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

Abstract

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 target gene was improved when shRNA was embedded into miRNA context.Thus,we constructed a new random shRNA library sized 1.8×107 based on miRNA context.Conclusion We successfully constructed a new large random shRNA library.