EHHADH Knockout SK-HEP-1 Polyclonal Cells are a CRISPR/Cas9-engineered polyclonal knockout cell population with targeted disruption of the human EHHADH gene. This polyclonal pool, generated using CRISPR/Cas9-mediated gene disruption, contains a heterogeneous collection of edited alleles, providing a robust loss-of-function model without the biases of monoclonal selection. The disruption abolishes expression of the L-bifunctional protein, a key peroxisomal enzyme, enabling researchers to interrogate peroxisomal fatty acid beta-oxidation and lipid metabolism in a physiologically relevant hepatic endothelial background.
The SK-HEP-1 host cell line is an adherent epithelial-like cell type originally isolated from the ascites of a patient with liver adenocarcinoma. Despite its tumor origin, SK-HEP-1 cells exhibit a stable endothelial phenotype and are widely used as a model for liver sinusoidal endothelial cells (LSECs). These cells recapitulate key aspects of the liver sinusoidal barrier, including transcellular transport, metabolic processing, and responses to inflammatory mediators, making them a relevant platform for studying hepatic endothelial biology and liver-specific metabolic pathways.
EHHADH encodes the peroxisomal L-bifunctional protein, which possesses enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities, catalyzing the second and third steps of peroxisomal fatty acid beta-oxidation. This enzyme converts enoyl-CoA esters into 3-ketoacyl-CoA intermediates, facilitating the breakdown of very long-chain fatty acids. The EHHADH gene is transcriptionally regulated by PPAR??, a nuclear receptor activated by fibrate drugs and endogenous ligands, positioning it within the PPAR signaling network. Within the peroxisomal matrix, the L-bifunctional protein functions in concert with factors such as ACOX1 (acyl-CoA oxidase 1) upstream and SCPx (sterol carrier protein X) and ACAA1 (acetyl-CoA acyltransferase 1) downstream. Its import into peroxisomes depends on the PEX5 and PEX7 receptors, which recognize its peroxisomal targeting signals. Disruption of EHHADH therefore impairs the peroxisomal beta-oxidation machinery, leading to accumulation of unmetabolized fatty acids and metabolic stress.
In the context of SK-HEP-1 hepatic endothelial-like cells, loss of EHHADH provides a relevant model to study lipid handling and peroxisomal function in liver sinusoidal endothelium. LSECs are exposed to high concentrations of fatty acids from the portal blood and play an active role in lipid metabolism and detoxification. EHHADH knockout in this cellular background can dissect the contribution of peroxisomal beta-oxidation to endothelial metabolic homeostasis, oxidative stress responses, and the maintenance of barrier integrity. This model also offers a platform to examine how impaired peroxisomal function influences LSEC phenotype in metabolic liver diseases and conditions such as Fanconi syndrome, where peroxisomal fatty acid oxidation defects are implicated.
This polyclonal knockout cell pool is well-suited for functional assays including western blotting and RT-qPCR to confirm gene disruption, immunofluorescence to assess peroxisomal morphology and EHHADH localization, and lipid droplet staining to visualize intracellular lipid accumulation. Metabolic phenotyping can be performed using peroxisomal beta-oxidation assays and fatty acid oxidation measurements to quantify the consequences of EHHADH loss. These cells are particularly valuable for drug metabolism studies exploring peroxisome-mediated activation or detoxification of therapeutic agents. For additional details and technical support, please contact Ascent Research.
