The DNAJC6 Knockout HAP1 Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout cell population engineered to disrupt DNAJC6 expression in the near-haploid human HAP1 cell line. This loss-of-function model eliminates the auxilin protein, a co-chaperone for Hsc70 that is essential for ATP-dependent disassembly of clathrin coats during endocytosis. Provided as a polyclonal pool, these cells serve as a robust tool for genetic and pharmacological studies without the need for clonal selection.
HAP1 is a human chronic myeloid leukemia (CML) cell line derived from KBM-7, characterized by a near-haploid karyotype and expression of the BCR-ABL1 fusion oncogene. The haploid genetic background simplifies genotype-phenotype correlations, making it an optimal system for targeted gene knockout studies. For DNAJC6 disruption, HAP1 cells provide a stable and well-characterized platform to dissect clathrin-mediated endocytosis without interference from the BCR-ABL1 signaling, allowing clean interrogation of auxilin-dependent processes.
DNAJC6 encodes auxilin, which recruits Hsc70 (HSPA8) to clathrin-coated vesicles. Auxilin stimulates Hsc70 ATPase activity to disassemble the clathrin lattice, a critical step in synaptic vesicle recycling and clathrin-mediated endocytosis. The protein directly interacts with clathrin heavy chain (CLTC), the AP2 adaptor complex (including AP2A1), and dynamin (DNM1). Upstream regulatory inputs include neuronal activity and synaptic signaling, while downstream consequences of auxilin loss encompass defective clathrin uncoating and reduced endocytic efficiency. Thus, DNAJC6 knockout impairs membrane trafficking by blocking vesicle uncoating.
Although HAP1 is non-neuronal, it retains the core endocytic machinery necessary to analyze auxilin??s cellular role, providing a simplified model for clathrin dynamics. The haploid state eliminates allelic variation, ensuring a clear readout of DNAJC6-dependent phenotypes. This model is particularly valuable for Parkinson disease (PARK19) research, as DNAJC6 mutations are linked to juvenile parkinsonism and ??-synuclein accumulation. In HAP1 cells, auxilin loss can be functionally probed using transferrin uptake assays to quantify endocytosis and immunofluorescence to visualize clathrin coat persistence, bridging in vitro findings to neuronal endocytic defects.
This polyclonal knockout product is ideally suited for high-throughput drug screening for synucleinopathies, detailed dissection of clathrin-mediated trafficking pathways, and mechanistic studies of Hsc70?Cauxilin complex formation. Compatible assays include Western blotting for auxilin quantification, RT-qPCR for transcript analysis, filter trap assays for ??-synuclein aggregation, and cell viability tests under proteotoxic stress. The model also supports large-scale genetic modifier screens to identify endocytosis regulators. For further information, please contact Ascent Research.
