The ECPAS Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed to disrupt the ECPAS gene in HEK293T human embryonic kidney cells. This heterogeneous pool provides a loss-of-function model for studying ECPAS-dependent proteasome regulation and protein quality control without clonal isolation, making it well-suited for pooled functional assays and high-content screening.
HEK293T cells are human embryonic kidney cells stably expressing the SV40 large T-antigen, derived from HEK293 cells. They are widely employed for transient gene expression and viral vector production owing to their high transfection efficiency and rapid proliferation, and they support episomal replication of plasmids containing the SV40 origin, further enhancing recombinant protein production. This genetic background offers a robust and experimentally tractable platform for knockout studies of the ubiquitin-proteasome system.
ECPAS encodes a scaffold protein critical for maintaining the structural integrity of the 26S proteasome. It interacts with the 19S regulatory particle, Hsp70, Hsp90, and ubiquitin, facilitating recognition and degradation of ubiquitinated substrates. ECPAS is activated by cellular stress signals, including heat shock, oxidative stress, and ubiquitination cues. Loss of ECPAS disrupts proteasome stability, impairing clearance of misfolded proteins, modulation of cell cycle regulators, and execution of apoptotic pathways, thereby compromising protein quality control.
In HEK293T cells, ECPAS knockout leads to accumulation of polyubiquitinated proteins and increased sensitivity to proteotoxic stress, such as treatment with the proteasome inhibitor MG132. This model recapitulates molecular features of proteasome dysfunction observed in neurodegenerative disorders and cancer. The high transfectability of HEK293T enables rescue experiments and high-throughput modifier screens to dissect ECPAS-dependent proteostasis mechanisms, including endoplasmic reticulum-associated degradation (ERAD).
Key research applications include investigating proteasome structure-function relationships, elucidating mechanisms of protein quality control, and modeling proteasome-related pathologies, including cancer and neurodegenerative disorders such as Alzheimer??s and Parkinson??s diseases. Researchers can employ Western blotting to detect polyubiquitinated proteins, proteasome activity assays using fluorogenic substrates, co-immunoprecipitation to assess 26S proteasome subunit interactions, immunofluorescence to visualize proteasome localization, and viability assays under proteotoxic stress. For further details or to discuss custom cell engineering services, please contact Ascent Research.
