Open Access Green as soon as Postprint is submitted to ZB.
Methods to study CARD11-BCL10-MALT1 dependent canonical NF-κB activation in jurkat T cells.
In: NF-κB Transcription Factors. Berlin [u.a.]: Springer, 2021. 125-143 (Methods Mol. Biol. ; 2366)
Jurkat T cells have been of central importance for the discovery of signalling mediators driving NF-κB activation in response to T cell antigen receptor (TCR)/CD28 co-stimulation. The critical function of the key regulators identified in Jurkat T cells has subsequently been verified in primary murine and human T cells. CRISPR/Cas9-mediated genomic editing techniques in combination with viral reconstitution are powerful tools that now enable the investigation of the exact molecular mechanisms that govern T cell signalling, especially the impact of protein-protein interactions, protein modifications, or cancer-associated gain- or loss-of-function mutations. As exemplified by the CARD11 gene encoding a key regulator of NF-κB signalling in T cells, we describe here the detailed workflow for the generation of CRISPR/Cas9 knockout (KO) Jurkat T cells and the subsequent reconstitution using a lentiviral transduction protocol. In addition, we explain the use of a stable NF-κB-dependent EGFP reporter system that enables a reliable quantification of NF-κB transcriptional activation in the reconstituted KO Jurkat T cells.
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Publication type Article: Edited volume or book chapter
Keywords Bcl10 ; Card11 ; Carma1 ; Cbm Complex ; Crispr/cas9 ; Egfp ; Jurkat ; Knockout ; Malt1 ; Nf-κb ; Reconstitution ; Reporter ; Signal Transduction ; T Cell Activation ; Transduction ; Viral Infection
ISSN (print) / ISBN 1064-3745
Book Volume Title NF-κB Transcription Factors
Journal Methods in Molecular Biology
Quellenangaben Volume: 2366, Pages: 125-143
Publishing Place Berlin [u.a.]
Reviewing status Peer reviewed