The ECHDC3 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the HAP1 human near-haploid chronic myelogenous leukemia (CML) cell line, designed to disrupt the ECHDC3 gene encoding mitochondrial enoyl-CoA hydratase. This polyclonal pool provides a robust loss-of-function model for investigating the role of ECHDC3 in fatty acid ??-oxidation and lipid metabolism, without the clonal heterogeneity often associated with single-cell-derived lines.
HAP1 cells originate from a male CML patient and are characterized by a near-haploid karyotype, with disomy only for chromosome 15, and are BCR-ABL1 positive. Their fibroblast-like adherent morphology and haploid genetic background facilitate complete gene disruption via CRISPR/Cas9, minimizing the likelihood of residual wild-type alleles and enabling unambiguous genotype-phenotype correlations in functional studies.
ECHDC3 encodes a mitochondrial enoyl-CoA hydratase that catalyzes the hydration of trans-2-enoyl-CoA to 3-hydroxyacyl-CoA, a critical step in the ??-oxidation of unsaturated fatty acids. This enzyme functions within the mitochondrial trifunctional protein complex, interacting with HADHA and HADHB, and is integrated into the fatty acid ??-oxidation pathway alongside CPT1, CPT2, ACADVL, and ACAA2. ECHDC3 expression is regulated by upstream factors including PPAR??, PPAR??, PGC-1??, and AMPK, and its activity yields downstream products such as 3-hydroxyacyl-CoA, acetyl-CoA, and ATP, while influencing lipid droplet dynamics. Disruption of ECHDC3 impairs unsaturated fatty acid catabolism, leading to potential accumulation of lipid intermediates and altered mitochondrial respiration.
The knockout of ECHDC3 in the HAP1 background creates a powerful model for dissecting mitochondrial lipid metabolism in the context of leukemia-like cells with a functional BCR-ABL1 fusion kinase. The near-haploid genome ensures that the CRISPR/Cas9-mediated disruption effectively abolishes ECHDC3 function across the polyclonal population, enabling studies of how impaired unsaturated fatty acid oxidation affects cellular energy homeostasis, lipid storage, and oxidative stress. This model is particularly relevant for investigating metabolic adaptations in cancer cells and the pathogenesis of insulin resistance, non-alcoholic fatty liver disease (NAFLD), and metabolic syndrome, where ECHDC3-mediated ??-oxidation plays a crucial role.
Researchers can employ these polyclonal knockout cells for a wide range of applications, including metabolic flux analysis using labeled fatty acids, Seahorse metabolic flux analysis, and lipidomic profiling to quantify acyl-CoA intermediates and lipid droplets. Compatible with Western blotting, RT-qPCR, immunofluorescence, Oil Red O staining, ATP assays, and targeted metabolomics, they are ideal for functional genomics and drug screening targeting metabolic pathways. For further details, contact Ascent Research.
