The AMACR Knockout HT29 Polyclonal Cells product comprises a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human colorectal adenocarcinoma cell line HT29, designed for loss-of-function studies of the AMACR gene. This heterogeneous population of gene-disrupted cells enables researchers to interrogate the functional consequences of AMACR ablation without clonal selection artifacts. The product serves as a robust model for investigating peroxisomal lipid metabolism and cancer biology.
The HT29 cell line, established from a primary colon adenocarcinoma, retains enterocytic differentiation capacity and is widely employed as a model of intestinal epithelium. These cells exhibit polarized morphology, mucus production, and expression of brush-border enzymes under appropriate culture conditions, making them particularly suitable for studying colorectal carcinogenesis and epithelial biology. HT29 cells harbor mutations in APC and TP53, which activate Wnt/??-catenin signaling, and they are responsive to metabolic and oncogenic stimuli commonly dysregulated in colorectal cancers.
AMACR encodes ??-methylacyl-CoA racemase, a peroxisomal enzyme that catalyzes the conversion of (2R)-methyl-branched-chain fatty acyl-CoAs to their (2S)-stereoisomers, an essential epimerization step required for subsequent ??-oxidation by peroxisomal acyl-CoA oxidases. This reaction generates propionyl-CoA and acetyl-CoA, feeding into central carbon metabolism and bile acid biosynthesis. AMACR expression is positively regulated by PPAR signaling, androgen receptor, and ??-catenin/TCF transcriptional complexes, linking its activity to oncogenic pathways frequently activated in colorectal and prostate cancers. The enzyme interacts with peroxisomal matrix proteins, including the PEX5 receptor that mediates its import into peroxisomes, and cooperates with D-bifunctional protein and 3-ketoacyl-CoA thiolase within the classic peroxisomal ??-oxidation spiral. Disruption of AMACR blocks racemization and impedes peroxisomal breakdown of branched-chain fatty acids, leading to accumulation of abnormal acylcarnitines and metabolic stress.
In the HT29 colorectal adenocarcinoma background, AMACR knockout models the metabolic vulnerability of colon cancer cells that rely on peroxisomal lipid metabolism for energy production and biomass synthesis. Given that HT29 cells exhibit high ??-catenin/TCF transcriptional activity, the loss of AMACR uncouples a downstream effector from this oncogenic signaling axis, providing a clean system to dissect the contribution of branched-chain fatty acid oxidation to tumor cell fitness. This polyclonal knockout pool captures the phenotypic heterogeneity of AMACR-disrupted cells, enabling robust evaluation of metabolic adaptations and therapeutic sensitivities without clonal bias.
The AMACR Knockout HT29 Polyclonal Cells product is ideally suited for a range of targeted investigations, including characterization of peroxisomal metabolism via fatty acid oxidation assays, acylcarnitine profiling by mass spectrometry, and catalase immunofluorescence to assess peroxisomal integrity. Researchers can employ cell viability assays under lipid-rich or nutrient-depleted conditions to uncover metabolic dependencies and screen for compounds that selectively target AMACR-deficient cancer cells. Additionally, these cells facilitate mechanistic studies of bile acid synthesis intermediates and the role of peroxisomal oxidation in colorectal cancer progression using colony formation assays and gene expression analysis by RT-qPCR and Western blot. For further information or custom cell engineering requests, please contact Ascent Research.
