应用GFP标记基因研究RNAi载体的抑制效果

目的 用绿色荧光蛋白(GFP)反映RNA干扰(RNAi)载体的转染率,以研究RNAi载体的抑制效果.方法 将GFP基因和RNAi片段(特异性针对Bcl-2基因)连接至pC1载体上,构建共表达载体GFP/RNAi及其对照载体,转染293细胞,用荧光显微镜、流式细胞仪检测细胞的GFP转染率,并用TR-PCR分析RNAi抑制Bcl-2 mRNA表达的效果.结果 在293细胞中,GFP的转染率可达85%以上,此时观察到RNAi有效的抑制了Bcl-2 mRNA的表达.结论 在RNAi实验时,可选用GFP等标记基因作为载体转染率的判断标准,以进一步判断RNAi的作用效果.构建的RNAi载体有效的抑制了Bcl-2的表达,为下一步抑制肿瘤细胞的生长提供科学资料.

Studying the suppression effect of RNAi by using GFP gene as a marker

Objective Studying the suppression effect of RNAi by using green fluorescence protein(GFP) to reflect the transfection rate.Methods GFP gene and RNAi segment specific to Bcl-2 gene was inserted into pC1 vector to construct the co-expression vector(GFP/RNAi),after 293 cells were treated with GFP/RNAi vector,the transfection rate was estimated from GFP by using fluorescence microscopy as well as FACS analysis,and the suppression of Bcl-2 mRNA expression was detected by RT-PCR.Results The transfection rate was above 85% from detection of GFP positive cells,and the Bcl-2 mRNA expression was also decreased to 50% as its control group.Conclusion The marker gene(such as GFP) may be used to reflect transfection rate in RNAi experiment,because the transfection rate is important for the inhibitive effect of RNAi.The suppression of Bcl-2 expression is crucial to inhibit proliferation of tumor cells.