RyR2-shRNA重组慢病毒载体的构建及其对离体心肌细胞RyR2基因表达的影响

The Construction of RyR2-shRNA Recombinant Lentivirus Vector and Its Effect on the Expression of RyR2 in Isolated Mouse Cardiac Myocytes

目的构建携带RyR2-shRNA的慢病毒载体,并检测其对小鼠离体心肌细胞中RyR2的基因沉默效果。方法设计并合成针对小鼠RyR2基因的shDNA,克隆入含U6启动子和EGFP报告基因的慢病毒载体psiHIV-U6-eGFP,构建表达RyR2-shRNA的慢病毒载体,通过将慢病毒转移质粒和包装质粒共转染293T细胞,得到重组慢病毒载体颗粒,然后离体感染小鼠心肌细胞,采用Real-timePCR检测RyR2在mRNA水平的沉默效应。结果经测序证实RNAi慢病毒载体构建成功并成功包装。流式细胞仪测得重组慢病毒感染小鼠心肌细胞的阳性率达80%以上。Real-timePCR证实,重组慢病毒Lenti-siRyR2感染的小鼠心肌细胞中RyR2在mRNA水平基因抑制率达90%以上。结论成功构建了表达RyR2-shRNA的重组慢病毒表达载体,并在小鼠心肌细胞中实现有效的基因沉默效应。

Objective To construct a combinant lentivirus vector that expresses RyR2-targating shRNA and detect the effect of specific gene silence of RyR2 in isolated mouse cardiac myocytes. Methods The shDNA oligonucleotides targeting to mouse RyR2 gene were designed and synthesized, then cloned into lentivirus expression vector psiHIV-U6-eGFP containing U6 promoter and enhanced green fluorescent protein （EGFP） report gene to generate shRNA lentivirus expressive vectors. 293T cells were co-transfected with the recombinant vector plasmids and the packaging plasmids to produce lentiviral vector particles. Neonatal mouse cardiac myocytes （NMCM） were isolated, cultured and infected by the lentiviral vector particles. The gene silencing effect was detected by real-time quantitive PCR on the level of mRNA. Results Four RNAi lentivirus expression vectors targeting mouse RyR2 gene were successfully constructed and confirmed by DNA sequencing. The recombinant lentivirus particles were packaged successfully to produce enough titer for the following experiments. The infection efficacy of NMCM were above 80% detected by Flow cytometry （FCM）. The result of real-time quantitive PCR showed the specific silencing effect of RyR2 in NMCM was up to 90% depress of mRNA. Conclusions The shRNA lentivirus expressive vectors targeting mouse RyR2 gene are successfully constructed. The effects of gene silencing of RyR2 are effective and stable in NMCM.