The ACOT8 Knockout HEK293T Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout cell population engineered to disrupt the ACOT8 gene in the HEK293T human embryonic kidney epithelial cell background. This product provides a genetically heterogeneous pool of cells carrying targeted gene disruptions, enabling loss-of-function investigations into peroxisomal lipid metabolism and associated signaling networks. The polyclonal format eliminates the need for single-cell clonal isolation and is suitable for pooled population studies, drug screening, and high-throughput assays where monoclonal homogeneity is not required.
HEK293T cells are a widely utilized human embryonic kidney epithelial cell line immortalized via the stable expression of the adenovirus 5 E1A and SV40 large T antigen. This modification confers exceptionally high transfectability and robust protein expression, making them a preferred host for lentiviral packaging, protein production, and functional genomics. Their epithelial origin and intrinsic metabolic capabilities render them a versatile platform for studying hepatocyte-like lipid metabolism and peroxisomal function, despite their non-hepatic derivation. The ACOT8 knockout in this background allows dissection of peroxisomal thioesterase activity in a well-characterized, easily manipulable cellular system.
ACOT8 encodes a peroxisomal thioesterase that terminates fatty acyl-CoA chain elongation by hydrolyzing acyl-CoAs to free fatty acids and coenzyme A, thereby sustaining peroxisomal fatty acid homeostasis. Its activity is governed by upstream regulators such as PPAR?? agonists, long-chain fatty acids, phytanic acid, and the peroxisomal import receptor PEX5, which facilitates its translocation into peroxisomes. Downstream, the liberation of free fatty acids and CoA feeds into bile acid biosynthesis and modulates the transcriptional activity of PPAR??, a master regulator of lipid metabolism. ACOT8 interacts directly with peroxisomal import machinery components PEX5 and PEX7, and functions within a network that includes ACOX1 (acyl-CoA oxidase 1), DBP (D-bifunctional protein), and the peroxisomal membrane transporter PMP70. Disruption of ACOT8 perturbs the balance of acyl-CoA substrates and products, leading to altered PPAR??-driven gene expression, impaired fatty acid degradation, and disrupted peroxisomal beta-oxidation.
In the HEK293T background, loss of ACOT8 creates a genetically engineered model to study peroxisomal dysfunction and its consequences on cellular lipid homeostasis. Although HEK293T cells are of kidney epithelial origin, they retain functional peroxisomes and engage in fatty acid oxidation and lipid signaling pathways, making them a useful surrogate for exploring hepatocyte-like metabolic processes. The ACOT8 knockout disrupts the hydrolysis of medium- and long-chain acyl-CoAs, potentially leading to accumulation of peroxisomal acyl-CoA esters and a concomitant reduction in free fatty acid pools. This metabolic imbalance is expected to attenuate PPAR?? transcriptional activity, as free fatty acids and their derivatives serve as endogenous ligands for PPAR??. Consequently, the knockout may impair expression of PPAR target genes involved in fatty acid oxidation and energy metabolism, ultimately affecting cellular proliferation and survival. This model thus enables detailed investigation of peroxisomal biology in a tractable, high-transfectability system.
This polyclonal ACOT8 knockout cell model is ideally suited for a range of experimental applications, including mechanistic studies of peroxisomal lipid metabolism, investigation of PPAR?? signaling dynamics, and functional analysis of bile acid precursor processing. Researchers can employ Western blotting or immunofluorescence to assess peroxisomal protein expression and localization, RT-qPCR to measure PPAR target gene induction, and fatty acid oxidation assays to quantify metabolic flux. LC-MS-based lipidomics can profile changes in cellular acyl-CoA species and free fatty acids, while cell proliferation assays allow evaluation of the knockout??s impact on growth in cancer-relevant contexts, particularly hepatocellular carcinoma and colorectal cancer research. For further technical details, custom requests, or bulk ordering information, please contact Ascent Research.
