PUBLICATION
A high-sensitivity, bi-directional reporter to monitor NF-κB activity in cell culture and zebrafish in real-time
- Authors
- Kuri, P., Ellwanger, K., Kufer, T.A., Leptin, M., Bajoghli, B.
- ID
- ZDB-PUB-161217-6
- Date
- 2017
- Source
- Journal of Cell Science 130(3): 648-657 (Journal)
- Registered Authors
- Bajoghli, Baubak, Kuri, Paola, Leptin, Maria
- Keywords
- Nod1, Tri-DAP, pSGNluc, proctodeum, zebrafish
- MeSH Terms
-
- Animals
- Animals, Genetically Modified
- Base Sequence
- Cell Culture Techniques/methods*
- Computer Systems*
- Cytokines/metabolism
- Embryo, Nonmammalian/metabolism
- Embryonic Development/drug effects
- Genes, Reporter*
- Green Fluorescent Proteins/metabolism
- HEK293 Cells
- Humans
- Inflammation/pathology
- Luciferases/metabolism
- NF-kappa B/metabolism*
- Peptidoglycan/metabolism
- Plasmids/metabolism
- Signal Transduction/drug effects
- Signal Transduction/radiation effects
- Tumor Necrosis Factor-alpha/pharmacology
- Ultraviolet Rays
- Zebrafish/embryology
- Zebrafish/metabolism*
- PubMed
- 27980067 Full text @ J. Cell Sci.
Citation
Kuri, P., Ellwanger, K., Kufer, T.A., Leptin, M., Bajoghli, B. (2017) A high-sensitivity, bi-directional reporter to monitor NF-κB activity in cell culture and zebrafish in real-time. Journal of Cell Science. 130(3):648-657.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping