Xi Ya-ming, Liu Ting, Meng Wen-tong, Wang Yu-hui, Gu Ling, Lu Xiao-xi, Gong Yu-ping
Department of Hematology, West China Hospital, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2005 Jul;36(4):460-3.
To construct eukaryotic expression vector of siRNA specific to bcr/abl and to initially investigate the effect of recombinant plasmid on bcr/abl and P210 protein expression in K562 cells.
siRNA (small interfering RNA) was designed according to the Tuschl's principle of RNAi-based medicine, and was converted into cDNA coding expression of shRNA (small hairpin RNAs)of siRNA for bcr/abl fusion gene. The cDNA was synthesized and inserted into plasmid pTER. The pTER117 and pTER363 of recombinant plasmid being eukaryotic expression vector was controlled by the H1 promoter of RNA polymerase III, identified by the restriction map and the sequence analysis, and transfected into K562 cells by Lipofectamine. Expression of bcr/abl mRNA was assayed by RT-PCR; expression of P210 protein was detected by immunohistochemistry.
The pTER117 and pTER363 of recombinant plasmid identified by the restriction map and the sequence analysis completely coincided with the designs. 24 hours after transfection in K562 cells, the recombinant plasmid could down regulate the expression of the bcr/abl mRNA and bcr/abl protein(P210) in K562 cells.
The siRNA eukaryotic expression vector against bcr/abl mRNA has been successfully conctructed, and it effectively inhibits the expression of bcr/abl in K562 cells.
构建针对bcr/abl的小干扰RNA（siRNA）真核表达载体，并初步探讨重组质粒对K562细胞中bcr/abl及P210蛋白表达的影响。
根据基于RNA干扰的医学中Tuschl原理设计siRNA，并将其转化为编码针对bcr/abl融合基因的小发夹RNA（shRNA）的cDNA进行表达。合成cDNA并插入质粒pTER。由RNA聚合酶III的H1启动子控制的真核表达载体重组质粒pTER117和pTER363，经酶切图谱和序列分析鉴定后，用脂质体转染K562细胞。用逆转录聚合酶链反应（RT-PCR）检测bcr/abl mRNA的表达；用免疫组织化学检测P210蛋白的表达。
经酶切图谱和序列分析鉴定的重组质粒pTER117和pTER363与设计完全一致。在K562细胞中转染24小时后，重组质粒可下调K562细胞中bcr/abl mRNA和bcr/abl蛋白（P210）的表达。
成功构建了针对bcr/abl mRNA的siRNA真核表达载体，其能有效抑制K562细胞中bcr/abl的表达。
