Cloning,Sequence Analysis and Transcription Activity of a Chicken U6 Promoter

WANG Yong-juan1,2,SHEN Peng-peng1,ZHANG Xin-yu1,XIA Xiao-li1,SUN Huai-chang1(1College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,Jiangsu;2Jiangsu Animal Husbandry and Veterinary College,Taizhou 225300,Jiangsu)

avian U6 promoter;cloning;sequence analysis;transcription activity

【Objective】 To cloning avian U6 promoter for construction of short interference RNA(siRNA) expression vectors in avian cells.【Method】 A putative chicken U6 promoter from chicken genomic DNA was amplified by PCR.After sequencing and bioinformatics analysis,the putative promoter was inserted into the reporter plasmid pEGFP-N1 together with GFP-specific short hairpin RNA(shRNA) and resulted in a siRNA expression vector pGFP-U6-shRNA.Then,using human H1 promoter-driven siRNA expression vector pGFP-H1-shRNA as the control,the simian-originated COS-1 cells or chicken embryonic fibroblast(DF-1) cells were cotransfected with the siRNA expression vector pGFP-U6-shRNA,and the GFP-positive cell numbers and total fluorescence were detected by fluorescence microscopy and flow cytometry.【Result】 The PCR product was 560bp long and located on chicken chromosome 28 with a percentage identity of 97.2% to the published chicken U6-3 promoter.Bioinformatics analysis showed that the putative chicken U6 promoter contained several Oct-1 motifs and a weak TATA box without other Pol Ⅲ promoter elements.In COS-1 cells,transfection with pGFP-U6-siRNA resulted in significant decreases in both the number of GFP-positive cells and total fluorescence,but significance was lower than that of transfection with pEGFP-H1-shRNA.While in DF-1 cells,transfection with pGFP-U6-shRNA resulted in significantly higher gene silencing effect than cotransfection with pEGFP-H1-shRNA.【Conclusion】 These data suggest that a chicken U6 promoter was successfully cloned,which can efficiently transcribe gene-specific siRNA expression in avian cells.