The ACSF3 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited heterogeneous population of human embryonic kidney 293T cells featuring a targeted disruption of the ACSF3 gene. This polyclonal knockout model enables loss-of-function studies without single-cell cloning, providing a robust tool for investigating mitochondrial malonate metabolism and related disorders.
Derived from the HEK293 lineage, HEK293T cells constitutively express the SV40 large T antigen, which enhances episomal replication of plasmids carrying the SV40 origin. Renowned for high transfectability and exceptional protein production, this host is ideal for studying mitochondrial enzyme function and producing viral vectors, making it a versatile platform for metabolic and genetic research.
ACSF3 encodes a mitochondrial matrix malonyl-CoA synthetase that catalyzes the conversion of malonate to malonyl-CoA, an essential substrate for mitochondrial fatty acid synthesis (mtFAS) and subsequent lipoic acid production. ACSF3 expression is regulated by mitochondrial biogenesis regulators PPARGC1A, TFAM, and NRF1, and its activity depends on malonate availability. The malonyl-CoA product feeds into the mtFAS pathway, where it interacts with MECR, OXSM, and ACPM to generate octanoyl-ACP, the precursor for lipoic acid. Lipoic acid serves as a cofactor for lipoylation of key metabolic enzymes, including PDH, ??-KGDH, and GCSH, placing ACSF3 upstream of critical cellular redox and energy processes.
In HEK293T cells, ACSF3 disruption halts mtFAS, causing malonate and methylmalonate accumulation and impairing lipoylation of PDH, ??-KGDH, and GCSH, thereby modeling combined malonic and methylmalonic aciduria (CMAMMA). This polyclonal knockout population allows investigation of mitochondrial dysfunction without clonal bias, making it suitable for studies of metabolic reprogramming and oxidative stress. The HEK293T background??s mitochondrial reliance under defined conditions further highlights the impact of lipoic acid deficiency on TCA cycle flux and cellular respiration.
Research applications include metabolomics profiling of malonate, methylmalonate, and lipoic acid; Seahorse respiration analysis; immunofluorescence for mitochondrial morphology; and knockout confirmation by Sanger sequencing or T7E1 assay. These cells are also valuable for complementation experiments and therapeutic screening. For further details or support, please contact Ascent Research.
