The DMXL1 Knockout HAP1 Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout cell population in the HAP1 near-haploid human cell line, generated for targeted disruption of the DMXL1 gene. This polyclonal format delivers a heterogeneous loss-of-function model, avoiding clonal artifacts and enabling population-level studies of DMXL1-dependent processes. It serves as a robust tool for deciphering endosomal trafficking, lysosomal biology, and signal transduction.
HAP1 is an adherent, fibroblast-like cell line derived from KBM-7 chronic myeloid leukemia cells, featuring a near-haploid karyotype that simplifies genetic manipulation and screening. The near-haploid genome reduces gene copy complexity, heightening sensitivity to gene disruption and making it optimal for knockout-based functional genomics in cell biology and drug discovery.
DMXL1, an essential component of the Rabconnectin-3 complex, orchestrates vacuolar ATPase (V-ATPase) assembly and endosomal trafficking, thereby controlling lysosomal acidification. Through interactions with RAB3GAP1, RAB3GAP2, WDR7, and V-ATPase subunits (ATP6V0A1, ATP6V1A), DMXL1 modulates endosomal pH. This regulation is pivotal for mTORC1 activation in response to nutrients and growth factors, leading to phosphorylation of RPS6KB1 and EIF4EBP1, and for Wnt signaling, where acidified vesicles sequester AXIN1 and GSK3??, stabilizing CTNNB1 and inducing target genes (AXIN2, MYC). Upstream cues such as WNT3A, amino acid sensing, and insulin/IGF-1 converge on DMXL1, while its deletion impairs autophagy, marked by accumulation of MAP1LC3B and SQSTM1/p62.
In the near-haploid HAP1 environment, DMXL1 knockout eliminates functional V-ATPase regulation, creating a precise model to dissect mTORC1?CWnt crosstalk and lysosomal-autophagy pathways. Loss of DMXL1 disrupts endosomal acidification and autophagic flux, recapitulating aspects of neurodevelopmental disorders and providing a system to explore cancer cell metabolic dependencies. Pathway components including Frizzled/LRP6/DVL receptors, AXIN1/GSK3?? destruction complex, and mTOR/RPTOR/RHEB can be interrogated without redundant allele interference.
This polyclonal knockout cell pool supports diverse assays: western blotting for phospho-RPS6KB1 and phospho-EIF4EBP1, RT-qPCR for AXIN2 and MYC, immunofluorescence for LC3 and LAMP2, and quantitative lysosomal pH measurements via LysoTracker and flow cytometry. Co-immunoprecipitation validates V-ATPase complex interactions, while ??-catenin reporter assays (TOPFlash) and bafilomycin A1 autophagy flux analyses provide functional readouts. Moreover, the haploid background enables genome-wide genetic screens to identify modulators of DMXL1-related phenotypes, making the cells invaluable for Wnt/mTOR drug screening and elucidating endolysosomal signaling in health and disease. For additional information, contact Ascent Research.
