小鼠NOMO1基因RNA干扰载体的构建及其功能的初步研究

Construction and function of mouse NOMO1 RNA interference vector

目的:构建小鼠NOMO1基因RNA干扰载体,比较其对P19细胞NOMO1基因的抑制效率,以研究NOMO1基因与P19细胞向心肌细胞方向分化的关系。方法:利用Designer3.0软件设计小鼠NOMO1基因干扰片段,合成shRNA序列,再行寡核苷酸双链的合成,退火后形成的寡核苷酸双链克隆到pGPU6/Hygro载体的黏性末端,连接产物转化到感受态细胞,增菌,质粒扩增,质粒DNA抽提,使用PstⅠ、BamHⅠ进行双酶切和DNA测序鉴定重组克隆。将小鼠NOMO1基因RNA干扰载体转染P19细胞,以RT-PCR技术验证NOMO1基因在P19细胞中的mRNA表达。以DMSO诱导分化方案诱导P19细胞向心肌细胞分化,采用定量RT-PCR技术检测P19细胞中α-MHC等基因mRNA的表达,评估NOMO1与P19干细胞向心肌细胞方向分化的关系。结果:双酶切证实shRNA正确插入质粒,测序结果表明插入的序列正确。载体转染的P19细胞筛选稳定表达株,经RT-PCR和Western blot验证4个位点设计的干扰质粒对NOMO1在P19细胞中的mRNA表达均具有较高的抑制效率。转染后P19细胞的α-MHC基因mRNA表达则显著下调(P<0.05)。结论:成功构建小鼠NOMO1基因RNA干扰载体,为研究NOMO1基因与P19细胞向心肌细胞方向分化的关系提供了稳定的转染细胞工具,NOMO1可能通过诱导α-MHC等基因表达,促进P19干细胞向中胚层的分化。

Objective：To construct the RNAi vectors targeting mouse NOMO1 gene and compare the silence effects of these small interference RNAs,and to study the effects of NOMO1 gene on the differentiation of P19 cells to cardiac myocytes.Methods：Short hairpin RNAs of mouse NOMO1 gene were designed by Designer3.0 software,synthesized and cloned into the pGPU6 / Hygro plasmid.Connection products were transfected into competent cells and identified by PstⅠ＋BamHⅠ double digestion and DNA sequencing.Then the vectors were transfected into P19 cell.The level of NOMO1 mRNA was evaluated by RT-PCR.The expression of α-MHC mRNA in P19 cell,which was induced by DMSO,was evaluated by RT-PCR.Results：shRNA was inserted into the pGPU6 / Hygro plasmid correctly,confirmed by double digestion and DNA sequencing.The strains of P19 cell stably expressing shRNA were selected.RT-PCR and Western blot showed that 4 shRNAs silenced the expression of NOMO1 gene markedly.α-MHC was downregulated in transfected P19 cell.Conclusion：pGPU6 / Hygro vectors carried mouse NOMO1 shRNA are constructed successfully.It provides us a useful tool for the study of NOMO1 gene function in the differentiation of P19 cell into cardiac myocyte.NOMO1 may be involved in α-MHC gene expression,and the differentiation of P19 stem cell into mesodermal cell.