GLT-1基因siRNA真核表达载体的构建及其在大鼠神经胶质细胞中的抑制效应  

Construction of Eukaryotic Expression Plasmid Expressing siRNA Targeting GLT-1 Gene and its Inhibitory Effect on GLT-1 Expression in Rats Glial Cells

作　　者：李哲[1,2] 王玮[1] 雷迎峰[3] 尹文[3] 王巍[1] 李惠民[4] 廖衡[4] 

机构地区：[1]第四军医大学唐都医院放射科 [2]解放军第451医院放射科,陕西西安710054 [3]第四军医大学基础部微生物学教研室 [4]解放军第451医院放射科

出　　处：《生物技术通讯》2007年第1期5-8,共4页Letters in Biotechnology

摘　　要：目的:研究特异性siRNA对大鼠神经胶质细胞中GLT-1基因的阻抑效果。方法:根据GLT-1基因的序列特点和RNAi设计原则,设计其shRNA的核苷酸片段。退火后将其克隆入pSupressorNeo,构建可表达大鼠GLT-1基因siRNA的重组真核表达质粒pSuppressorNeo-GLT-1;利用脂质体法将其转染神经胶质细胞后,用RT-PCR、Western印迹及免疫荧光法等方法检测转染的神经胶质细胞中GLT-1基因的表达水平。结果:瞬时转染的神经胶质细胞中GLT-1基因的表达受到明显抑制,GLT-1蛋白含量明显下降。结论:pSuppressor-Neo-GLT-1质粒构建成功,瞬时转染神经胶质细胞后可以明显抑制GLT-1基因的表达。Objective： To study the inhibitory effect of siRNA targeting GLT-1 gene in rats glial cells. Methods： Acorrding to the GLT-1 gene sequence and mRNA structure, the hairpin siRNA templates were designed. The annealing product was cloned into pSuppressor vector, the recombinant pSuppressorNeo-GLT-1 was constructed. The glial cells were transfected by pSuppressorNeo-GLT-1 with LipofectAMINE2000. The inhibitory effect of GLT-1 mRNA was detected by RT-PCR and Western-blot and the inhibitory effect of GLT-1 protein expression was detected by indirect immunofluorescency. Results： The distinct inhibition of the expression of GLT-1 in the transfected glial cells was observed. Conclusion： The plasmid vector expressing siRNA targeting GLT-1 gene was successfully constructed. The results of transient transfection showed the GLT-1 gene expression level significantly decrease in the transfected cells.

关 键 词：GLT-1基因 RNA干扰 神经胶质细胞 转染 

分 类 号：Q782[生物学—分子生物学] Q786