Real-time imaging of ligand-induced IKK activation in intact cells and in living mice

Abstract

The transcription factor NF-kappaB is a key regulator of cellular activation, proliferation and apoptosis. Defects in the NF-kappaB pathway contribute to a broad array of malignant, neurodegenerative and chronic inflammatory diseases. IKK-dependent IkappaB alpha degradation by the 26S proteasome is a critical NF-kappaB regulatory control point, which is emerging as an important target for drug development. To directly monitor regulation of IKK activation in intact organisms, we engineered an IkappaB alpha-firefly luciferase (IkappaB alpha-FLuc) fusion reporter. In cultured cells and living animals, the reporter provided a continuous, noninvasive readout of the kinetics of ligand-induced IKK activation and the pharmacodynamics of selective inhibitors of both IKK and the 26S proteasome. This IkappaB alpha-FLuc reporter now permits continuous readout of IKK activation in vivo, facilitates development and validation of target-specific therapeutics, and complements conventional NF-kappaB transcriptional reporters for more complete temporal and regional investigations of the NF-kappaB signaling pathway in health and disease.

Real-time imaging of β-catenin dynamics in cells and living mice

“…The WT cDNA was then fused to codon-optimized firefly (Photinus pyralis) luciferase (FLuc) from the pGL-3 control plasmid (Promega) by using a strategy described in ref. 30. Construction of the pFLuc control plasmid is described in ref.…”

“…Construction of the pFLuc control plasmid is described in ref. 30. In the final constructs, ␤-cat-FLuc and FLuc expression were driven by an immediate-early CMV promoter.…”

Interrogation of Inhibitor of Nuclear Factor κB α/Nuclear Factor κB (IκBα/NF-κB) Negative Feedback Loop Dynamics

“…Previously, we demonstrated that fusing ⌱B␣ to the firefly luciferase gene (⌱B␣-FLuc) enabled quantitative monitoring of ⌱B␣ degradation (which directly correlates with IKK activity) in vitro and in vivo (26). We then placed the fusion reporter under the control of an NF-B-responsive promoter (B 5 3 ⌱B␣-FLuc) and showed that it recapitulated the endogenous ⌱B␣ negative feedback loop (Fig.…”

Generation of a highly inducible Gal4 → Fluc universal reporter mouse for in vivo bioluminescence imaging

“…To image firefly luciferase, the media was changed to MEBSS containing 150 g/ml D-luciferin and images acquired with a Ͼ590 nm bandpass filter. Data analysis was performed as described (29,30).…”

“…Animal care and euthanasia were approved by the Washington University Medical School Animal Studies Committee. Bioluminescence imaging was performed on the IVIS under 2.5% isoflurane anesthesia at the indicated time points as described (29,30). After imaging, animals were killed by cervical dislocation.…”