基于微小RNA架构的新型随机短发夹RNA文库构建

Construction of A New Random Short Hairpin RNA Library Based on microRNA Context

目的构建高效发夹状随机短发夹RNA(shRNA)文库。方法构建由polⅡ启动子(CMV)驱动的增强绿色荧光蛋白(EGFP)下游连接shRNA的表达载体,转染细胞后收取蛋白,用Western blot和荧光素酶报告基因系统检测该载体表达的shRNA对靶基因表达的抑制效率。在由CMV启动的EGFP下游连接shRNA的表达载体基础上,构建EGFP下游连接基于微小RNA(miRNA)架构的shRNA表达载体,转染细胞后收取RNA,用实时定量荧光PCR法检测该载体表达的shRNA对靶基因表达的抑制效果。基于EGFP下游连接基于miRNA架构的shRNA表达载体实时定量荧光PCR的检测结果,构建基于miRNA架构的大容量随机shRNA文库。结果由polⅡ启动子(CMV)驱动发夹状shRNA转录时,shRNA要位于一个大的转录本之后才能被有效转录。基于miRNA架构环境可提高shRNA的干扰效率。构建了一个容量为1.8×10~7的基于miRNA架构的大容量随机shRNA文库。结论构建了一个大容量的新型随机shRNA文库。

Objective To build an efficient random short hairpin RNA（shRNA） library.Methods shRNA expression vector was constructed with enhanced green fluorescent protein（EGFP） in the upstream of shRNA,driven by pol Ⅱ promoter（CMV）.After the constructs were transfected into cells,the proteins were collected.The inhibition efficiency of shRNA was determined by Western blot and dual luciferase reporter system.After the shRNA expression vector was constructed with EGFP in the upstream of shRNA,driven by pol Ⅱ promoter（CMV）,shRNA was further embedded into microRNA（miRNA） context.The constructs were transfected into cells,and then the inhibition efficiency of shRNA against target genes was evaluated by quantificational real-time polymerase chain reaction.According to the result of quantificational real-time polymerase chain reaction,a new random shRNA library was constructed based on miRNA context.Results shRNA downstream of a large transcript was transcripted efficiently by pol Ⅱ promoter（CMV）.The efficiency of shRNA interference on targetgene was improved when shRNA was embedded into miRNA context.Thus,we constructed a new random shRNA library sized 1.8×10~7 based on miRNA context.Conclusion We successfully constructed a new large random shRNA library.