Cybb Knockout RAW 264.7 Cell Line

Cybb Knockout RAW 264.7 Cell Line is a CRISPR/Cas9-edited knockout cell line derived from the mouse RAW 264.7 macrophage line, engineered for targeted disruption of the Cybb gene. This genetic modification eliminates functional expression of gp91phox (NOX2), the catalytic subunit of phagocyte NADPH oxidase. The cell line provides a defined loss-of-function model for investigating NOX2-dependent processes without confounding variables from pharmacological inhibition.

RAW 264.7 is a widely used mouse leukemic monocyte/macrophage cell line capable of phagocytosis, cytokine production, and antigen presentation, key features of innate immune responses. These cells respond to Toll-like receptor ligands, Fc receptor engagement, and inflammatory cytokines, making them suitable for studying macrophage signaling pathways. The cell line’s robustness and well-characterized responses to stimuli such as LPS and IFN-?? facilitate reproducible experimental systems.

Cybb encodes gp91phox, the catalytic core of the NOX2 enzyme complex. This complex, assembled with regulatory subunits including p22phox (Cyba), p47phox (Ncf1), p67phox (Ncf2), p40phox (Ncf4), and Rac GTPases, catalyzes the transfer of electrons from NADPH to molecular oxygen, generating superoxide anion. Subsequent dismutation yields hydrogen peroxide, which can be further converted by myeloperoxidase to hypochlorous acid. Upstream activators such as IFN-??, GM-CSF, LPS, TNF-??, and phorbol esters trigger complex assembly and enzyme activation via pathways involving TLR4, Fc?? receptors, and protein kinase C. Downstream effects include activation of redox-sensitive transcription factors NF-??B and AP-1, and production of pro-inflammatory cytokines. Thus, Cybb knockout disrupts both the direct microbicidal respiratory burst and redox-dependent signal transduction.

In RAW 264.7 macrophages, Cybb disruption models X-linked chronic granulomatous disease (CGD), characterized by defective superoxide production and impaired killing of bacteria and fungi. This cell line enables dissection of NOX2 contributions to phagocytosis, intracellular killing, and cytokine responses without the compensatory adaptations seen in whole-organism knockouts. It also allows investigation of non-phagosomal ROS signaling roles of NOX2 in macrophage polarization and inflammatory gene expression.

Typical uses include functional analysis of NOX2 in macrophage biology, host-pathogen interaction studies with bacterial and fungal pathogens, screening of NADPH oxidase inhibitors, and examination of ROS-dependent signaling pathways. Representative assays compatible with this model include luminol-enhanced chemiluminescence for total ROS, DHE fluorescence for superoxide, flow cytometric measurement of respiratory burst using dihydrorhodamine 123, western blotting for gp91phox, phagocytosis and bacterial killing assays, cytokine ELISAs, and NF-??B luciferase reporter gene assays. For further technical details or to request this product, please contact Ascent Research.