A high-sensitivity, bi-directional reporter to monitor NF-κB activity in cell culture and zebrafish in real-time

Abstract

NF-κB transcription factors play major roles in numerous biological processes including development and immunity. Here, we engineered a novel bi-directional NF-κB-responsive reporter, pSGNluc, in which a high-affinity NF-κB promoter fragment simultaneously drives expression of luciferase and GFP. Treatment with TNFα induced a strong, dose-dependent luciferase signal in cell culture. The degree of induction over background was comparable to that of other NF-κB-driven luciferase reporters, but the absolute level of expression was at least 20-fold higher. This extends the sensitivity range of otherwise difficult assays mediated exclusively by endogenously expressed receptors, as we show for Nod1 signalling in HEK293 cells. To measure NF-κB activity in the living organism, we established a transgenic zebrafish line carrying the pSGNluc construct. Live in toto imaging of transgenic embryos revealed the activation patterns of NF-κB signalling during embryonic development and as responses to inflammatory stimuli. Altogether, by integrating qualitative and quantitative NF-κB reporter activity, pSGNluc is a valuable tool for studying NF-κB signaling at high spatiotemporal resolution in cultured cells and living animals which goes beyond the possibilities provided by currently available reporters.