DMXL1 Knockout HEK293T Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout cell population designed for loss?of?function studies of DMXL1. This polyclonal pool comprises a heterogeneous mix of HEK293T cells carrying targeted disruptions of the DMXL1 gene, offering a robust system that bypasses clonal selection biases and reflects population?level editing. The format is well?suited for high?content screening, bulk functional assays, and comparative analysis against wild?type cells.
The HEK293T host cell line is a human embryonic kidney epithelial derivative that stably expresses SV40 large T antigen, enabling episomal replication of SV40?origin plasmids and thus facilitating high?efficiency transfection and protein expression. Widely adopted in cell biology, HEK293T cells provide a reproducible and manipulable platform for investigating membrane trafficking, signal transduction, and lysosomal biology, making them an ideal context for probing DMXL1 function.
DMXL1 encodes a large scaffold protein that functions as a regulatory subunit of the vacuolar H+-ATPase (V-ATPase), a multi-subunit proton pump essential for organelle acidification. DMXL1 physically interacts with V-ATPase core components ATP6V0A1 and ATP6V1A, the RAB3GAP complex (RAB3GAP1, RAB3GAP2), and LAMTOR1, which anchors the Ragulator complex on lysosomes. These interactions are indispensable for V-ATPase assembly and its coupling to mTORC1 activation. When DMXL1 is disrupted, V-ATPase activity declines, lysosomal acidification is reduced, and mTORC1 fails to phosphorylate S6K1 efficiently. Simultaneously, TFEB remains dephosphorylated and constitutively nuclear, upregulating lysosomal and autophagy genes, yet autophagic clearance is blocked, leading to accumulation of LC3-II and p62.
HEK293T cells offer an experimentally tractable human epithelial system to dissect the DMXL1?CV-ATPase?CmTORC1 axis. Their high transfectability allows transient or stable expression of DMXL1 variants (including patient-derived mutations) to test rescue of lysosomal and signaling defects. The cell line??s rapid proliferation supports high-throughput applications, such as genetic screens for modifiers of DMXL1-dependent phenotypes. This knockout model holds particular relevance for studying monogenic neurodevelopmental disorders like DMXL1-associated developmental and epileptic encephalopathy, hypogonadotropic hypogonadism, and central hypothyroidism, and may also inform broader lysosomal storage disease mechanisms.
Detailed phenotypic profiling can be performed using LysoSensor-based ratiometric pH measurements to quantify lysosomal acidity, western blot analysis of LC3?II turnover and phospho?S6K1 levels as mTORC1 readouts, and co?immunoprecipitation to assess V-ATPase complex integrity. Immunofluorescence staining for LAMP1 and p62 visualizes lysosomal clustering and autophagic substrate accumulation. This polyclonal knockout population is compatible with pooled CRISPR screens, small-molecule library screening for DMXL1-related epileptic encephalopathy, and studies investigating the interplay between endosomal maturation and mTORC1 signaling. For further technical information or custom requests, please contact Ascent Research.
