The ALKBH2 Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-mediated polyclonal knockout cell population featuring targeted disruption of the ALKBH2 gene in the HT29 human colorectal adenocarcinoma cell line. This loss-of-function model is designed to support investigations into DNA alkylation repair mechanisms and cellular responses to genotoxic stress. The polyclonal nature of the knockout population ensures representation of diverse editing events, providing a robust tool for functional studies without the clonal selection biases inherent in single-cell-derived lines.
The HT29 cell line is derived from a 44-year-old female with colorectal adenocarcinoma and is widely used as a model of colorectal cancer. These cells exhibit epithelial morphology and retain properties relevant to barrier function and differentiation research. Critically, HT29 cells harbor mutations in the tumor suppressor APC, TP53, and oncogenic KRAS, recapitulating common genetic alterations in colorectal tumors. This genetic landscape makes HT29 a valuable system for studying the interplay between DNA repair defects and cancer progression.
ALKBH2 encodes a dioxygenase that catalyzes iron(II)- and 2-oxoglutarate-dependent oxidative demethylation of 1-methyladenine and 3-methylcytosine in DNA, directly reversing alkylation damage. ALKBH2 functions downstream of DNA damage signaling, with its expression transcriptionally regulated by FOXM1 and activated by ATM/ATR-mediated pathways in response to genotoxic stress. The protein interacts with proliferating cell nuclear antigen (PCNA) and collaborates with ALKBH3 and other repair factors to maintain genomic stability. Its enzymatic activity prevents alkylation-induced mutagenesis, thereby protecting cells from carcinogenic transformation.
Disruption of ALKBH2 in HT29 cells impairs the direct reversal of alkylated DNA adducts, leading to increased genomic instability and hypersensitivity to alkylating agents such as methyl methanesulfonate (MMS). Given the background mutations in APC, TP53, and KRAS, this knockout model accentuates the phenotypic consequences of defective DNA repair in a colorectal cancer context. It provides a relevant platform for dissecting the interplay between alkylation damage accumulation and tumor cell survival, and for evaluating therapeutic strategies targeting DNA repair vulnerabilities in colorectal adenocarcinoma.
Researchers can utilize these ALKBH2 knockout polyclonal cells in a variety of assays including alkylation sensitivity testing via MMS treatment, comet assays for DNA strand breaks, immunofluorescence for ??-H2AX foci as a marker of double-strand breaks, and colony formation assays to assess cell survival. They are also suitable for mutational signature analysis and biomarker discovery related to alkylation-induced carcinogenesis. This model supports drug sensitivity screening and mechanistic studies of base excision repair and direct damage reversal pathways. For additional information, please contact Ascent Research.
