Construction of a practical RNA polymerase I fluorescence reporting system

Abstract

Objective To construct an practical RNA polymerase I fluorescent reporting system with stable transfection of cells expressing influenza A virus RNPs and being quantifiable in-vitro based on pMDT-18 plasmid vector. Methods Human RNA polymerase I promoter, mice terminator and UTR of NP gene from A/Puerto Rico/8/34 were redesigned and synthesized respectively. Chimeric PCR was used to combine enhanced green fluorescent protein (eGFP) with the above parts to form the reporting element consisting of Mice terminator-3’ UTR-eGFP-5’UTR-human RNA polymerase I promoter. The reporting element was cloned into pMDT-18 plasmid vector without any promoter to build a practical RNA polymerase I fluorescent reporting plasmid. HeLa cells with stable expression of influenza A/Puerto Rico/8/34 virus RNPs were transfected. The expression of fluorescence was observed and the relative intensity of fluorescence was measured. Results The reporting system expressed no fluorescence after transfection of HeLa cells. After transfection of the HeLa cells with stable expression of influenza A/Puerto Rico/8/34 virus RNPs, fluorescence were expressed at 24, 36 and 48 hours after transfection with increasing fluorescence intensity over time. Conclusions A practical RNA polymerase I fluorescent reporting system was successfully constructed. The system may be used to increase the rescue efficiency of influenza reverse genetics system, to facilitate the researches on the mechanism of influenza RNA polymerase, to explore the effect of UTR gene polymorphism on gene duplication and transcription, and to provide a reliable platform for scientists in the fields of viral molecular mechanism research. Key words: RNA Polymerase I; Influenza virus; Green fluorescent protein