Abstract
Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine produced by activated macrophages and lymphocytes and involved in many inflammatory diseases. Preventing the production or action of TNF-α is a potent therapeutic strategy for these inflammatory diseases. Since there is a lack of rapid and effective assay for examining the expression TNF-α in macrophages, we attempt to establish a reporter system to assess TNF-α 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-α promoter and 3′-untranslated region (3′-UTR). The TNF-α-luciferase reporter cell line is used for assessing the expression of TNF-α gene induced by LPS in the presence or absence of chemicals that inhibit the biosynthesis of TNF-α such as dexamethasone and emodin, and also for measuring change of expression of TNF-α gene under downregulation of the expression of steroid receptor coactivator-3, a modulator for TNF-α. The luciferase activity correlated well with the ELISA results for TNF-α production, therefore, the TNF-α-luciferase reporter cell line is a sensitive, effective tool for studying the expression of TNF-α gene.
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Acknowledgment
We would like to thank Xiaoliang Wang and Yuan Jiang for their technique assistance. This work was supported by China National Natural Science Foundation Grants 30600566 and 30770455.
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Chen, Q., Zhao, Y., Cheng, Z. et al. Establishment of a cell-based assay for examining the expression of tumor necrosis factor alpha (TNF-α) gene. Appl Microbiol Biotechnol 80, 357–363 (2008). https://doi.org/10.1007/s00253-008-1552-9
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DOI: https://doi.org/10.1007/s00253-008-1552-9