The HACD3 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population derived from HEK293T human embryonic kidney cells, designed to disrupt the HACD3 gene. This heterogeneous loss-of-function model allows study of HACD3??s role in very long-chain fatty acid metabolism without clonal selection, ensuring robust and reproducible experiments.
HEK293T cells are a widely used human embryonic kidney epithelial line that stably expresses the SV40 large T antigen, which promotes episomal plasmid replication and high transient protein expression. Their ease of transfection, rapid growth, and well-characterized proteome make them ideal for generating gene-edited derivatives for biochemical and cell biology research, including lipid metabolism studies.
HACD3 (3-hydroxyacyl-CoA dehydratase 3) is an integral component of the endoplasmic reticulum-associated very long-chain fatty acid (VLCFA) elongation cycle. It catalyzes dehydration of 3-hydroxyacyl-CoA to trans-2-enoyl-CoA, a step essential for VLCFA synthesis (??C22). HACD3 functions downstream of ELOVL elongases (ELOVL1-7) and cooperates with HACD family members (HACD1-4), KAR reductases, and TER trans-2,3-enoyl-CoA reductases. Its expression is regulated by SREBP transcription factors and PPAR agonists, integrating signals from lipid availability and mTOR signaling. Disruption of HACD3 impairs production of VLCFA-derived lipids like ceramides and sphingolipids, affecting membrane integrity and cell signaling. HACD3 also interacts with TER complex components, underscoring its central role in the elongation machinery.
In HEK293T cells, HACD3 knockout provides a model to study defective VLCFA synthesis and its downstream effects. This system enables investigation of altered lipid profiles on membrane organization, protein trafficking, and ER stress responses. Loss of HACD3 likely leads to accumulation of 3-hydroxyacyl-CoA intermediates and reduced VLCFA-derived lipids, which can be monitored via lipidomics and cell viability assays. Additionally, the model facilitates exploration of HACD3??s role in neurodegenerative disorders where VLCFA metabolism is perturbed.
Applications include functional dissection of the elongation cycle using radioactive substrate assays, lipidomic profiling of VLCFAs and sphingolipids, Western blotting and RT-qPCR to assess HACD3 depletion, drug screening for metabolic disease modulators, and investigation of mTOR-SREBP-lipid homeostasis crosstalk. For further details, please contact Ascent Research.
