Description

The Btk Knockout RAW 264.7 Cell Line is a CRISPR/Cas9-edited knockout cell line in which the Bruton??s tyrosine kinase (Btk) gene has been disrupted in the RAW 264.7 macrophage background. This cell-based model provides a defined loss-of-function system for investigating Btk-dependent signaling in murine monocyte/macrophage-like cells. The knockout cell line is delivered as a ready-to-use live cell product, eliminating the need for researchers to perform gene editing themselves. It enables consistent, reproducible experiments in innate immunity, inflammation, and TLR signaling without the variables introduced by transient knockdown or pharmacological inhibition.

The parental RAW 264.7 cell line is an Abelson murine leukemia virus-transformed macrophage cell line originally derived from BALB/c mice. These cells exhibit many of the functional characteristics of activated macrophages, including robust phagocytic activity, cytokine secretion in response to Toll-like receptor (TLR) stimulation, and the ability to polarize into pro-inflammatory or anti-inflammatory phenotypes. As a widely used model in immunology, RAW 264.7 cells are particularly valued for studies of pattern recognition receptor signaling, inflammasome biology, and host-pathogen interactions. Their genetic tractability and well-characterized responses to LPS and other TLR ligands make them an ideal host for targeted gene disruption.

Btk encodes a non-receptor tyrosine kinase that functions downstream of multiple immune receptors. In macrophages, Btk is activated downstream of TLR4, Fc?? receptors, and the B cell receptor?Clike signaling complex, and it phosphorylates critical effectors such as PLC??2 to initiate calcium mobilization and NF-??B activation. Btk interacts with adaptor proteins MyD88 and TIRAP, bridging TLR4 engagement to TRAF6-dependent signaling cascades. It also participates in NLRP3 inflammasome activation through physical association with NLRP3 and MyD88. Representative pathway components include TLR4, MyD88, TRAF6, NF-??B, PLC??2, PI3K, AKT, and NLRP3. Btk??s activity is regulated upstream by Src family kinases including Lyn and Syk, and it transduces signals to MAPKs and the PI3K/AKT axis, controlling pro-inflammatory gene expression and metabolic reprogramming.

Disruption of Btk in the RAW 264.7 macrophage context profoundly alters innate immune signaling. Btk-deficient macrophages exhibit impaired TLR4-induced production of pro-inflammatory cytokines such as TNF-?? and IL-6, reduced NF-??B activation, and defective NLRP3 inflammasome assembly. These functional changes are accompanied by altered phagocytic capacity and perturbed M1/M2 polarization. The Btk Knockout RAW 264.7 Cell Line thus serves as a powerful tool for dissecting the kinase??s role in macrophage effector functions and for validating Btk as a therapeutic target in inflammatory diseases. Its use facilitates investigation into the molecular mechanisms by which Btk coordinates multiple signaling branches to regulate the intensity and duration of innate immune responses.

This knockout cell line supports a wide array of experimental applications, including characterization of TLR and Fc??R signaling pathways, analysis of NLRP3 inflammasome activation by Western blotting for Btk and phospho-Btk, quantification of cytokine release via ELISA (e.g., TNF-??, IL-6), and qPCR profiling of inflammatory gene expression. Phagocytosis assays, cell viability and apoptosis studies, and drug sensitivity testing with Btk inhibitors such as ibrutinib can all be conducted with this model. It is particularly valuable for preclinical evaluation of Btk-targeted therapies in macrophage-driven inflammation and for functional studies of X-linked agammaglobulinemia?Crelated signaling defects. For more detailed information, please contact Ascent Research.