The ACOT8 Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-engineered polyclonal knockout cell population derived from the HT29 human colorectal adenocarcinoma line, featuring targeted disruption of the ACOT8 gene encoding peroxisomal acyl-CoA thioesterase 8. This loss-of-function model is specifically designed for studying peroxisomal lipid metabolism and its implications in colorectal cancer, providing a mixed genetic background that avoids clonal artifacts in downstream functional assays.
The parental HT29 cell line originates from a moderately differentiated colorectal adenocarcinoma and is characterized by adherent growth and enterocytic differentiation. As a widely utilized intestinal epithelial model, HT29 cells enable investigation of colon cancer biology, including metabolic reprogramming, proliferation, and drug responses, making them an ideal host for interrogating lipid-related pathways in a disease-relevant setting.
ACOT8 encodes a peroxisomal acyl-CoA thioesterase that hydrolyzes medium- and long-chain fatty acyl-CoAs to free fatty acids and coenzyme A (CoA), thereby regulating peroxisomal beta-oxidation and cellular CoA homeostasis. Its expression is transcriptionally activated by PPAR?? and PPAR?? in response to fatty acids like oleic acid. Downstream, the liberated free fatty acids and CoA modulate the activity of acyl-CoA synthetases (ACSL) and the expression of fatty acid-binding protein 1 (FABP1). ACOT8 interacts with peroxisomal biogenesis factor PEX19 and other thioesterase family members, embedding it within the peroxisomal beta-oxidation machinery that includes acyl-CoA oxidase 1 (ACOX1), L-bifunctional protein (EHHADH), and peroxisomal thiolase (ACAA1), ultimately producing acetyl-CoA and shortened acyl-CoAs. Through these interactions, ACOT8 critically influences lipid homeostasis and energy metabolism signaling.
In the HT29 colorectal adenocarcinoma context, lipid metabolism is frequently reprogrammed to sustain malignant proliferation. ACOT8 disruption in these cells provides a direct means to evaluate the contribution of peroxisomal fatty acid processing to cancer cell metabolic fitness. The knockout is anticipated to perturb beta-oxidation flux, alter free fatty acid and CoA pools, and impact lipid droplet dynamics, thereby offering insights into the intersection of peroxisomal dysfunction and colorectal cancer progression, which is particularly relevant given the links between metabolic syndrome, peroxisomal disorders, and intestinal lipid dysregulation.
This ACOT8 knockout polyclonal cell population is ideally suited for a broad array of research applications, including peroxisomal beta-oxidation assays using radiolabeled palmitate, LC-MS-based acyl-CoA profiling, and BODIPY lipid droplet staining to monitor neutral lipid storage. It can be employed for cell proliferation assays, drug screening with PPAR agonists or other metabolic modulators, lipidomic profiling, and immunofluorescence analysis of peroxisomal markers. Western blotting and RT?qPCR further enable validation of ACOT8 ablation and assessment of downstream targets such as FABP1. These applications position the model as a versatile platform for investigating peroxisomal and colorectal cancer lipid metabolism. For further information, please contact Ascent Research.
