Establishment of a cell-based assay for examining the expression of tumor necrosis factor alpha (TNF-alpha) gene

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine produced by activated macrophages and lymphocytes and involved in many inflammatory diseases. Preventing the production or action of TNF-alpha is a potent therapeutic strategy for these inflammatory diseases. Since there is a lack of rapid and effective assay for examining the expression TNF-alpha in macrophages, we attempt to establish a reporter system to assess TNF-alpha gene expression through measuring luciferase activity. In this study, mouse macrophage cell line RAW 264.7 was stably transfected with a luciferase reporter pGL3-TNFPro-UTR, which contains TNF-alpha promoter and 3'-untranslated region (3'-UTR). The TNF-alpha-luciferase reporter cell line is used for assessing the expression of TNF-alpha gene induced by LPS in the presence or absence of chemicals that inhibit the biosynthesis of TNF-alpha such as dexamethasone and emodin, and also for measuring change of expression of TNF-alpha gene under downregulation of the expression of steroid receptor coactivator-3, a modulator for TNF-alpha. The luciferase activity correlated well with the ELISA results for TNF-alpha production, therefore, the TNF-alpha-luciferase reporter cell line is a sensitive, effective tool for studying the expression of TNF-alpha gene.