GFP-p65 knock-in mice as a tool to study NF-kappaB dynamics in vivo.

The nuclear factor kappaB (NF-kappaB) signaling pathway regulates immune and inflammatory responses and is implicated in the pathogenesis of multiple diseases. The principal mechanism controlling NF-kappaB activation depends on the association of NF-kappaB transcription factor dimers with IkappaB molecules, which prevents the accumulation of NF-kappaB in the nucleus and the activation of target gene transcription. Monitoring the nucleocytoplalsmic shuttling of NF-kappaB factors is a reliable method to study the dynamic regulation of NF-kappaB activity. Here, we generated knock-in mice expressing a fusion protein between the green fluorescent protein (GFP) and the p65/RelA NF-kappaB subunit (GFP-p65) from the endogenous p65 genomic locus. Homozygous GFP-p65 mice developed normally and showed normal NF-kappaB activation, demonstrating that the GFP-p65 fusion protein functionally substitutes for wild-type p65. Live imaging of primary cells from GFP-p65 mice allowed real-time monitoring of p65 nucleocytoplasmic shuttling upon NF-kappaB activation. Therefore, the GFP-p65 knock-in mice constitute an invaluable tool for studying the dynamic regulation of NF-kappaB.