Cloning Of Chicken Rna Polymerase Ⅲ Type Ⅲ Promoter7SK And Characterization Of Its Shrna Transcription Activity In Vitro

Aim of Investigtaion: Present promoters for shRNA expression are primarily highlyefficient human and mouse type Ⅲ RNA polymerase Ⅲ promoter, namely U6and7SK.Their transcriptional role in chicken cells, tissues even to biological entity are under widelyinvestigation. However, the exploration of avian7SK promoter was only limited to a fewreports. As a highly efficient promoter in mammalian cells,7SK has already showedadvantage over U6of the same type, and was also reported experimentally with commercialvector application. Therefore, employing avian7SK promoter as a molecular tool playssignificant role in the investigation of avian gene function, avian cell, embryo development,and avian transgenic application for selectively disease resistant breeding and medical bioreactors.Methods: Based on chicken genome template and the results of bioinformatics analysis,chicken7SK promoter core sequence was amplified. After sequencing and bioinformaticsanalysis, the cloned chicken7SK promoter core sequence was inserted into pcDNA3.1(+)vector to replace the original CMV and T7promoter, and the modified recombinant plasmidwas named as pch7SK vector. Then, the DNA sequence encoding the predicted shRNAtargeting EGFP gene was inserted into pch7SK vector to construct the RNAi vectorpch7SK-shEGFP with specific shEGFP target. The mouse U6promoter was subcloned, andRNAi vector pmU6-shEGFP was constructed in the same way of pch7SK-shEGFP. DF-EGFPand Vero-EGFP cell stably expressing EGFP was established by lentiviral infection of avianDF-1and mammalian Vero cells. Setting the pch7SK as control vector, the constructed RNAivector was transfected into DF-EGFP and Vero-EGFP cells to assess promoter efficiency ofdriving shRNA expression. EGFP positive cells were observed using fluorescencemicroscopy, and mean fluorescence intensity (MFI) in the cell culture was measured by flowcytometry. The relative EGFP mRNA expression level was measured by Real-time PCR aswell.Results: The cloned chicken7SK core sequence was99%homologous to the publisheddata, including multiple Oct-1motif and SPH domain, without CACCC box, and it containsbasic RNA pol Ⅲ promoter elements of PSE and TATA box. All the RNAi groups showed significant MFI reduction. In the pch7SK-shEGFP transfected DF-EGFP cell culture, the MFIreduction ratio was smaller than that of pmU6-shEGFP tranfected cell culture. In thepmU6-shEGFP transfected Vero-EGFP cell culture,the MFI was reduced significantly thanthe pch7SK-shEGFP transfected cells.Conclusions: The chicken7SK promoter core sequence was successfully cloned, and it iscapable of efficiently expressing shRNA with relative gene specific siRNA expression inavian and mammalian cells.