ACADSB Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from HT29 colorectal adenocarcinoma cells. This tool enables loss-of-function studies of ACADSB, the gene encoding short/branched-chain specific acyl-CoA dehydrogenase. The polyclonal pool contains a heterogeneous mix of gene disruptions generated by CRISPR/Cas9, providing a robust population-level knockout model without clonal selection. Disruption of ACADSB abolishes the enzyme’s catalytic activity, suitable for investigating metabolic pathways and mitochondrial biology.
HT29 is a well-characterized human colorectal adenocarcinoma cell line with epithelial morphology and known oncogenic mutations in APC and TP53. These cells are widely used as a model for colon cancer, displaying typical features of colorectal tumors including dysregulated Wnt and p53 signaling. The epithelial nature and tumorigenic properties make HT29 an ideal host for studying the interplay between branched-chain amino acid metabolism and cancer cell biology.
ACADSB encodes a mitochondrial flavoenzyme that catalyzes the alpha,beta-dehydrogenation of short/branched-chain acyl-CoA esters, using FAD as a cofactor and transferring electrons to electron transfer flavoprotein (ETF). It functions in the isoleucine degradation pathway, converting 2-methylbutyryl-CoA to tiglyl-CoA, and participates in fatty acid beta-oxidation. ACADSB is regulated by transcription factors PPARA, PPARG, PPARGC1A, and HNF4A, and responds to fasting and glucagon signaling. The enzyme couples to the electron transport chain via ETF, ETFDH, and ubiquinone, transferring electrons to Complex III. Thus, ACADSB sits at the intersection of amino acid catabolism and oxidative phosphorylation; its knockout disrupts electron flux.
In the HT29 colorectal cancer context, ACADSB knockout creates a metabolic liability by impairing degradation of branched-chain amino acids, leading to accumulation of acylcarnitines and 2-methylbutyrylglycine. Since cancer cells often rewire metabolism, this model reveals dependencies on mitochondrial fatty acid oxidation and amino acid utilization for energy and biosynthesis. Loss of ACADSB activity may also trigger stress responses and alter redox balance, increasing susceptibility to metabolic inhibitors. Studying this polyclonal population enables assessment of bulk metabolic reprogramming without clonal variation.
This knockout model is suited for investigating branched-chain amino acid metabolism in colorectal cancer, mitochondrial fatty acid oxidation, and energy homeostasis. It can be employed in metabolic profiling using LC-MS-based acylcarnitine analysis, cellular respiration assays (Seahorse), and viability assays for metabolic inhibitor testing. Additionally, it models short/branched-chain acyl-CoA dehydrogenase deficiency (2-methylbutyrylglycinuria) and aids drug sensitivity screening targeting metabolic vulnerabilities. Common analytical methods include Western blotting for ACADSB, RT-qPCR, colony formation, and apoptosis assays. For more information, please contact Ascent Research.
