The HADH Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population featuring disruption of the HADH gene, which encodes mitochondrial short-chain 3-hydroxyacyl-CoA dehydrogenase. As a pooled population of edited cells, this product provides a versatile loss-of-function model for studying HADH-dependent fatty acid oxidation and its metabolic consequences without the need for clonal isolation.
HEK293T cells, derived from human embryonic kidney cells, constitutively express the SV40 large T antigen and are widely used for transient protein expression and viral packaging. Despite their non-hepatic origin, HEK293T cells possess functional mitochondrial ??-oxidation machinery and express key enzymes including HADH, making them a suitable host for metabolic studies when coupled with gene knockout. Their robust growth and transfectability facilitate multiplexed genetic and metabolic assays.
HADH catalyzes the NAD+-dependent oxidation of L-3-hydroxyacyl-CoA to 3-ketoacyl-CoA in the mitochondrial matrix, a critical step in short-chain fatty acid ??-oxidation. This reaction is integrated within a broader metabolic network involving upstream regulators PPARA/PGC-1?? and insulin, interacting factors HADHB and electron transfer flavoprotein, and pathway partners ACADVL, HADHA, HADHB, CPT1A, and ACADM. The generated NADH and acetyl-CoA fuel the TCA cycle and oxidative phosphorylation, linking HADH activity to cellular energy status. Disruption of HADH impairs this oxidation step, leading to altered acylcarnitine profiles and redox imbalances that can affect insulin secretion and energy homeostasis.
In the HEK293T background, HADH knockout enables systematic investigation of mitochondrial short-chain fatty acid oxidation and its impact on insulin signaling. The loss-of-function model can be used to examine how ??-oxidation defects influence NADH/NAD+ ratios, acetyl-CoA supply, and TCA cycle flux. Additionally, the HEK293T platform supports complementation experiments to validate target-specific phenotypes, providing a tractable system for dissecting the roles of HADH in metabolic regulation.
This polyclonal knockout cell population is applicable to research on fatty acid oxidation disorders, inherited 3-hydroxyacyl-CoA dehydrogenase deficiency, hyperinsulinemic hypoglycemia, and insulin resistance. Typical assays include western blotting for HADH expression, enzyme activity measurements, acylcarnitine profiling by mass spectrometry, RT-qPCR of metabolic genes, and fatty acid oxidation flux analysis using radiolabeled or stable isotope tracers. The model also lends itself to small-molecule screening for metabolic modulators. For additional product information, custom modifications, or technical support, please contact Ascent Research.
