The HADHA Knockout HEK293T Polyclonal Cells product delivers a CRISPR/Cas9-edited polyclonal knockout cell population with targeted loss of HADHA function. This knockout model disrupts the gene encoding the alpha subunit of the mitochondrial trifunctional protein (MTP), a key enzyme in long-chain fatty acid beta-oxidation. The polyclonal format provides a heterogeneous population of gene-edited cells, ensuring robust and representative biological responses without the artifacts of clonal selection.
The HEK293T host cell line is a derivative of human embryonic kidney 293 cells, stably expressing the SV40 large T antigen. This antigen permits episomal replication of plasmids carrying the SV40 origin, making these cells exceptionally efficient for transient transfection and lentivirus production. As adherent epithelial cells, HEK293T cells exhibit rapid growth and high transfection efficiency, simplifying the introduction of additional genetic perturbations or reporter constructs for detailed metabolic studies.
HADHA encodes the ??-subunit of MTP, which catalyzes hydration, dehydrogenation, and thiolysis in long-chain fatty acid ??-oxidation as a complex with the ??-subunit HADHB. Its transcription is regulated by PPARA and PPARD via PGC-1??, while AMPK and SIRT1 modulate activity in response to energy status. Knockout ablates acetyl-CoA and NADH production, causing accumulation of long-chain acylcarnitines. The enzyme operates downstream of CPT1A, CPT2, and ACADVL, and precedes ECHS1 and ACAA2 in the ??-oxidation spiral.
In HEK293T cells, HADHA disruption completely abolishes mitochondrial long-chain fatty acid oxidation, forcing a metabolic shift toward glucose and glutamine utilization. This results in reduced acetyl-CoA levels, impaired oxidative phosphorylation, and compromised ATP production, particularly pronounced under glucose-restricted conditions that unmask the reliance on fatty acid-derived energy. The consequent accumulation of long-chain acylcarnitines and lipid intermediates can induce lipotoxic stress and mitochondrial dysfunction, phenocopying key features of human mitochondrial trifunctional protein deficiency, a disorder associated with hypoglycemia, cardiomyopathy, and acute fatty liver of pregnancy.
These polyclonal knockout cells are suited for modeling mitochondrial trifunctional protein deficiency, exploring PPAR signaling, and developing therapies for related metabolic disorders. Standard validation employs Western blotting and immunofluorescence, while functional analyses rely on radiolabeled fatty acid oxidation flux assays, acylcarnitine profiling, and Seahorse respirometry. Further metabolic characterization includes ATP measurement, qPCR, and viability testing under glucose restriction. For additional technical information or custom cell engineering, please contact Ascent Research.
