The Acod1 Knockout RAW 264.7 Cell Line is a CRISPR/Cas9-edited knockout cell line derived from the mouse macrophage cell line RAW 264.7, featuring targeted disruption of the Acod1 gene. This product serves as a loss-of-function model for investigating the biological functions of aconitate decarboxylase 1 and the immunometabolic roles of itaconate.
The RAW 264.7 cell line, established from an Abelson murine leukemia virus-induced tumor in a BALB/c mouse, is a widely used macrophage model that exhibits robust phagocytic activity, cytokine secretion, and responsiveness to Toll-like receptor ligands such as lipopolysaccharide (LPS). These cells recapitulate many aspects of primary macrophage biology, including activation of NF-??B signaling, production of inflammatory cytokines, and metabolic reprogramming, making them a suitable host for studying innate immune mechanisms and itaconate-mediated regulation.
Acod1 catalyzes the decarboxylation of cis-aconitate to produce itaconate, a metabolite that accumulates in activated macrophages and exerts potent anti-inflammatory effects. Upstream, Acod1 expression is induced by LPS/TLR4 signaling via NF-??B, IRF1, and STAT1 transcription factors, as well as by TNF-?? and IFN-??. Downstream, itaconate covalently modifies KEAP1 at critical cysteine residues, leading to NRF2 stabilization and activation of antioxidant gene programs, including HO-1 and NQO1. Itaconate also inhibits succinate dehydrogenase (SDH), limiting mitochondrial reactive oxygen species production, and alkylates GAPDH to suppress glycolysis, while simultaneously blocking NLRP3 inflammasome assembly, thereby reducing caspase-1 activation and IL-1?? secretion.
In the RAW 264.7 macrophage context, Acod1 knockout eliminates itaconate synthesis, enabling precise dissection of its role in dampening inflammatory responses. This deficiency disrupts the typical TCA cycle rewiring that occurs upon immune activation and abolishes itaconate-dependent feedback inhibition of the NLRP3 inflammasome and oxidative stress. Consequently, the knockout cell line is particularly valuable for studying the interplay between metabolism and inflammation, including mechanisms of sepsis, inflammatory bowel disease, rheumatoid arthritis, and macrophage responses to bacterial or viral pathogens.
Researchers can employ the Acod1 Knockout RAW 264.7 Cell Line in a variety of experimental paradigms, including Western blotting and RT-qPCR for signaling protein and cytokine analysis, ELISA for secreted cytokines, immunofluorescence for NF-??B or NRF2 localization, and flow cytometry for surface macrophage markers. Metabolic investigations benefit from liquid chromatography-mass spectrometry (LC-MS) for itaconate quantification, Seahorse flux analysis for mitochondrial respiration, and ROS measurements. Additionally, functional assays such as co-immunoprecipitation of KEAP1-NRF2 interactions, NRF2 reporter assays, bacterial killing assays, and NLRP3 inflammasome activation assays can be performed. This knockout cell line supports drug screening for anti-inflammatory agents and detailed immunometabolic studies. For further information, please contact Ascent Research.
