Chmp4b Knockout RAW 264.7 Cell Line

The Chmp4b Knockout RAW 264.7 Cell Line is a CRISPR/Cas9-edited knockout cell line designed for loss-of-function studies of the Chmp4b gene. This model utilizes the RAW 264.7 mouse macrophage cell line, engineered to disrupt expression of CHMP4B, a core component of the endosomal sorting complexes required for transport (ESCRT)-III machinery. The disruption is achieved via CRISPR/Cas9-mediated gene targeting, providing a stable and heritable knockout background for investigating ESCRT-dependent cellular processes. This cell line serves as a powerful tool for researchers studying membrane dynamics, autophagy, and immune cell function.

The parental RAW 264.7 cell line is a widely used mouse macrophage model derived from BALB/c mice. These cells exhibit key characteristics of professional phagocytes, including robust phagocytic activity, cytokine production, and responsiveness to inflammatory stimuli. As a macrophage line, RAW 264.7 cells are integral to studies of innate immunity, inflammation, and tissue homeostasis. Their genetic tractability and well-characterized signaling pathways make them an ideal host for gene disruption studies, particularly those involving intracellular trafficking and immune effector functions.

CHMP4B functions as a core subunit of the ESCRT-III complex, which polymerizes into helical filaments to drive membrane scission in diverse contexts such as multivesicular body (MVB) formation, autophagosome closure, and cytokinesis. The protein interacts with other ESCRT-III subunits including CHMP2A, CHMP3, and IST1, and is regulated by the AAA-ATPase VPS4, which disassembles ESCRT-III polymers, and by upstream factors such as ALIX and CC2D1A. Downstream, CHMP4B-mediated membrane remodeling is essential for intraluminal vesicle generation, autophagic flux, and the final abscission step of cell division. Thus, Chmp4b knockout disrupts these critical membrane remodeling events.

In the context of RAW 264.7 macrophages, loss of CHMP4B profoundly impacts ESCRT-dependent processes that are vital for immune function. Macrophages rely on ESCRT machinery for phagolysosomal maturation, cytokine secretion, and autophagic clearance of intracellular pathogens. Chmp4b disruption is expected to impair MVB biogenesis, alter autophagy, and potentially affect membrane repair mechanisms, thereby modulating inflammatory responses and antigen presentation. This knockout model therefore allows dissection of the specific contribution of ESCRT-III to macrophage biology and pathology.

This cell line is suitable for a range of advanced research applications, including the study of ESCRT-mediated processes in immune cells, autophagy regulation, and mechanisms of viral budding and neurodegenerative disease modeling. Researchers can employ assays such as Western blotting to confirm CHMP4B depletion, immunofluorescence and live-cell imaging to monitor membrane dynamics, LC3 puncta analysis to assess autophagy, and flow cytometry to evaluate phagocytosis and cytokine production. The Chmp4b Knockout RAW 264.7 Cell Line thus provides a valuable resource for exploring the intersection of membrane trafficking and innate immunity. For more details, please contact Ascent Research.