The ATP6V0A2 Knockout HT29 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population with targeted disruption of the ATP6V0A2 gene. This polyclonal format provides a heterogeneous mixture of gene-edited cells, reducing clonal selection artifacts. The model is designed for investigating the role of the vacuolar ATPase (V-ATPase) a2 subunit in lysosomal and endosomal acidification. The CRISPR/Cas9-mediated gene disruption offers a dependable loss-of-function tool.
The host HT29 cell line is a human colon adenocarcinoma epithelial model originally derived from a primary tumor of a 44-year-old female patient. HT29 cells are extensively utilized in colorectal cancer studies due to their retention of epithelial features and ability to recapitulate proliferation, differentiation, and drug responses. This background provides a physiologically relevant context for dissecting V-ATPase contributions to colorectal cancer pathology.
ATP6V0A2 encodes the a2 subunit of the V0 domain within the multi-subunit V-ATPase complex, which pumps protons into lysosomes and late endosomes, maintaining their acidic lumen. This activity is essential for lysosomal hydrolase function, autophagosome maturation, and endocytic trafficking. Upstream regulation involves mTORC1, EGFR signaling, nutrient sensing, and hypoxia-inducible factors. Gene disruption impairs acidification, leading to accumulation of LC3-II and p62, diminished cathepsin activity, and defective endosomal sorting mediated by Rab7, RILP, and ARF6. Consequently, mTORC1 effectors S6K and 4E-BP1 are dysregulated, altering TFEB nuclear translocation and reducing matrix metalloproteinase secretion.
In colorectal cancer, V-ATPase overexpression contributes to an acidic tumor microenvironment and facilitates invasion and metastasis. This HT29 knockout model enables investigation of how loss of V-ATPase affects cancer cell proliferation, migration, and drug sensitivity. It also serves as a cellular model for autosomal recessive cutis laxa type IIA, related to ATP6V0A2 mutations, enabling studies on lysosomal dysfunction in neoplastic and genetic disease contexts.
This polyclonal knockout population is amenable to diverse assays. Lysosomal pH can be quantified using LysoSensor dyes, and autophagy flux assessed by immunoblotting for LC3-II and p62 with lysosomal inhibitors. Immunofluorescence for LAMP1 and LC3 visualizes lysosomal and autophagic compartments. RT-qPCR confirms ATP6V0A2 disruption. Cell proliferation, wound-healing migration, and transwell invasion assays reveal phenotypic changes. Drug sensitivity testing further supports translational research. For further information, contact Ascent Research.
