The L2HGDH Knockout HT29 Polyclonal Cells represent a CRISPR/Cas9-mediated gene-disrupted polyclonal population of the HT29 human colorectal adenocarcinoma cell line, engineered for loss-of-function studies of L-2-hydroxyglutarate dehydrogenase (L2HGDH). By disrupting the L2HGDH locus, this polyclonal knockout model abolishes functional enzyme expression, enabling the investigation of downstream consequences of L-2-hydroxyglutarate (L-2HG) accumulation without the confounding effects of clonal variation inherent in single-cell-derived lines.
The parental HT29 cell line is a well-established adherent epithelial model of human colorectal adenocarcinoma characterized by endogenous mutations in the tumor suppressors TP53 and APC. These genetic alterations recapitulate key features of colorectal cancer pathogenesis, including constitutive Wnt pathway activation and impaired DNA damage responses. HT29 cells are widely employed to study intestinal epithelial cell biology, oncogenic signaling pathways, and therapeutic responses, providing a physiologically relevant platform for investigating metabolic enzyme deficiencies in a cancer context.
L2HGDH encodes a mitochondrial FAD-dependent dehydrogenase that catalyzes the oxidation of the oncometabolite L-2HG to alpha-ketoglutarate (??-KG). Loss of L2HGDH function leads to intracellular L-2HG accumulation, which competitively inhibits ??-KG-dependent dioxygenases, including the DNA demethylase TET2 and the JmjC-domain histone demethylases KDM4A and KDM6A. This inhibition induces global alterations in DNA methylation and histone modification landscapes. Furthermore, L2HGDH expression is regulated by hypoxia through HIF1A-mediated transcriptional repression, linking metabolic state to epigenetic remodeling and hypoxic signaling networks.
In the HT29 colorectal cancer background, L2HGDH loss exacerbates L-2HG?Cmediated epigenetic dysregulation superimposed on the pre-existing oncogenic landscape driven by APC and TP53 mutations. The interplay between elevated L-2HG and aberrant Wnt/??-catenin signaling may further promote malignant phenotypes, offering a powerful system to study how oncometabolites synergize with classical oncogenic pathways. This model is particularly suited for exploring mechanisms by which metabolic reprogramming influences colorectal tumorigenesis, including effects on cell proliferation, differentiation, and metastatic potential.
This polyclonal knockout cell population is suitable for a broad spectrum of mechanistic and phenotypic investigations. Key applications include cancer metabolism analysis via LC-MS/MS quantification of L-2HG, DNA methylation profiling using reduced representation bisulfite sequencing, and chromatin immunoprecipitation sequencing to assess histone modification changes. Functional assays such as cell proliferation, Wnt reporter assays, metabolic flux analysis, and migration/invasion assays enable direct assessment of oncogenic behaviors. The model also supports exploration of the hypoxia?CHIF1A axis and screening of small-molecule inhibitors targeting L-2HG?Cdriven pathways. For additional technical information, please contact Ascent Research.
