The ACACA Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from HEK293T, engineered to disrupt the endogenous ACACA gene. ACACA encodes acetyl-CoA carboxylase alpha (ACC??), the rate-limiting enzyme in de novo lipogenesis that catalyzes the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. The polyclonal format provides a heterogeneous pool of edited cells, enabling loss-of-function studies without the confounding effects of clonal selection. This product serves as a versatile tool for dissecting the roles of ACACA in metabolic regulation, signal transduction, and lipid metabolism.
HEK293T cells are a clonal derivative of the HEK293 line that stably expresses the SV40 large T antigen, allowing high-level episomal replication of plasmids containing the SV40 origin of replication. Originating from human embryonic kidney epithelial cells, HEK293T is renowned for its exceptionally high transfection efficiency, making it a preferred host for transient protein expression, viral packaging, and recombinant protein production. Its robust growth characteristics and well-documented genetic background enhance its suitability for generating knockout models, facilitating reliable and reproducible experimental outcomes in cell-based assays.
ACC??, encoded by ACACA, is a biotin-dependent enzyme that catalyzes the rate-limiting step of fatty acid synthesis: the conversion of acetyl-CoA to malonyl-CoA. Its activity is allosterically activated by citrate and inhibited via phosphorylation by AMP-activated protein kinase (AMPK); insulin signaling relieves this inhibition through protein phosphatase 2A (PP2A)-mediated dephosphorylation. Transcriptional control by sterol regulatory element-binding protein 1c (SREBP1c) further adjusts ACC?? expression in response to nutritional status. Malonyl-CoA serves not only as a substrate for fatty acid synthase (FASN) but also as a potent inhibitor of carnitine palmitoyltransferase 1 (CPT1), thereby linking lipogenesis to the regulation of mitochondrial fatty acid oxidation. This central metabolic node integrates signals from AMPK, insulin, mTOR, and citrate to coordinate lipid synthesis with cellular energy homeostasis.
In the HEK293T cellular context, ACACA disruption permits detailed analysis of de novo lipogenesis, leveraging the line’s high transfection efficiency for complementation assays and signaling analysis. The polyclonal knockout population balances target-gene disruption with maintenance of cellular heterogeneity, making it suitable for both population-level metabolic assays and single-cell based analyses. This model is particularly valuable for exploring how ACACA-mediated lipid synthesis impacts membrane biogenesis, energy storage, and lipid-derived signaling molecules in an experimentally tractable system.
This ACACA knockout model is well-suited for a broad range of biomedical research applications, including the investigation of metabolic disorders such as obesity, type 2 diabetes, non-alcoholic fatty liver disease, and metabolic syndrome, as well as the study of aberrant lipogenesis in cancer metabolism. Representative experimental approaches include Western blotting for ACACA and phospho-AMPK, RT-qPCR quantification of ACACA mRNA, 14C-acetate incorporation assays to measure fatty acid synthesis, Seahorse metabolic flux analysis, and Oil Red O staining for lipid droplet accumulation. By employing these techniques, researchers can dissect ACACA-dependent metabolic rewiring and its implications in health and disease. For additional information or custom-engineered cell products, please contact Ascent Research.
