title: A high-sensitivity bi-directional reporter to monitor NF-κB activity in cell culture and zebrafish in real time.
abstract: Nuclear factor (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 (also known as TNF) 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 signaling 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 signaling during embryonic development and as responses to inflammatory stimuli. Taken together, 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 that goes beyond the possibilities provided by currently available reporters.
authors.affiliation: Directors' Research Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Paola
authors.initials: P
authors.lastname: Kuri
authors.affiliation: Department of Immunology, Institute of Nutritional Medicine, University of Hohenheim, 70593 Stuttgart, Germany.
authors.forename: Kornelia
authors.initials: K
authors.lastname: Ellwanger
authors.affiliation: Department of Immunology, Institute of Nutritional Medicine, University of Hohenheim, 70593 Stuttgart, Germany.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Kufer
authors.affiliation: Directors' Research Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany bajoghli@embl.de maria.leptin@embo.org.|Institute of Genetics, University of Cologne, Zülpicherstrasse 47a, 50674 Cologne, Germany.|EMBO, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Leptin
authors.affiliation: Directors' Research Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany bajoghli@embl.de maria.leptin@embo.org.
authors.forename: Baubak
authors.identifier: 0000-0002-7368-7523
authors.initials: B
authors.lastname: Bajoghli
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods*; D003199:Computer Systems; D016207:Cytokines / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism; D047108:Embryonic Development / Q000187:drug effects; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007249:Inflammation / Q000473:pathology; D008156:Luciferases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000528:radiation effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014466:Ultraviolet Rays; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism*
chemical_list: D016207:Cytokines; D016328:NF-kappa B; D010457:Peptidoglycan; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D008156:Luciferases
keywords: Nod1; Proctodeum; Tri-DAP; Zebrafish; pSGNluc