The ALDH2 Knockout HT29 Polyclonal Cells product consists of a CRISPR/Cas9-edited population of human HT29 colorectal adenocarcinoma cells carrying a targeted disruption of the ALDH2 gene. This polyclonal knockout model preserves the inherent genetic and phenotypic heterogeneity of the parental line, enabling robust loss-of-function investigations without the clonal selection biases associated with single-cell-derived knockouts. The edited pool provides a versatile platform for studying the role of the mitochondrial aldehyde dehydrogenase ALDH2 in colon cancer biology, ethanol metabolism, and responses to cytotoxic aldehydes.
HT29 cells are a human colon adenocarcinoma line widely used to model intestinal epithelial barrier function. They harbor mutations in APC, TP53, and BRAF V600E, and retain the ability to differentiate into enterocyte-like cells. These cells form polarized monolayers with measurable transepithelial electrical resistance (TEER), facilitating studies of barrier integrity, carcinogenesis, and drug response. Their inherent heterogeneity enables investigation of cancer cell subpopulations and differentiation states.
ALDH2 is a mitochondrial enzyme that catalyzes NAD+-dependent oxidation of acetaldehyde, 4-hydroxynonenal (4-HNE), and malondialdehyde. Its expression is transcriptionally regulated by NRF2, while its activity depends on NAD+ and is modulated by PKC??-mediated phosphorylation. ALDH2 functions downstream of alcohol dehydrogenase (ADH) and CYP2E1, converting acetaldehyde to acetyl-CoA, and interacts with HSP70 and HSP60 for mitochondrial import. By clearing reactive aldehydes, ALDH2 limits protein carbonylation, suppresses NF-??B activation, and maintains AMPK/mTOR homeostasis, thereby reducing mitochondrial ROS. The KEAP1?CNRF2?CALDH2 axis is a critical antioxidant defense pathway.
In the HT29 background, ALDH2 knockout provides a relevant model for ethanol-related colorectal carcinogenesis and aldehyde toxicity. The BRAF V600E, mutant TP53, and APC defects create a permissive context for aldehyde-induced DNA damage and genomic instability. Researchers can study how ALDH2 loss affects enterocytic differentiation, cancer stem cell properties, and redox signaling. HT29 cells also form tight junctions, enabling investigation of barrier dysfunction under ethanol or nitroglycerin stress, where ALDH2 is required for nitroglycerin bioactivation.
This polyclonal knockout model supports a wide range of assays, including acetaldehyde degradation kinetics, MitoSOX-based mitochondrial ROS measurement, and DNA damage markers. ALDH2 enzymatic activity, Western blotting, and RT-qPCR confirm target disruption, while downstream signaling effects on NF-??B, AMPK, and mTOR are assessed after 4-HNE challenge. Functional applications encompass colony formation, wound healing, alkaline phosphatase differentiation assays, and TEER-based barrier integrity tests with ethanol or nitroglycerin. Drug metabolism and resistance studies are also feasible. For technical inquiries, contact Ascent Research.
