The HIBADH Knockout HT29 Polyclonal Cells product is a polyclonal population of HT29 human colorectal adenocarcinoma cells in which the HIBADH gene has been disrupted using CRISPR/Cas9 genome editing. This polyclonal knockout cell pool provides a heterogeneous mixture of edited alleles, enabling researchers to study loss-of-function effects of 3-hydroxyisobutyrate dehydrogenase without single-cell clonal selection. The polyclonal format retains genetic diversity that may more closely mirror the heterogeneity observed in tumor populations, making it suitable for investigating the overall impact of HIBADH disruption on cancer cell metabolism.
The HT29 host cell line is a well-established epithelial cell model derived from a human colorectal adenocarcinoma. HT29 cells are widely used in cancer biology for studying intestinal epithelial biology, signal transduction, and metabolic alterations in colorectal cancer. Their ability to form polarized monolayers and differentiate under specific culture conditions makes them a versatile platform for investigating nutrient metabolism and mitochondrial function in a cancer context. The HT29 background provides a relevant system for examining how HIBADH inactivation influences colorectal cancer cell behavior.
HIBADH encodes mitochondrial 3-hydroxyisobutyrate dehydrogenase, a critical enzyme in the valine degradation pathway. This enzyme catalyzes the NAD+-dependent oxidation of 3-hydroxyisobutyrate to methylmalonate semialdehyde, which is subsequently converted by ALDH6A1 into propionyl-CoA, a precursor for succinyl-CoA entry into the tricarboxylic acid (TCA) cycle. HIBADH functions downstream of the branched-chain aminotransferase BCAT2 and the branched-chain ??-keto acid dehydrogenase (BCKDH) complex, and it is transcriptionally regulated by PPARGC1A, a master regulator of mitochondrial biogenesis. The enzyme’s activity is dependent on NAD+ availability and substrate supply. In the knockout model, disruption of HIBADH impairs this conversion, leading to accumulation of 3-hydroxyisobutyrate and reduced flux of propionyl-CoA and succinyl-CoA, which can alter TCA cycle activity and cellular energy homeostasis.
In the context of colorectal cancer, HIBADH knockout offers a valuable tool to dissect the role of branched-chain amino acid (BCAA) catabolism in tumor metabolism. Colorectal cancer cells often rewire metabolic pathways to support proliferation and survival; valine degradation may contribute to anaplerotic supply of TCA cycle intermediates. HT29 cells with HIBADH disruption can be employed to investigate how loss of this mitochondrial dehydrogenase affects metabolic reprogramming, mitochondrial respiration, and cell growth under nutrient-limited conditions. This model also facilitates exploration of potential synthetic lethal interactions with other metabolic vulnerabilities in colorectal cancer.
This polyclonal knockout product is suitable for a range of research applications, including western blotting and RT-qPCR to confirm target disruption, enzymatic activity assays to validate loss of dehydrogenase function, metabolic flux analysis using labeled valine to trace catabolic intermediates, cell proliferation assays, and mitochondrial respiration measurements with Seahorse analyzers. Researchers may use this cell model to advance understanding of metabolic disorders such as 3-hydroxyisobutyrate dehydrogenase deficiency and to identify biomarkers or therapeutic targets in colorectal cancer. For further details, technical support, or to discuss custom projects, please contact Ascent Research.
