The ATP6V1G2 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population with targeted disruption of the ATP6V1G2 gene in HEK293T cells. This heterogeneous knockout pool enables functional studies without clonal selection biases, preserving genetic diversity and facilitating investigation of V-ATPase biology in a population context.
HEK293T cells are a highly transfectable human embryonic kidney epithelial line derived from HEK293, transformed with adenovirus 5 DNA and expressing SV40 large T-antigen. They are widely used for heterologous protein expression and lentivirus production, offering an ideal background for studying endocytosis, signal transduction, and organelle function.
ATP6V1G2 encodes the G2 subunit of the V-ATPase V1 sector, essential for assembly of the proton pump that acidifies lysosomes, endosomes, and secretory vesicles. V-ATPase activity drives mTORC1 recruitment to lysosomes via the Ragulator-Rag complex, linking nutrient and energy status to cell growth. The gene is transcriptionally regulated by TFEB, MITF, and TFE3 downstream of mTORC1 and AMPK. Its acidification function controls autophagic flux (LC3, p62), ??-secretase-mediated Notch signaling, Wnt pathway activation through LRP6 maturation, and lysosomal hydrolase activity. ATP6V1G2 interacts with multiple V1 subunits (ATP6V1A, ATP6V1B2, etc.), V0 subunits, and the RAVE complex.
In HEK293T cells, ATP6V1G2 knockout disrupts V-ATPase-dependent endolysosomal acidification, impairing mTORC1 signaling and autophagy. The polyclonal nature of this knockout avoids clonal artifacts, providing a heterogeneous loss-of-function model to study dominant-negative effects or compensatory mechanisms. This system is particularly suited for dissecting how pH dynamics influence signaling and trafficking in an epithelial context amenable to high-level recombinant expression.
Applications include characterizing V-ATPase assembly via co-immunoprecipitation, probing mTORC1 activity using phospho-S6K/4E-BP1 blots, analyzing autophagy with LC3/p62 markers, and assessing lysosomal pH with LysoTracker. The cells support immunofluorescence for LAMP1/2, RNA-seq profiling, drug screening for V-ATPase modulators, and complementation with mutant ATP6V1G2 to model diseases such as epileptic encephalopathy. For further information, please contact Ascent Research.
