The Mdh2 Knockout RAW 264.7 Cell Line is a CRISPR/Cas9-edited gene-disrupted cell line derived from the murine RAW 264.7 macrophage cell line. This product provides a stable loss-of-function model for mitochondrial malate dehydrogenase 2 (MDH2), generated through CRISPR/Cas9-mediated genomic disruption of the Mdh2 locus. The knockout cell line is supplied as a viable culture, enabling direct use in functional studies of central carbon metabolism and immune cell biology.
RAW 264.7 cells, originating from a BALB/c mouse monocyte/macrophage transformed by Abelson murine leukemia virus, serve as a widely used macrophage model. These adherent cells retain key features of innate immune cells, including phagocytic activity, cytokine secretion, and responsiveness to inflammatory stimuli. Their relevance to macrophage biology makes them suitable for investigating metabolic regulation in immune responses.
MDH2 is a mitochondrial enzyme that catalyzes the reversible oxidation of malate to oxaloacetate, utilizing NAD+ as a cofactor. As a pivotal component of the tricarboxylic acid (TCA) cycle, MDH2 maintains mitochondrial NADH/NAD+ redox balance and supports anaplerotic reactions. Its activity is regulated by upstream factors such as PGC-1??, HIF-1??, c-MYC, and the mitochondrial deacetylase SIRT3. MDH2 functions in concert with citrate synthase, aconitase 2 (ACO2), isocitrate dehydrogenase 2 (IDH2), oxoglutarate dehydrogenase (OGDH), succinyl-CoA ligase (SUCLG2), succinate dehydrogenase (SDH), and fumarate hydratase (FH), ultimately coupling with glutamic-oxaloacetic transaminase 2 (GOT2) to drive the malate-aspartate shuttle. Disruption of MDH2 leads to altered TCA cycle flux, NADH/NAD+ imbalance, reduced mitochondrial ATP production, and diminished aspartate and citrate export.
In macrophages, MDH2-dependent metabolic pathways intersect with immune effector functions. Wild-type RAW 264.7 cells undergo metabolic reprogramming upon activation, shifting from oxidative phosphorylation to glycolysis, in part through HIF-1?? and c-MYC signaling. The Mdh2 knockout disrupts mitochondrial malate oxidation, impairing NADH regeneration and aspartate biosynthesis, which can attenuate pro-inflammatory cytokine production and phagocytosis. This model allows dissection of how TCA cycle integrity influences macrophage polarization and the interplay between mitochondrial metabolism and innate immune signaling.
This knockout cell line is particularly suited for immunometabolism research, including studies on metabolic reprogramming in tumor-associated macrophages, TCA cycle dysfunction in inflammation, and drug screening targeting mitochondrial metabolism. Experimental approaches may include western blotting and RT-qPCR for expression analysis, Seahorse extracellular flux assays to measure mitochondrial respiration and glycolysis, LC-MS-based metabolomics and NADH/NAD+ quantification, as well as ELISA for cytokine detection and phagocytosis assays. The model provides a robust platform for exploring MDH2 contributions to macrophage biology. For additional information or custom requests, please contact Ascent Research.
