This product consists of a CRISPR/Cas9-edited polyclonal knockout cell population targeting EHHADH in Huh-7 cells. The knockout abolishes enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity, the peroxisomal enzyme responsible for the second and third steps of long-chain 2-methyl-branched fatty acid and bile acid intermediate degradation. The polyclonal nature ensures a heterogeneous knockout pool suitable for functional genomics and metabolic assays.
Huh-7 is a human hepatocellular carcinoma cell line derived from a well-differentiated tumor in a 57-year-old Japanese male. These cells maintain hepatocyte-like features, including active peroxisomal beta-oxidation and the capacity to host hepatitis C virus replication, making them a pertinent model for studying hepatic peroxisomal metabolism.
EHHADH catalyzes the hydration and dehydrogenation of substrates such as pristanic acid, DHCA, and THCA, generating acetyl-CoA and propionyl-CoA. Its expression is regulated by PPAR??, RXR??, and PGC-1?? under fasting or high-fat diet conditions. The enzyme interacts with PEX5 and SCP-2 for peroxisomal import and substrate presentation. Knockout of EHHADH leads to accumulation of pristanic acid and C27-bile acid intermediates, impairing lipid metabolism and energy homeostasis, and perturbing mitochondrial fatty acid oxidation through altered acetyl-CoA and NADH pools.
Loss of EHHADH in Huh-7 cells models L-bifunctional enzyme deficiency, recapitulating elevated pristanic acid and bile acid precursor levels. In the hepatocellular carcinoma background, this knockout also allows exploration of the crosstalk between peroxisomal dysfunction and cancer metabolism, including effects on cell proliferation and stress signaling.
Key applications include functional analysis of peroxisomal beta-oxidation via [14C]-pristanic acid assays, LC-MS/MS metabolomic profiling of bile acid intermediates, metabolic flux analyses with Seahorse technology, and drug screening for peroxisomal disorder therapies. The cell population is also suited for studying lipid metabolism in hepatocarcinoma. For additional details, please contact Ascent Research.
