The DNAJC6 Knockout HEK293T Polyclonal Cells product is a CRISPR/Cas9-edited polyclonal population derived from the widely used HEK293T human embryonic kidney cell line, engineered to disrupt the DNAJC6 gene. This polyclonal knockout cell population provides a heterogeneous pool of gene-edited cells, enabling loss-of-function studies without the constraints of clonal selection. The CRISPR/Cas9-mediated gene disruption targets the DNAJC6 locus, generating a versatile knockout model while preserving the advantageous features of the parental HEK293T line.
HEK293T cells are a derivative of the HEK293 lineage, stably expressing the SV40 large T antigen. This expression permits episomal replication of transfected plasmids containing the SV40 origin of replication, thereby enhancing protein production in transient transfection assays. As adherent, epithelial-derived cells with robust growth characteristics and high transfection efficiency, HEK293T cells serve as a standard platform for recombinant protein expression, lentivirus and retrovirus production, and a broad spectrum of molecular and cellular assays. Their embryonic kidney origin provides a context for studying general eukaryotic cell biology processes, including endocytosis and signal transduction.
DNAJC6 encodes auxilin, a co-chaperone that recruits the constitutively expressed heat shock cognate 70 (Hsc70/HSPA8) to clathrin-coated vesicles. Auxilin stimulates Hsc70-mediated ATP hydrolysis, driving the uncoating of clathrin cages from endocytic vesicles, a critical step in clathrin-mediated endocytosis and synaptic vesicle recycling. This process is regulated upstream by the leucine-rich repeat kinase 2 (LRRK2), a kinase associated with Parkinson disease pathology. Auxilin functions within a macromolecular complex that includes clathrin heavy chain, the adaptor protein AP-2, and the GTPase dynamin. Loss of auxilin disrupts the clathrin disassembly cycle, providing a direct link between endocytic dysfunction and neurodegeneration.
In the HEK293T background, which lacks neuron-specific synaptic machinery, the DNAJC6 knockout model is particularly valuable for dissecting the general mechanistic aspects of clathrin-mediated endocytosis and chaperone-dependent vesicle trafficking. Although HEK293T cells are not neuronal, they express the core endocytic machinery and Hsc70, enabling detailed biochemical studies of auxilin function. The polyclonal nature of the knockout population introduces genetic heterogeneity that can reveal dominant effects of gene disruption while mitigating clonal artifacts. This model thus serves as a reductionist system to investigate how auxilin-dependent uncoating impacts overall endocytic flux and to identify small molecules that may compensate for auxilin loss.
Typical applications include modeling molecular deficits relevant to juvenile Parkinson disease and other neurodegenerative disorders, screening for pharmacological modulators of clathrin uncoating, and dissecting the Hsc70-auxilin interaction. Representative assays compatible with this polyclonal knockout system include Western blotting and immunofluorescence to confirm DNAJC6 ablation, co-immunoprecipitation to assess protein interactions, in vitro clathrin uncoating assays, and transferrin uptake assays to monitor endocytic efficiency. The cells can also be adapted for synaptic vesicle recycling assays with exogenous neuronal components. For additional technical details, please contact Ascent Research.
