shRNA transcribed by RNA Pol II promoter induce RNA interference in mammalian cell.
RNA interference is a powerful tool for gene functional analysis in mammals. Permanent gene suppression can be achieved by siRNAs as stem-loop precursors transcribed from RNA Pol III promoter such as H1 and U6 based on vector. This approach, however, has a major limitation: inhibition can not be controlled in a time or tissue specific manner because the RNA Pol III promoter is not time or tissue specific. To overcome these limitations, we designed a strategy that allows synthesis of small hairpin RNAs in a GFP-fused form mediated by RNA Pol II promoter CMV to efficiently and specifically knock down expression of both exogenous and endogenous genes in mammalian cells. As assayed by both fluorescence observing and quantitative RT-PCR, the protein and mRNA products of exogenous gene RFP were efficiently and specifically inhibited; quantitative RT-PCR and western blotting results respectively demonstrated that endogenous lamin B2 mRNA and protein was suppressed without global down-regulation of protein synthesis. Furthermore, GFP-fused shRNA efficacy for RNAi is dependent on target position based on this vector system. This method may provide a novel approach for the application of RNAi technology in suppressing gene expression in mammalian system.
Yuan
Jing
J
State Key Laboratory for Agrobiotechnology, China Agricultural University, No. 2 Yuanmingyuan West Rd, Haidian District, Beijing, 100094, P.R. China.
Wang
Xiaobo
X
Zhang
Ying
Y
Hu
Xiaoxiang
X
Deng
Xuemei
X
Fei
Jing
J
Li
Ning
N
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics; D004257:DNA Polymerase II / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D034921:Lamin Type B / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics*
D034921:Lamin Type B; D011993:Recombinant Fusion Proteins; D012313:RNA; D004257:DNA Polymerase II