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.
Medicine Healthcare Apparatus