Construction of red fluorescent protein reporter gene vector driven by mouse HMGB1 promoter and expression in mammalian cells

Abstract

Objective To construct the vector of mouse HMGB1 promoter-driven red fluorescent protein reporter gene so as to supply a tool for the study on the expression regulation of HMGB1 gene in mammalian cells and related signal transduction mechanism. Methods The mouse HMGB1 promoter sequence was subcloned into a red fluorescent protein reporter gene vector, pDsRed1-1. The recombinant vector pDsRed1-1-HMGB1p was then transfected into NIH3T3 cells by liposome, and the intracellular activity of HMGB1 promoter was observed under a fluorescence microscope in normal condition or after tumor necrosis factor-α (TNF-α) stimulation. Results The recombinant plasmid was confirmed by enzyme digestion and DNA sequence analysis. The vector was expressed with red fluorescence at a low level in the rest NIH3T3 cells, but the expression was highly increased by the stimulation with TNF-α. Conclusion A red fluorescent protein reporter gene vector driven by mouse HMGBI promoter is constructed successfully, which can be expressed in mammalian cells with a physiological response to TNF-α stimulation, thus providing an important and convenient tool for the study on the regulatory mechanisms of HMGB 1 gene expression. Key words: High mobility group box 1; Promoter; Red fluorescent protein; Reporter gene