UL27基因shRNA表达载体对293细胞转染效率的优化

Optimization of UL27 shRNA Expression Plasmid Transfection Efficiency by 293 Cell

旨在优化Lipofectamine2000试剂介导UL27基因重组质粒p GPU6/GFP/Neo-UL27shRNA转染HEK293细胞的条件,为研究UL27基因重组质粒对HSV-2的干扰作用奠定基础。用Lipofectamine2000试剂介导p GPU6/GFP/Neo-UL27shRNA重组质粒转染293细胞,采用流式细胞仪检测不同的转染时间下的转染效率,同时荧光显微镜和流式细胞仪检测不同比例条件下的转染效率,MTT法检测细胞的存活率。在质粒与转染试剂复合物的配比为0.8μg∶2μL,转染48 h,转染效率最高并且对细胞毒性最小。优化p GPU6/GFP/Neo-UL27shRNA转染293细胞的条件,提高了转染效率。

Abstact：In order to obtain highly transfection efficiency , the recombinant plasmid pGPU 6/GFP/Neo-UL27 shRNA was transfected into 293 cells by Lipofectamine2000 as mediation.The recombinant plasmid pGPU6/GFP/Neo-UL27 shRNA was transfected into 293 cells mediated by Lipofectamine 2000.After 48 h observing expression of the recombinant plasmid in the cells by fluorescence microscope , the transfection efficiency was calculated by using flow cytometry.293 cells were transfected with the recombinant plasmid pGPU 6/GFP/Neo-UL27 shRNA carrying green fluorescent protein （ GFP） gene in mediation of Lipofectamine2000 , based on which transfection time , ratio of plasmid mass to lipofectamine 2000 volume of medium were optimized.The expression of GFP was observed by fluorescent microscopy , and the transfection efficacy was calculated by using flow cytometry.The cell survival rate was detected with MTT method.When the ratio of plasmid mass to Lipofectamine 2000 volume was 0.8 μg：2μL, the positive rate of GFP reached a peak value 48 h after transfection （ P〈0.05 ） , and the cell apoptosis rate was low.The condition for transfection of 293 cells in mediation of Lipofectamine 2000 was optimized , and the transfection efficacy increased.