KappaBle fluorescent reporter mice enable low-background single-cell detection of NF-κB transcriptional activity in vivo

Abstract

Nuclear factor-κB (NF-κB) is a transcription factor with a key role in a great variety of cellular processes from embryonic development to immunity, the outcome of which depends on the fine-tuning of NF-κB activity. The development of sensitive and faithful reporter systems to accurately monitor the activation status of this transcription factor is therefore desirable. To address this need, over the years a number of different approaches have been used to generate NF-κB reporter mice, which can be broadly subdivided into bioluminescence- and fluorescence-based systems. While the former enables whole-body visualization of the activation status of NF-κB, the latter have the potential to allow the analysis of NF-κB activity at single-cell level. However, fluorescence-based reporters frequently show poor sensitivity and excessive background or are incompatible with high-throughput flow cytometric analysis. In this work we describe the generation and analysis of ROSA26 knock-in NF-κB reporter (KappaBle) mice containing a destabilized EGFP, which showed sensitive, dynamic, and faithful monitoring of NF-κB transcriptional activity at the single-cell level of various cell types during inflammatory and infectious diseases.

Dendritic cell functions in vivo: A user's guide to current and next‐ generation mutant mouse models

“…While long half-lives can allow for long-term tracking similar to fate mapping [95], short half-lives are required for real-time assessment of the activation of signalling pathways in the target cells and of the resulting functions. Here, the addition of a degradation-inducing PEST sequence has been successfully used to significantly reduce the half-life of a fluorescent protein [96][97][98]. Reliability of real-time assessment of functional activities heavily relies on faithfulness of the reporter fluorescence regarding cellular specificity as well as the time course of fluorescence.…”

“…While long half‐lives can allow for long‐term tracking similar to fate mapping [95], short half‐lives are required for real‐time assessment of the activation of signalling pathways in the target cells and of the resulting functions. Here, the addition of a degradation‐inducing PEST sequence has been successfully used to significantly reduce the half‐life of a fluorescent protein [96–98].…”

Generation of reporter mice for detecting the transcriptional activity of nuclear factor of activated T cells

“…Since the significance of NFAT on various intracellular events has been indicated and novel drugs for regulating NFAT activity are being developed [2], the establishment of a simplified system for monitoring NFAT activity has been desirable. Recently, fluorescence-based reporter mice by which the activity of another transcription factor, NFκB, can be monitored at a single-cell level were generated [5]. Here we reported the generation of NFAT reporter mice useful for analyzing stimulationinduced NFAT activation in T cells.…”