The ACAD8 Knockout HT29 Polyclonal Cells represent a CRISPR/Cas9-mediated gene-disrupted population derived from the HT29 human colorectal adenocarcinoma cell line. This polyclonal knockout pool contains a heterogeneous mixture of cells with loss-of-function mutations in the ACAD8 locus, providing a robust in vitro model for studying valine catabolism and mitochondrial fatty acid ??-oxidation without relying on single-cell clones. The knockout product is supplied as a ready-to-use, polyclonal population that retains the intrinsic genetic heterogeneity of Cas9-edited pools, enabling researchers to assess ACAD8-dependent phenotypes in a physiologically relevant context.
The parental HT29 cell line originates from a colorectal adenocarcinoma of a 44-year-old female and serves as a well-established model of the human intestinal epithelium. HT29 cells are widely used to investigate epithelial barrier function, transepithelial transport, and metabolic homeostasis in the gut. Their ability to differentiate into enterocyte-like cells under appropriate culture conditions makes them particularly suitable for studying how metabolic perturbations influence intestinal physiology.
ACAD8 encodes mitochondrial isobutyryl-CoA dehydrogenase, a flavoenzyme that catalyzes the dehydrogenation of isobutyryl-CoA to methacrylyl-CoA, a key step in the valine degradation pathway. This reaction requires electron transfer via the electron transfer flavoprotein (ETF) and ETF dehydrogenase (ETFDH) to the mitochondrial respiratory chain. ACAD8 expression is positively regulated by the transcriptional coactivators PPAR?? and PGC-1??, which orchestrate mitochondrial biogenesis and fatty acid oxidation programs. The enzymatic activity of ACAD8 directly feeds acetyl-CoA into the tricarboxylic acid (TCA) cycle, linking branched-chain amino acid catabolism to cellular energy production. Disruption of ACAD8 therefore impairs valine catabolism, leading to accumulation of isobutyryl-CoA and reduced carbon flux into the TCA cycle, with downstream effects on mitochondrial respiration.
In the context of HT29 intestinal epithelial cells, ACAD8 knockout creates a relevant model for exploring the intersection between branched-chain amino acid metabolism and gut barrier function. The HT29 line is frequently employed in studies of inflammatory bowel disease, colorectal cancer metabolism, and drug-induced gastrointestinal toxicity. Loss of ACAD8 activity in these cells can trigger metabolic stress, potentially altering epithelial integrity and proliferation. This model thus enables investigation of how mitochondrial valine catabolism supports the energetic demands of the intestinal epithelium and how its failure contributes to metabolic acidosis and organic acidurias, such as isobutyryl-CoA dehydrogenase deficiency.
This knockout product is well-suited for a variety of advanced experimental applications, including modeling metabolic disorders, probing cancer metabolism reprogramming, and screening compounds for mitochondrial toxicity. Researchers can validate the knockout by Western blotting or RT-qPCR, assess mitochondrial respiratory capacity using Seahorse flux analyzers, quantify isobutyryl-CoA accumulation via LC-MS, and measure transepithelial electrical resistance (TEER) to evaluate barrier function under metabolic stress. The polyclonal nature allows for population-level functional studies without clonal bias, making it ideal for drug metabolism and epithelial biology research. For additional technical specifications and ordering details, please contact Ascent Research.
