【摘要】 目的构建并筛选能高效沉默大鼠垂体瘤转化基因(rPTTG)的shRNA慢病毒重组载体。方法设计并合成4组特异性针对大鼠PTTG基因的shRNA序列将其构建到慢病毒载体pGCL-GFP中;评估慢病毒重组载体在大鼠肾细胞中的转染效率并运用real time PCR技术检测各组重组载体对目的基因的沉默效果。结果PCR及测序结果显示,目的片段插入正确,重组载体构建成功,慢病毒重组载体转染效率达70%以上;4组shRNA序列均有基因敲减效果,并且第4组shRNA(4#shRNA)序列效果最为明显(>80%)。结论成功构建了高效沉默rPTTG基因的慢病毒载体;验证了慢病毒载体作为RNAi载体工具转染效率的可靠性;实验显示4#shRNA能高效抑制内源性rPT-TG基因表达。更多还原

【Abstract】 Objective To construct and screen the shRNAs-expressing lentiviral recombinants targeting the rat PTTG gene.Methods Four groups of shRNA sequences specifically targeting the rPTTG gene were designed and synthesized.These shRNAs were inserted into lentiviral vectors pGCL-GFP.The recombinants′ transfection efficiency was evaluated after infecting NRK cell line with the lentiviral particles.Real time PCR was employed to assess the gene silencing efficacy of these recombinants.Results Real time PCR and sequencing showed that shRNAs were correctly inserted into the lentiviral vector and recombinants were constructed successfully. Transfection efficiency of lenviral recombinants was more than 70%.All of these four shRNAs could achieve gene knockdown effect,and 4# shRNA had the most significant gene silence effect among them(>80%).Conclusions shRNAs-expressing lentiviral recombinants targeting the rPTTG gene were successfully constructed and screened.Lentivirus can operated as RNAi vectors to achieve effective transfection.4# vshRNA could inhibit PTTG expression successfully.更多还原