IVD Knouckout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed for loss-of-function studies of isovaleryl-CoA dehydrogenase (IVD) in a human colorectal adenocarcinoma background. Generated via CRISPR/Cas9-mediated gene disruption, this polyclonal product provides a heterogeneous pool of IVD-null HT29 cells, eliminating the need for single-cell cloning while maintaining parental genetic diversity. This format is particularly suited for functional assays where population-averaged readouts are informative, such as metabolic profiling and proliferation analyses. These cells are suitable for a wide range of in vitro assays, enabling robust investigation of leucine catabolism and mitochondrial function.
The HT29 host cell line is a well-established human colorectal adenocarcinoma model with epithelial morphology, originally derived from a primary tumor of a female patient. These cells exhibit a moderately differentiated phenotype and can undergo enterocytic differentiation in response to metabolic cues or pharmacological agents, such as sodium butyrate. HT29 cells are extensively employed in cancer biology to investigate tumorigenesis, apoptosis, chemoresistance, and metabolic reprogramming. Their robust mitochondrial activity and relevance to intestinal epithelial physiology make them an ideal host for interrogating the metabolic consequences of IVD deficiency in a cancer-relevant context.
IVD encodes isovaleryl-CoA dehydrogenase, a mitochondrial flavoenzyme that catalyzes the FAD-dependent oxidation of isovaleryl-CoA to 3-methylcrotonyl-CoA in the third step of the leucine degradation pathway. This reaction is integral to branched-chain amino acid catabolism and feeds electrons into the ETF-ETF dehydrogenase system, linking isovaleryl-CoA oxidation to mitochondrial ATP generation. The IVD protein interacts directly with FAD and electron transfer flavoprotein, and its activity is post-translationally regulated by the NAD+-dependent deacetylase SIRT3. Transcriptional control of IVD is mediated by PPARGC1A (PGC-1??). Downstream, 3-methylcrotonyl-CoA carboxylase converts 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA, yielding acetyl-CoA and TCA cycle intermediates. Inactivating IVD disrupts this metabolic cascade, leading to accumulation of isovaleryl-CoA and isovaleric acid, which are characteristic of isovaleric acidemia.
In the HT29 colorectal adenocarcinoma system, IVD knockout serves as a physiologically relevant model for studying the interplay between leucine catabolism and cancer cell bioenergetics. Colorectal cancer cells often display altered mitochondrial metabolism, and IVD loss may exacerbate reliance on alternative substrates, sensitizing cells to nutrient stress. This model recapitulates features of isovaleric acidemia, including impaired leucine oxidation and accumulation of toxic metabolites. By leveraging the epithelial origin and mitochondrial competence of HT29 cells, researchers can investigate how IVD deficiency impacts respiration, redox homeostasis, and proliferation, providing insights into metabolic disorders and cancer vulnerabilities.
This polyclonal knockout product supports metabolomic profiling via LC-MS to quantify isovaleric acid, mitochondrial respiration assays (Seahorse), ATP measurement, and cell proliferation assays. Western blotting and RT-qPCR confirm IVD disruption, while enzyme activity assays directly assess residual function. These applications support detailed investigations into branched-chain amino acid metabolism, mitochondrial dysfunction, and the metabolic adaptations of colorectal cancer cells, making this product valuable for both inborn error of metabolism research and oncology. For additional product details, please contact Ascent Research.
