The HADHA Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population derived from HT29 colorectal adenocarcinoma cells, engineered to disrupt the HADHA gene. This heterogeneous model enables investigation of long-chain fatty acid oxidation (FAO) deficiency in an intestinal epithelial context. The polyclonal nature preserves genetic diversity of the edited pool, allowing robust loss-of-function studies without clonal artifacts.
HT29 cells originate from a primary colorectal adenocarcinoma of a 44-year-old female and serve as a widely used model for intestinal epithelial function and colorectal cancer. These adherent epithelial cells retain features such as polarity and tight junction formation, and exhibit aberrant Wnt/??-catenin signaling. The HT29 background provides a disease-relevant platform to study metabolic adaptation in colorectal tumor microenvironments.
HADHA encodes the alpha subunit of mitochondrial trifunctional protein (MTP), which heterotrimerizes with HADHB to catalyze hydration of long-chain 2-enoyl-CoA and dehydrogenation of 3-hydroxyacyl-CoA in FAO. HADHA expression is regulated by PPARA and PPARGC1A, and its activity is modulated by AMPK and SIRT1 under energy stress. The MTP complex operates with CPT1A, CPT2, SLC25A20, and ACADVL for fatty acid import and initial oxidation, and with ETF/ETFDH to transfer electrons to the respiratory chain. Disruption thus impairs NADH, FADH?, and acetyl-CoA production, compromising ATP synthesis, TCA cycle, and ketogenesis.
In HT29 colon cancer cells, HADHA knockout disrupts mitochondrial long-chain FAO, causing energy deficiency and accumulation of toxic intermediates like acylcarnitines. This metabolic stress forces rewiring toward glycolysis or glutaminolysis, making the model valuable for studying metabolic reprogramming and context-specific vulnerabilities such as sensitivity to mitochondrial stress. Additionally, it allows assessment of how FAO deficiency impacts intestinal epithelial differentiation, barrier function, and apoptotic responses to dietary lipids.
Applications include 3H-palmitate FAO assays, Seahorse respirometry, acylcarnitine profiling by LC-MS, ATP measurement, and cell viability under metabolic stress. The model is suitable for drug screening for mitochondrial fatty acid oxidation disorders. Complementary analyses encompass immunoblotting, RT-qPCR, apoptosis upon palmitate treatment, and migration/invasion assays. For further information, please contact Ascent Research.
