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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.

SUBMITTER: Tortola L

PROVIDER: S-EPMC9259492 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Sensing of pathogens by innate immune cells is essential for the initiation of appropriate immune responses. Toll-like receptors (TLRs), which are highly sensitive for various structurally and evolutionary conserved molecules derived from microbes have a prominent role in this process. TLR engagement results in the activation of the transcription factor NF-κB, which induces the expression of cytokines and other inflammatory mediators. The exquisite sensitivity of TLR signalling can be exploited for the detection of bacteria and microbial contaminants in tissue cultures and in protein preparations. Here we describe a cellular reporter system for the detection of TLR ligands in biological samples. The well-characterized human monocytic THP-1 cell line was chosen as host for an NF-ᴋB-inducible enhanced green fluorescent protein reporter gene. We studied the sensitivity of the resultant reporter cells for a variety of microbial components and observed a strong reactivity towards TLR1/2 and TLR2/6 ligands. Mycoplasma lipoproteins are potent TLR2/6 agonists and we demonstrate that our reporter cells can be used as reliable and robust detection system for mycoplasma contaminations in cell cultures. In addition, a TLR4-sensitive subline of our reporters was engineered, and probed with recombinant proteins expressed in different host systems. Bacterially expressed but not mammalian expressed proteins induced strong reporter activity. We also tested proteins expressed in an E. coli strain engineered to lack TLR4 agonists. Such preparations also induced reporter activation in THP-1 cells highlighting the importance of testing recombinant protein preparations for microbial contaminations beyond endotoxins. Our results demonstrate the usefulness of monocytic reporter cells for high-throughput screening for microbial contaminations in diverse biological samples, including tissue culture supernatants and recombinant protein preparations. Fluorescent reporter assays can be measured on standard flow cytometers and in contrast to established detection methods, like luciferase-based systems or Limulus Amebocyte Lysate tests, they do not require costly reagents.

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

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