The DNAJC10 Knockout HEK293T Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout cell population derived from the HEK293T human embryonic kidney line, targeting the DNAJC10 gene which encodes the ER-resident oxidoreductase and co-chaperone ERdj5. This loss-of-function model is designed for functional analysis of DNAJC10 in endoplasmic reticulum-associated degradation (ERAD) and protein quality control. The polyclonal nature preserves genetic diversity while ensuring consistent gene disruption across the population, avoiding artifacts associated with clonal selection.
HEK293T cells are an epithelial cell line transformed with adenovirus type 5 DNA and expressing SV40 large T-antigen, which facilitates episomal replication of plasmids with SV40 origin. Their high transfection efficiency and robust protein secretory capacity make them ideal for studying ER biology, including the unfolded protein response (UPR) and ERAD. The cells possess intact ER quality control machinery, providing a physiologically relevant host for investigating DNAJC10 function.
DNAJC10/ERdj5 reduces disulfide bonds in misfolded ER proteins, a critical step in terminally misfolded substrate recognition and disassembly prior to retrotranslocation. It interacts with HSPA5/BiP and the SEL1L-SYVN1/HRD1 E3 ligase complex to target substrates for VCP/p97-mediated extraction and ubiquitin-proteasome degradation. Under ER stress, the UPR sensors IRE1, PERK, and ATF6 activate transcription factors XBP1 and ATF4, upregulating DNAJC10 expression to enhance ERAD capacity and alleviate stress.
Disruption of DNAJC10 in HEK293T cells impairs ERAD and sensitizes cells to ER stress inducers such as tunicamycin and thapsigargin. The resulting accumulation of misfolded cargo allows detailed kinetic studies of substrate retention, retrotranslocation, and degradation. The polyclonal knockout model facilitates dose-response and time-course analyses of UPR activation and can be used to interrogate compensatory mechanisms involving alternative ER oxidoreductases. This system is particularly valuable for dissecting the interplay between ER stress and apoptosis.
Key applications include mechanistic studies of ER stress signaling, protein quality control, and ERAD substrate selection. Compatible assays encompass Western blotting for UPR markers (HSPA5, DDIT3), RT-qPCR for XBP1 splicing, co-immunoprecipitation with BiP, proteasome activity assays, flow cytometry for apoptosis, and immunofluorescence for ER morphology. Additionally, the cells support high-throughput screening of ER stress modulators and are relevant to cancer biology, neurodegenerative disease, and drug discovery targeting protein misfolding disorders. For inquiries, contact Ascent Research.
