IDH3B Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population engineered from the human HT29 colorectal adenocarcinoma line. This research tool features CRISPR/Cas9-mediated disruption of the IDH3B gene, creating a loss-of-function model to investigate the role of the mitochondrial isocitrate dehydrogenase beta subunit in cancer cell metabolism. The polyclonal nature ensures representation of diverse editing events, facilitating robust functional studies without clonal selection artifacts.
The HT29 cell line, originally isolated from a primary colorectal adenocarcinoma, is widely employed as a model for colon cancer biology. Under appropriate culture conditions, HT29 cells can differentiate into polarized intestinal enterocyte-like cells, making them valuable for studying intestinal epithelial barrier function, drug absorption, and transporter-mediated efflux. Their tumorigenic properties and well-characterized signaling networks further support their use in anticancer drug screening and metabolic reprogramming research.
IDH3B encodes the beta subunit of the NAD-dependent isocitrate dehydrogenase complex, which catalyzes the oxidative decarboxylation of isocitrate to ??-ketoglutarate within the mitochondrial TCA cycle, generating NADH for oxidative phosphorylation. IDH3B functions within a heterotetrameric complex alongside IDH3A and IDH3G, and its activity is transcriptionally regulated by factors such as PGC-1??, c-MYC, and HIF-1?? downstream of mTOR and NRF1 signaling. Disruption of IDH3B impairs TCA cycle flux, reducing NADH and ??-ketoglutarate production, which subsequently alters ATP synthesis, AMPK activation, and mitochondrial reactive oxygen species (ROS) levels, potentially triggering apoptosis via cytochrome c release and caspase-3 activation.
In the HT29 colorectal cancer context, IDH3B knockout provides a physiologically relevant platform to dissect metabolic adaptations in tumor cells. Loss of IDH3B forces a shift from mitochondrial respiration to aerobic glycolysis and glutaminolysis, mimicking metabolic reprogramming observed in aggressive colon cancers. This model thus enables investigation of how TCA cycle dysfunction influences tumor growth, survival under nutrient stress, and sensitivity to metabolic inhibitors, offering insights into potential therapeutic vulnerabilities.
Researchers can employ these polyclonal knockout cells in diverse assays, including Western blotting and RT-qPCR for knockout validation, LC?MS-based TCA metabolite profiling, NADH/NAD+ ratio measurements, Seahorse metabolic flux analysis to quantify oxygen consumption and extracellular acidification rates, and cell proliferation or apoptosis assays using MTT and Annexin V staining. The model supports applications such as mitochondrial metabolism studies in colorectal cancer, identification of metabolic vulnerabilities, and validation of drug targets for metabolic therapy. For technical support or ordering information, please contact Ascent Research.
