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

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。

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

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.