ACSF3 Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population in which the ACSF3 gene has been disrupted to create a loss-of-function model. This heterogeneous pool avoids clonal artifacts and is suited for bulk functional assays, enabling robust investigation of ACSF3-dependent mitochondrial processes without clonal selection bias.
The HT29 host cell line is a human colorectal adenocarcinoma epithelial line derived from a 44-year-old female. These cells exhibit an epithelial morphology, produce mucin, and perform absorptive and secretory functions, serving as a well-established intestinal epithelial model. Their metabolic adaptability and extensive use in cancer biology make HT29 cells an ideal background for studying mitochondrial fatty acid synthesis and lipoylation.
ACSF3 encodes a mitochondrial acyl-CoA synthetase that activates fatty acids for de novo mitochondrial fatty acid synthesis, generating the octanoyl substrate required for lipoic acid biosynthesis. ACSF3 is regulated by PPARGC1A (PGC-1??), NRF1, and PPARA in response to mitochondrial stress. It functions within the mitochondrial fatty acid synthase complex alongside MCAT, OXSM, MECR, and ACP. The octanoyl product is transferred to LIAS, which catalyzes lipoic acid assembly and its covalent attachment to key mitochondrial enzymes, including DLAT, DBT, and GCSH. This lipoylation is essential for TCA cycle and branched-chain amino acid catabolism. Disruption of ACSF3 abolishes this cascade, leading to loss of protein lipoylation and subsequent metabolic dysfunction.
In the HT29 colorectal cancer context, ACSF3 knockout recapitulates metabolic features of combined malonic and methylmalonic aciduria (CMAMMA), such as accumulation of malonate and methylmalonate and impaired mitochondrial respiration. This model enables exploration of how defects in mitochondrial fatty acid synthesis affect cancer cell metabolism, proliferation, and response to metabolic stress. It also provides a relevant platform to dissect the intersection between lipoic acid metabolism and colorectal cancer pathophysiology.
This polyclonal knockout cell product supports a wide range of assays, including western blotting for lipoylated proteins (DLAT, DBT, GCSH), RT-qPCR for ACSF3 expression, Seahorse mitochondrial respiration analysis, LC-MS metabolomics for malonate and methylmalonate, immunofluorescence for mitochondrial morphology, and cell proliferation assays. Research applications include disease modeling of CMAMMA, investigation of mitochondrial lipoylation in cancer, drug screening for mitochondrial disorders, and detailed analysis of TCA cycle and branched-chain amino acid metabolism. For additional information, please contact Ascent Research.
