The ECI1 Knockout HEK293T Polyclonal Cells are a carefully designed CRISPR/Cas9-edited polyclonal knockout cell population that disrupts the ECI1 gene in a human embryonic kidney HEK293T background. This polyclonal format provides a genetically heterogeneous pool of cells harboring targeted ECI1 disruptions, enabling robust loss-of-function studies without clonal selection bias. The knockout model serves as a versatile platform for investigating mitochondrial enoyl-CoA isomerase function in unsaturated fatty acid ??-oxidation and broader lipid metabolism pathways.
HEK293T cells are a widely used host line derived from human embryonic kidney cells, immortalized by transformation with adenovirus type 5 DNA and constitutively expressing the SV40 large T-antigen. These epithelial cells are favored for their exceptionally high transfection efficiency and robust capacity for transient protein expression and viral vector production. Their well-characterized biology and ease of genetic manipulation make them an ideal chassis for generating gene-edited models, particularly for studying metabolic pathways where the parental line exhibits basal fatty acid oxidation activity.
ECI1 encodes a mitochondrial enoyl-CoA isomerase that catalyzes the isomerization of 3-cis or 3-trans ??3-enoyl-CoA intermediates to 2-trans-enoyl-CoA, an essential step in the ??-oxidation of unsaturated fatty acids. ECI1 functions downstream of key regulators including PPAR?? and PGC-1??, and is responsive to long-chain fatty acids and nutritional cues. It interacts transiently with enoyl-CoA hydratase (ECHS1) and operates within the mitochondrial ??-oxidation enzyme network, which includes carnitine palmitoyltransferases (CPT1/CPT2), acyl-CoA dehydrogenases, HADH, ACAA2, and DECR1. Disruption of ECI1 blocks this isomerization, leading to accumulation of ??3-enoyl-CoA species and impaired production of acetyl-CoA and ATP from unsaturated fats.
In the HEK293T context, ECI1 knockout creates a well-defined model for dissecting the cellular consequences of impaired unsaturated fatty acid catabolism. Loss of ECI1 function uncouples the degradation of unsaturated fatty acids from energy production, potentially triggering lipid overload, altered mitochondrial respiration, and metabolic reprogramming. This system is particularly suited to probing mitochondrial stress responses, lipid droplet dynamics, and the interplay between fatty acid oxidation and other pathways. Unlike primary cells, HEK293T offers the advantages of scalability and genetic tractability, enabling high-throughput approaches.
This polyclonal knockout model is applicable in diverse experimental settings, including acylcarnitine profiling via LC-MS, Seahorse respirometry with unsaturated fatty acid substrates, and fluorescence-based quantification of lipid droplets using BODIPY staining. It supports studies in metabolic disorders, mitochondrial dysfunction, fatty acid oxidation deficiencies, and cancer metabolism, and can be employed in drug screening campaigns targeting metabolic liabilities. Standard validation assays such as RT-qPCR, western blot, ATP quantification, and ROS measurement further characterize the knockout phenotype. For additional details or tailored applications, please contact Ascent Research.
