Luciferase reporter construction and analysis of promoter region of mouse interferon-γ receptor β gene / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE To functionally analyze the promoter using dual luciferases reporter assay system by cloning the promoter region of mouse interferon-y receptor 2 (Ifngr2) gene. METHODS The 5' sequence (-1344-+48) containing the transcription start site of the mouse Ifngr2 gene was acquired from the Eukaryotic Promoter Database, and the gene fragment was cloned into the luciferase reporter vector. Series of 5' deleted truncated variants were further constructed to obtain six luciferase reporter vectors with different lengths of promoters. The transcriptional activity of different Ifngr2 promoter truncations was determined by the dual luciferase analysis. In addition, site-directed mutation was applied to explore the effect of NF-kB on mouse Ifngr2 transcription. RESULTS The constructed mouse Ifngr2 promoter luciferase reporter vector was confirmed by gene sequencing and alignment. Promoter deletion analysis showed that the-132-97 and-1059-727 regions were important for mouse Ifngr2 gene transcription. Site-directed mutation indicated that the NF-kB binding region was essential for the transcription of Ifngr2 gene. CONCLUSION Mouse Ifngr2 gene promoter luciferase vectors are successfully constructed. Functional analysis of the mouse Ifngr2 gene promoter reveals an NF-kB binding site, which is essential for transcription of Ifngr2 gene.