CRISPR/Cas9-mediated gene disruption at the ECI2 locus in Huh-7 human hepatocellular carcinoma cells produces a polyclonal knockout population designed for investigating mitochondrial fatty acid oxidation. This heterogeneous pool of edited cells enables population-level study of ECI2 loss-of-function without clonal selection, preserving genetic diversity while enabling robust interrogation of metabolic phenotypes.
The host cell line Huh-7, established in 1982 from a well-differentiated hepatocellular carcinoma of a 57-year-old Japanese male, retains hepatitis B virus DNA sequences. Huh-7 cells are widely used for studying hepatic metabolism, viral hepatitis, and liver cancer biology, providing a physiologically relevant human hepatocyte-derived model for dissecting lipid and energy homeostasis.
ECI2 encodes mitochondrial enoyl-CoA delta isomerase 2, which catalyzes the isomerization of 3-cis-??5-enoyl-CoA to 2-trans-??4-enoyl-CoA, a critical step in unsaturated fatty acid beta-oxidation. ECI2 expression is regulated by PPAR??, PPARGC1A, and SIRT1, and is responsive to nutritional cues such as fasting and high-fat diet. The isomerase functions within the mitochondrial matrix, acting downstream of the carnitine shuttle (CPT1A, CPT2, SLC25A20) and in coordination with acyl-CoA dehydrogenases, enoyl-CoA hydratases, and the trifunctional protein (HADHA/HADHB). Together with ACADL, ECHS1, ACAA2, and HADH, ECI2 drives the production of acetyl-CoA, ATP, and maintenance of mitochondrial respiration. Its disruption impairs unsaturated fatty acid degradation, leading to accumulation of long-chain acylcarnitines and reduced metabolic flux.
In the Huh-7 hepatocellular carcinoma context, ECI2 knockout specifically blocks unsaturated fatty acid utilization, allowing dissection of substrate-dependent metabolic dependencies in liver cancer. This defect is relevant to hepatic steatosis, metabolic syndrome, and hepatocellular carcinoma progression, as it may uncover compensatory pathways and reveal vulnerabilities associated with lipid metabolic reprogramming.
Typical research applications include studying mitochondrial fatty acid oxidation defects, modeling fatty acid oxidation disorders, and screening for modulators of hepatic lipid metabolism. Validation of ECI2 knockout by western blotting and RT-qPCR is recommended, followed by functional assays such as cellular oxygen consumption rate (Seahorse assay), acylcarnitine profiling by LC-MS/MS, and fatty acid oxidation flux using radiolabeled oleate or palmitate. Additional phenotyping may include lipid droplet staining (Oil Red O, BODIPY), cell proliferation, and apoptosis assays, complemented by RNA-seq for global metabolic pathway analysis. This polyclonal knockout product serves as a versatile tool for investigating mitochondrial beta-oxidation biology in a human liver cancer background. For further details, please contact Ascent Research.
