南方医科大学学报2011，Vol.31Issue(1)：86-89,4.
人单磷酸腺苷激活的蛋白激酶催化亚单位α2基因shRNA载体的鉴定和表达
Identification and expression of shRNA vectors targeting human AMPKα2
摘要
Abstract
Objective To construct pGPU6/GFP/Neo-shRNA expression vector targeting human AMPKα2 gene and evaluate its silencing effect in SH-SY5Y cell line. Methods The oligonucleotides designed by Ambion online CAD software targeting AMPKα2 were cloned into the pGPU6/GFP/Neo vector. After confirmation by DNA sequencing and enzyme digestion analysis, the recombinant vectors were transfected into the SH-SY5Y cell line via lipofectamine and the positive clones were selected using G418. The expression levels of AMPKα2 mRNA and protein in the transfected cells were detected by RT-PCR and Western blotting, respectively. Results Four shRNA vectors were successfully constructed as confirmed by DNA sequencing and the enzyme digestion analysis. Among the 4 recombinant vectors, pGPU6/GFP/Neo-shRNA AMPKα2(3) showed the strongest gene silencing effect and down-regulated the protein expression of AMPKα2 by 63% in the transfected cells. Conclusion Transfection with pGPU6/GFP/Neo-shRNA AMPKα2(3) results in effective inhibition of AMPKα2 gene expression in SH-SY5Y cells, which provide a means for studying AMPK-mediated cell injury.关键词
局麻药/RNAi干扰/单磷酸腺苷激活的蛋白激酶催化亚单位α2Key words
local anesthetic/ RNA interference/ AMPK分类
生物科学引用本文复制引用
卢俊,徐世元,崔睿,张庆国,雷洪伊..人单磷酸腺苷激活的蛋白激酶催化亚单位α2基因shRNA载体的鉴定和表达[J].南方医科大学学报,2011,31(1):86-89,4.基金项目
国家自然科学基金(30972843) （30972843）
