人MEF2C基因shRNA慢病毒载体构建及其对Daoy细胞MEF2C mRNA表达的影响

Construction of shRNA lentiviral vector targeting human MEF2C gene and its influence on the expression of MEF2C mRNA in Daoy cell line

目的构建人肌细胞增强因子2C(MEF2C)基因短发卡RNA(shRNA)慢病毒载体,观察其对髓母细胞瘤Daoy细胞MEF2C mRNA表达的影响。方法设计MEF2C基因特异性siRNA靶点,构建于慢病毒pGLV3/H1/GFP/Puro载体,筛选有效shRNA慢病毒载体,继而在293T细胞中包装成病毒颗粒,将其感染髓母细胞瘤Daoy细胞,Real-time PCR检测MEF2C mRNA。结果构建的MEF2C基因shRNA慢病毒载体测序正确,包装获得的shR-NA慢病毒颗粒感染Daoy细胞后其MEF2C mRNA表达量较阴性对照组下降了81.1%。结论成功构建了MEF2C基因shRNA慢病毒表达载体并包装成慢病毒颗粒,其能够有效抑制MEF2C mRNA的表达。

Objective To construct a short hairpin RNA（shRNA） lentivirus vector of of human MEF2C gene,and to detect its interference efficiency on the expression of MEF2C mRNA in Daoy cell line.Methods The specific siRNA sequences targeting human MEF2C gene were synthesized and cloned into pGLV-3 lentiviral vector.The lentivirus particles were packaged and transmitted into Daoy cell line after screening for the valid siRNA.The expression of MEF2C mRNA was determined by Real-time PCR.Results PCR and DNA sequencing demonstrated that MEF2C-shRNA plasmids was correctly constructed.The Daoy cell line was infected with the packaged virus particles and the expression of MEF2C mRNA was inhibited about 81.1%,compared with the negative control group.Conclusion The recombinant lentiviral MEF2C-shRNA vector has been successfully constructed and packaged into lentiviral particles targeting human MEF2C gene,which can effectively inbit the expression of MEF2C mRNA.