The ACAA2 Knockout HEK293T Polyclonal Cells consist of a CRISPR/Cas9-edited polyclonal knockout cell population with targeted disruption of the ACAA2 gene. Derived from the HEK293T human embryonic kidney line, this product provides a heterogeneous pool of cells carrying ACAA2 inactivation, enabling loss-of-function studies of mitochondrial ??-oxidation.
HEK293T cells are a widely used adherent line expressing the SV40 large T antigen, which enhances episomal replication and transfection efficiency. They serve as a versatile platform for recombinant protein production, viral packaging, and transient gene expression, making them ideal for genetic knockout applications.
ACAA2 encodes mitochondrial 3-ketoacyl-CoA thiolase, the enzyme responsible for the thiolytic cleavage of 3-ketoacyl-CoA to produce acetyl-CoA and a chain-shortened acyl-CoA, the terminal step of mitochondrial fatty acid ??-oxidation. This reaction is essential for energy production, ketone body generation, and the degradation of branched-chain amino acids such as valine, leucine, and isoleucine. ACAA2 is transcriptionally regulated by PPAR?? and PGC-1??, post-translationally modulated by AMPK, and induced during fasting or high-fat dietary intake. The thiolase cooperates closely with VLCAD, the mitochondrial trifunctional protein (including LCHAD), and requires NAD+ and CoA-SH as cofactors. It operates within a pathway network that includes HADHA, HADHB, ECHS1, EHHADH, and the peroxisomal ACAA1. Downstream, acetyl-CoA fuels the TCA cycle and serves as a precursor for ketone bodies, linking fatty acid oxidation to central carbon metabolism.
Within HEK293T cells, ACAA2 knockout is anticipated to impair mitochondrial ??-oxidation, causing a metabolic shift toward enhanced glycolysis to meet energy demands. This alteration may trigger changes in intracellular lipid accumulation, mitochondrial reactive oxygen species levels, and ATP homeostasis, mirroring metabolic phenotypes observed in cancer and inborn errors of fatty acid oxidation. The polyclonal nature of the knockout population, combined with the high transfectability of HEK293T, allows for rapid integration of reporter constructs or rescue experiments, making it a practical model for dissecting ACAA2-dependent metabolic adaptations and screening for modulators of lipid metabolism.
Key applications include measuring fatty acid oxidation flux via radiolabeled palmitate tracing, quantifying acetyl-CoA and ATP levels, assessing mitochondrial respiration with Seahorse analysis, and performing untargeted metabolomics and lipidomics. The cells are suitable for investigating disorders of mitochondrial fatty acid oxidation, metabolic reprogramming in tumor biology, and the pharmacological targeting of lipid catabolism. Typical validation includes western blotting for ACAA2 and RT-qPCR for downstream metabolic targets. For inquiries regarding this product, please contact Ascent Research.
