The BTD Knockout HT29 Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout population derived from the HT29 human colorectal adenocarcinoma cell line, engineered to disrupt the BTD gene encoding biotinidase. This loss-of-function model enables investigation of biotin recycling and its metabolic consequences in a cancer-relevant epithelial background. The polyclonal format ensures heterogeneity of editing events, reflecting a varied knockout pool suitable for population-level studies of BTD deficiency.
HT29 is a widely characterized cell line originally established from a primary colorectal adenocarcinoma of a 44-year-old female patient. These epithelial cells serve as a robust in vitro model for colorectal cancer research, displaying typical adenocarcinoma features and retaining key signaling and metabolic pathways. The HT29 background provides a physiologically relevant platform to examine the intersection of biotin metabolism and colorectal tumor biology.
Biotinidase, the product of the BTD gene, catalyzes the cleavage of biotin from biocytin and biotinylated peptides, releasing free biotin for reuse in holocarboxylase synthesis by HLCS (holocarboxylase synthetase). This recycling mechanism sustains the activity of critical biotin-dependent carboxylases: acetyl-CoA carboxylase (ACC), pyruvate carboxylase (PC), propionyl-CoA carboxylase (PCC), and 3-methylcrotonyl-CoA carboxylase (MCC). These carboxylases function in essential metabolic pathways including fatty acid synthesis, gluconeogenesis, and amino acid catabolism. Consequently, BTD acts upstream of these carboxylases by maintaining the intracellular pool of biotin, a necessary cofactor for their enzymatic activity. The regulation of BTD expression is influenced by biotin availability, and its disruption can impair carboxylase-dependent metabolic flux.
In the context of colorectal cancer, the HT29 model with BTD knockout allows dissection of how biotin recycling contributes to the metabolic plasticity of tumor cells. Cancer cells often exhibit altered metabolic dependencies, and the disruption of biotin salvage pathways may sensitize them to biotin deprivation or reveal vulnerabilities in lipid synthesis and energy metabolism. This knockout system provides a valuable tool to investigate whether biotinidase deficiency limits tumor growth, alters redox balance, or affects proliferation through impaired ACC, PC, PCC, and MCC activity.
Typical research applications include western blotting and RT-qPCR to assess compensation or off-target effects, biotinidase activity assays and biotin quantification to confirm functional knockout, metabolic flux analysis to track carboxylase pathway activities, and proliferation or cell cycle assays under biotin-free or low-biotin conditions to evaluate metabolic dependency. These polyclonal knockout cells are also suited for studying mechanisms of biotinidase deficiency-related neurological symptoms. For ordering, technical support, or custom inquiries, please contact Ascent Research.
