脂质体LTX介导高效转染山羊胎儿成纤维细胞(gFFCs)条件的优化

摘要

探讨获得外源基因高效转染山羊胎儿成纤维细胞(gFFCs)的方法，为筛选出体细胞核移植的供核细胞，制备转基因山羊提供技术基础。该研究以绿色荧光蛋白基因(GFP)为报告基因，采用脂质体LipofectaminTM LTX+PLUSTMReagent介导，对细胞接种量、质粒DNA用量、脂质体与质粒DNA比例、脂质体-DNA复合物与细胞作用时间进行优化，荧光显微镜下统计转染后24h的转染效率。结果显示：在24孔培养板中，每孔接种细胞6×104个，24h后进行转染，质粒DNA每孔0.6μg，脂质体与质粒DNA比例为4.5：1.0，脂质体-DNA复合物与细胞作用时间为6 h时，转染效率最高，达到81.2％。%To prepare donor cells for somatic cell nuclear transplantation and produce transgenic goat, a method of foreign gene efficiently transfecting into goat fetal fibroblast cells (gFFCs) was established. Using GFP as a reported gene., four factors including the number of inoculated cells, dose of plasmid DNA, ratio of liposome to plasmid DNA and exposure time of the cells to DNA-liposome complexes were investigated and optimized by Lipofectamin TMLTX and PLUSTM Reagent. After 24 h, transfection efficiency was evaluated as the percentage of transfected cells under fluorescence microscope. The results showed that the gFFCs cultured in 24 wells with 6×104 cells per well, transfected 24 h later, 0.6μg plasmid DNA per well, and ratio of liposome to plasmid DNA 4.5 : 1.0, and exposure of the cells to DNA-liposome complexes for 6 h resulted in the highest transfection efficiency, which was 81.2%.