应用载体介导的RNAi技术抑制Bcl-2的表达

RNAi-mediated gene silencing of Bcl-2 expression

目的 应用载体介导的RNAi技术特异地干扰抗凋亡基因bcl 2在HeLa细胞中的表达。方法 构建利用H1启动子转录功能性siRNA的载体 ;在H1启动子下游分别插入含不同bcl 2基因特异性序列的 64nt寡核苷酸片段 ;将构建的质粒转染HeLa细胞 ,以间接免疫荧光和免疫印迹检测转染后细胞中Bcl 2的表达水平。结果 3种含不同bcl 2特异性序列的质粒转染后均能干扰该基因在细胞中的表达 ,但干扰的效果存在差异 ,从 45 %～ 83 5 %。结论 本研究建立的载体介导的RNAi技术可成功地用于干扰Bcl

Objective To silence the expression of antiapoptotic gene bcl 2 in HeLa cells using vector based RNA interference (RNAi) technique. Methods A vector used to transcribe functional short interfering RNA (siRNA) was constructed, and 64 nt oligonucleotide fragment was inserted into the downstream of the H1 promoter. Plasmids containing different bcl 2 target sequences were transfected into HeLa cells, and then the expression level of Bcl 2 was detected by indirect immunofluorescence assay (IFA) and Western blotting. Results Vector based RNAi had advantages over antisense RNA and synthesized siRNA because it could be delivered to the target cell more efficiently, and its effect could last longer. bcl 2 expression could be inhibited by plasmid expressed siRNA. Three different targeting sequences were selected from bcl 2 gene, and the inhibitory effect ranged from 45% to 83 5%. Conclusion Bcl 2 expression in HeLa cells can be inhibited significantly using plasmid based RNAi.