The EHHADH Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population carrying targeted disruption of the human EHHADH gene. This heterogeneous pool of HEK293T-derived cells harbors loss-of-function mutations that eliminate EHHADH protein function, creating a reliable model for studying peroxisomal beta-oxidation. The polyclonal format ensures robust representation of knockout alleles while avoiding clone-specific artifacts.
HEK293T cells, a derivative of human embryonic kidney epithelial cells immortalized with SV40 large T-antigen, are widely utilized for their high transfection efficiency and robust metabolic activity. Their renal epithelial origin makes them particularly suited for investigating peroxisomal and mitochondrial functions in kidney physiology. Despite oncogenic transformation, HEK293T cells maintain active peroxisomal pathways, making them a relevant host for dissecting EHHADH-dependent metabolism.
EHHADH encodes a peroxisomal bifunctional enzyme with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities, catalyzing the second and third steps of peroxisomal fatty acid beta-oxidation. This enzyme converts trans-2-enoyl-CoA to 3-ketoacyl-CoA via a 3-hydroxyacyl-CoA intermediate, yielding NADH and acetyl-CoA. EHHADH is transcriptionally regulated by PPAR??, PPAR??, and HNF4??, placing it under the control of lipid-sensing nuclear receptors. It interacts with ACOX1 and ACAA1 within the beta-oxidation complex, and its import into peroxisomes depends on PEX5.
In HEK293T knockout cells, loss of EHHADH disrupts peroxisomal beta-oxidation, recapitulating metabolic defects observed in peroxisomal disorders and renal Fanconi syndrome. The deficiency leads to accumulation of very-long-chain and medium-chain fatty acids, impaired NADH production, and altered PPAR-driven transcriptional programs. This model allows researchers to dissect kidney-specific metabolic adaptations and explore the interplay between peroxisomal function and cellular energetics in an epithelial context.
These cells are ideal for palmitate oxidation assays, metabolomic profiling, and peroxisomal enzyme activity measurements. Complementing these are RT-qPCR and Western blotting for expression analysis, immunofluorescence for organelle integrity, and NADH/NAD+ ratio determination. The knockout model also facilitates drug screening for PPAR??/?? agonists and investigations into metabolic syndrome. For further technical details, please contact Ascent Research.
