Abstract:

【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.

Key words: avian U6 promoter, cloning, sequence analysis, transcription activity