The ACADS Knockout HT29 Polyclonal Cells provide a ready-to-use CRISPR/Cas9-mediated gene-disrupted polyclonal population in the HT29 human colorectal adenocarcinoma cell line, designed for loss-of-function studies of the ACADS gene. This polyclonal knockout product contains a heterogeneous pool of cells harboring ACADS-targeting edits, enabling robust and reproducible modeling of short-chain acyl-CoA dehydrogenase deficiency in an intestinal epithelial context.
HT29 cells were originally established from a primary colorectal adenocarcinoma of a 44-year-old female patient and are widely utilized as an intestinal epithelial model of colorectal cancer. This adherent cell line retains characteristics of enterocytic differentiation and tumorigenic potential, and is extensively applied in studies of nutrient metabolism, mitochondrial function, and oncogenic signaling. The HT29 background provides a clinically relevant platform for dissecting the role of fatty acid oxidation in colorectal adenocarcinoma biology.
The ACADS gene produces mitochondrial short-chain acyl-CoA dehydrogenase (SCAD), which catalyzes the initial ??,??-dehydrogenation of C4?CC6 acyl-CoAs such as butyryl-CoA and hexanoyl-CoA in the fatty acid ??-oxidation pathway. This FAD-dependent reaction yields trans-2-enoyl-CoA and FADH2; electrons are transferred via electron transfer flavoprotein (ETF) to the respiratory chain, supporting ATP synthesis and preventing toxic acylcarnitine accumulation. ACADS transcription is regulated by PPAR?? and PGC-1?? in response to nutritional cues, including fasting/glucagon-mediated activation and insulin/feeding suppression, with cAMP signaling modulating expression. Downstream, acetyl-CoA, NADH, and FADH2 feed into the TCA cycle and oxidative phosphorylation. SCAD functions in concert with enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase, while the carnitine shuttle (CPT1, CPT2, translocase) ensures mitochondrial substrate import.
In the colorectal adenocarcinoma context, ACADS disruption impairs the oxidation of short-chain fatty acids, such as butyrate, which is a significant energy source for colonocytes and a known modulator of tumor cell behavior. HT29 cells are capable of utilizing both glycolysis and oxidative phosphorylation; therefore, ACADS knockout cells enable dissection of metabolic reprogramming and unveil dependencies on fatty acid ??-oxidation for tumor survival under nutrient-limited conditions. This model is particularly relevant for investigating links between SCAD deficiency and colorectal cancer pathogenesis, as well as for evaluating metabolic liabilities associated with mitochondrial fatty acid oxidation disorders.
Typical experimental workflows include quantification of ACADS protein and mRNA by Western blotting and RT-qPCR, metabolic profiling of short-chain acylcarnitines by LC-MS/MS, and functional assessment of fatty acid oxidation rates using radioactive tracers or Seahorse extracellular flux analysis. Complementary phenotypic assays, such as MTT/CCK8 proliferation, colony formation, and ATP measurement, allow comprehensive evaluation of the metabolic consequences of ACADS loss. These polyclonal knockout cells are also suitable for drug metabolism and toxicity screens targeting fatty acid oxidation pathways. For further information or custom cell engineering services, please contact Ascent Research.
