Fiche publication
Date publication
mars 2026
Journal
Methods in cell biology
Auteurs
Membres identifiés du Cancéropôle Est :
Dr MICHEAU Olivier
,
Pr PLENCHETTE Stéphanie
,
Pr CHLUBA Johanna
Tous les auteurs :
Radoua A, Alrustom B, Wang J, Bordessoules M, Chluba J, Plenchette S, Micheau O
Lien Pubmed
Résumé
The generation of isogenic knockout (KO) cell lines for intracellular proteins using non-viral CRISPR-Cas9 approaches has long been technically demanding and time-consuming. Here, we describe a streamlined and cost-effective method based on ptARgenOM, an all-in-one mammalian expression vector designed for efficient delivery of the CRISPR-Cas9 system. This vector co-expresses the guide RNA (gRNA) and Cas9 endonuclease, which is fused to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein (EGFP) and the puromycin N-acetyltransferase. This design enables transient, expression-dependent antibiotic selection and fluorescence-based enrichment of successfully transfected cells, facilitating the rapid generation of isogenic KO populations or clones. The method is particularly well-suited, though not limited, to functional studies involving intracellular components of the cell death machinery, including both the extrinsic and intrinsic apoptotic signaling pathways. We illustrate the utility of this system by targeting and deleting FADD, Caspase-8, and RIPK1. This approach can be easily adapted to any intracellular target protein, offering a robust platform for gene function analysis in mammalian cells.
Mots clés
CRISPR-Cas9, Cell death, Cell sorting, GFP, Gene editing, Gene function analysis, Isogenic knockout, Non-viral, Plasmid, Transient expression
Référence
Methods Cell Biol. 2026 03 9;208:115-147
