤维细胞
转染
脂质体法
plasmid DNA
sheep fibroblasts
transfection
cationic liposome method
