The ATP6V0A1 Knockout HEK293T Polyclonal Cells constitute a CRISPR/Cas9-engineered polyclonal knockout cell population targeting the ATP6V0A1 gene. This product provides a heterogeneous mixture of HEK293T cells with disrupted expression of the a1 isoform of vacuolar ATPase (V-ATPase) V0 subunit, a critical component of the proton translocation machinery. The polyclonal format avoids clonal biases, offering a robust tool for investigating V-ATPase-dependent acidification, membrane trafficking, and signaling pathways in a population-averaged context.
The host cell line, HEK293T, is an immortalized human embryonic kidney epithelial line expressing SV40 large T antigen. Widely used for protein expression, viral production, and signal transduction studies, HEK293T cells exhibit high transfection efficiency and rapid growth. Their kidney epithelial origin provides a physiologically relevant system for examining ion homeostasis and organelle acidification, where V-ATPase function is essential. Stable expression of SV40 large T antigen enhances episomal replication of plasmids containing the SV40 origin, making these cells particularly suitable for transient overexpression or complementation experiments.
ATP6V0A1 encodes the a1 subunit of the V-ATPase V0 domain, which forms the proton pore. V-ATPase activity acidifies lysosomes, endosomes, and autolysosomes, facilitating autophagy and cargo degradation. Upstream regulators MITF and TFEB promote ATP6V0A1 transcription under nutrient stress, while mTORC1 senses V-ATPase function at the lysosomal membrane via RAG GTPases. V-ATPase also interacts with LRP5/6 to modulate Wnt signalosome assembly and ??-catenin stabilization. Consequently, ATP6V0A1 disruption impairs lysosomal hydrolase activity, mTORC1 signaling, and Wnt pathway output, linking nutrient sensing to cellular growth and differentiation.
In HEK293T cells, loss of ATP6V0A1 results in a phenotype measurable by standard assays. Reduced LysoTracker staining confirms impaired lysosomal acidification, while increased LC3 puncta indicate defective autophagy. Attenuated mTORC1 activity is detected by decreased phospho-S6K western blot, and altered Wnt signaling is assessed via TOPFlash/FOPFlash luciferase reporters. The polyclonal knockout population is ideal for bulk biochemical experiments and pooled screens, as it averages out clonal variation and avoids single-clone artifacts.
This model is applicable to research on lysosomal storage disorders, neurodegeneration, and cancer, where dysregulated V-ATPase and autophagy are key. Osteoclast function studies benefit from ATP6V0A1’s role in acidifying the resorption lacuna, relevant to infantile malignant osteopetrosis. The intersection of V-ATPase with mTOR and Wnt pathways makes these cells useful for drug discovery screening of autophagy modulators or Wnt inhibitors. For further information, please contact Ascent Research.
