The ACAD11 Knockout HT29 Polyclonal Cells consist of a CRISPR/Cas9-edited polyclonal population derived from the HT29 colorectal adenocarcinoma cell line, serving as a loss-of-function model for the ACAD11 gene. ACAD11 encodes a mitochondrial acyl-CoA dehydrogenase essential for the initial dehydrogenation step in long-chain fatty acid beta-oxidation. The polyclonal nature of these cells, generated through CRISPR/Cas9-mediated gene disruption, provides a genetically diverse pool that reflects population-level metabolic responses without clonal selection artifacts, making it well-suited for bulk metabolic assays.
HT29 is a widely utilized human colorectal adenocarcinoma cell line with epithelial morphology and established genetic alterations in APC, TP53, and KRAS, conferring a metabolic profile characteristic of aggressive cancer metabolism. These cells engage both glycolytic and oxidative pathways, enabling dissection of how fatty acid oxidation contributes to colorectal cancer bioenergetics and anabolic demands. The HT29 background is extensively validated for studies of intestinal epithelial biology, drug transport, and metabolic adaptation, offering a robust platform for targeted gene knockout experiments.
ACAD11 resides in the mitochondrial matrix where it initiates the beta-oxidation cycle of long-chain acyl-CoA esters, working in concert with electron transfer flavoproteins ETFA, ETFB, and ETFDH. Transcription of ACAD11 is positively regulated by the fatty acid-sensing nuclear receptor PPARA and its coactivator PPARGC1A, aligning its expression with metabolic demands. Functional disruption of ACAD11 by CRISPR/Cas9 impairs fatty acid-derived ATP production, elevates long-chain acylcarnitine species, and disturbs cellular redox balance, linking this gene to broader mitochondrial oxidative pathways involving ACADVL, HADHA, and HADHB.
In the context of HT29 colorectal cancer cells, ACAD11 knockout creates a metabolic vulnerability that illuminates the role of long-chain fatty acid oxidation in sustaining tumor cell energy metabolism. Colorectal tumors often rewire lipid utilization to support proliferation; thus, the ACAD11-deficient model allows investigation of how loss of this dehydrogenase impacts mitochondrial respiration, shifts substrate oxidation preferences, and alters lipid storage dynamics. The polyclonal format captures heterogeneous compensatory adaptations, providing a more physiologically relevant system for studying cancer metabolic flexibility and potential therapeutic targeting.
This knockout cell population is amenable to a range of functional assays, including RT-qPCR and Western blotting for target validation, Seahorse-based respirometry for mitochondrial function, LC-MS acylcarnitine profiling for fatty acid oxidation flux, and lipid droplet staining to assess neutral lipid accumulation. Applications include modeling mitochondrial fatty acid oxidation disorders, exploring metabolic reprogramming in colorectal cancer, and screening compounds that modulate lipid metabolism. For further details or technical assistance, please reach out to Ascent Research.
