The Sqstm1 Knockout RAW 264.7 Cell Line is a CRISPR/Cas9-edited knockout cell line that disrupts the mouse Sqstm1 gene, creating a loss-of-function model for the multifunctional autophagy receptor and signaling scaffold p62/SQSTM1. By targeting the Sqstm1 locus, this cell line enables precise investigation of p62-dependent processes without off-target interference, serving as an essential tool for dissecting autophagy and related pathways.
The parental RAW 264.7 cell line is a well-characterized murine macrophage model originally derived from BALB/c mouse ascites tumor. These cells exhibit robust phagocytic activity and are widely employed to study innate immunity, inflammatory responses, and tissue homeostasis. Their macrophage identity makes them particularly suitable for exploring how p62 integrates autophagy with immune signaling in a physiologically relevant phagocytic context.
SQSTM1/p62 functions as a central hub connecting selective autophagy with cell signaling. Through its UBA domain, it binds ubiquitinated cargo??including protein aggregates and damaged mitochondria??and delivers them to forming autophagosomes via interaction with the LC3/GABARAP family through its LIR motif. Simultaneously, p62 acts as a signaling scaffold: it oligomerizes TRAF6 to activate the IKK complex, leading to NF-??B p65 nuclear translocation, and sequesters Keap1, thereby stabilizing the antioxidant transcription factor Nrf2. These activities are modulated by upstream stimuli such as oxidative stress, inflammatory cytokines (TNF-??, IL-1??), and Toll-like receptor ligands, and profoundly influence downstream mTORC1 activation and autophagic flux.
In RAW 264.7 macrophages, p62 is critical for coordinating phagocytosis, cytokine secretion, and the balance between pro-inflammatory and antioxidant responses. Disruption of Sqstm1 in this cell line provides a powerful model to examine how loss of p62-mediated selective autophagy and signaling deregulation contribute to macrophage dysfunction. This is particularly relevant for studying diseases where macrophages play key roles, including cancer, neurodegenerative disorders such as Alzheimer’s and Parkinson’s, Paget’s disease of bone, metabolic syndrome, and liver disease. By removing p62, researchers can dissect its specific contributions to NF-??B-driven inflammation and Nrf2-mediated cytoprotection in a cell type highly dependent on these pathways.
This knockout cell line supports a wide range of experimental applications, including mechanistic studies of autophagy flux using western blot for p62 and LC3-II, fluorescent imaging of p62 puncta and LC3 colocalization, and co-immunoprecipitation to map p62 interaction networks. It is also valuable for assessing NF-??B and Nrf2 transcriptional activity via luciferase reporters, measuring phagocytic capacity, and profiling cytokine secretion by ELISA. Furthermore, it serves as a screening platform for autophagy modulators and a disease-relevant model for investigating protein aggregate clearance. For additional information on this product and technical support, please contact Ascent Research.
