Mammalian Pol III Promoter H1 can Transcribe shRNA Inducing RNAi in Chicken Cells

Abstract

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms. RNAi based on DNA vector is not sufficiently established in chicken species. The present study was performed to evaluate RNAi induced by shRNA transcribed from mammalian Pol III promoter H1 in the chicken cells by using a dual fluorescence reporter assay, a plasmid encoding GFP and a plasmid encoding RFP. The evaluation of RNAi efficiency was performed in two kinds of chicken cell type: primary CEF cells and chicken DT-40 cells by lipofection. GFP- and RFP-expressing cells were observed under fluorescent microscopy, and their mRNAs content were analyzed by quantitative RT-PCR. The intensity of the green fluorescence generated by GFP was greatly suppressed by human H1 promoter transcribed GFP-shRNA. Quantitative RT-PCR analysis showed that normalized GFP mRNA expression was reduced to 37 and 32 in primary CEF and DT-40 cells, respectively. In contrast to GFP, the intensity of the red fluorescence generated by RFP protein and the RFP mRNA levels remained unchanged. Consequently, it was concluded that the RNAi induced by shRNA transcribed from mammalian Pol III promoter H1 is applicable to suppress the gene expression specifically and efficiently in chicken cells.