靶向SynDIG1的shRNA慢病毒载体的构建及功能鉴定

Construction of shRNA Lentivirus Vectors Targeting SynDIG1 and Functionl Identification

为了构建靶向突触分化诱导基因1(SynDIG1)的shRNA慢病毒表达载体,设计出SynDIG1的siRNA的靶点序列,并合成含干扰序列的双链DNA发卡结构shRNA,合成的Oligo经过退火分别与双酶切处理后的pLKO.1-GFP载体连接。将构建的重组质粒pLKO.1-GFP-SynDIG1 shRNA转化到DH5α感受态细菌中,过夜培养后挑选阳性克隆子,扩增后提取DNA进行质粒测序,得到两个序列正确的重组质粒。用这些重组质粒转染HEK293T细胞以及大鼠海马神经元细胞,蛋白免疫印迹及细胞免疫荧光检测SynDIG1 shRNA对外源性和内源性SynDIG1表达的敲减作用。进一步用重组质粒转染HEK293T细胞产生慢病毒颗粒,用病毒颗粒感染DIV2和DIV14的神经细胞,免疫印迹检测SynDIG1的表达。结果表明这两个重组质粒均能够有效地抑制外源性和内源性SynDIG1的表达,对HEK293T中瞬时表达的SynDIG1敲减率达到75%,其慢病毒颗粒感染对早期神经细胞中内源性SynDIG1的表达抑制也很明显。用pLKO.1-GFP成功构建了能够有效抑制外源性和内源性SynDIG1表达的重组质粒,为探讨RNA干扰技术抑制神经细胞SynDIG1基因表达的相关研究奠定基础,为研究SynDIG1基因在神经传导和突触发育及其可塑性调节中的作用提供了有力工具。

To construct a shRNA lentivirus expression vector targeting SynDIG1(synapse differentiation induction gene),the target sequence of SynDIG1 siRNA was designed and sequence double-stranded DNA hairpin structure including interference shRNA was synthesized.The synthesized oligo was ligated with vector pLKO.1-GFP after annealing and double enzyme digestion.Recombinant plasmid pLKO.1-GFP-SynDIG1 shRNA then transformed into DH5 alpha bacteria.Positive clones were selected and amplificated after overnight culture,then sequenced after extraction of plasmid DNA.Two correct recombinant plasmids were obtained and used to transfect HEK293T cells and rat hippocampal neurons,the knockdown effect of SynDIG1 shRNA on exogenous and endogenous SynDIG1 expressions was detected by Western blot and cellular immunofluorescence test.The HEK293T cells were further transfected with packaging plasmids to produce lentivirus particles,which was used to infect nerve cells at DIV2 and DIV14,and the expression of SynDIG1 was detected by Western blot.The results showed both of recombinant plasmids could inhibit the expressions of exogenous and endogenous SynDIG1 effectively.In HEK293T cells the knockdown rate of SynDIG1 was up to 75%.Lentivirus infection also significantly inhibited the expression of endogenous SynDIG1 in inchoate neurons.Recombinant plasmids were successfully constructed with pLKO.1-GFP and it can effectively inhibit the expressions of exogenous and endogenous SynDIG1.This technique laied the foundation for the function study of SynDIG1 gene by using RNA interference to inhibit its expression in nerve cells,it also provided a powerful tool for studying the role of SynDIG1 gene in nerve conduction and synaptic development and the regulation of synaptic plasticity.