EDEM3 Knockout Huh-7 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the human hepatocellular carcinoma cell line Huh-7, designed to disrupt the EDEM3 gene. This loss-of-function model enables investigation of EDEM3??s role in ER-associated degradation (ERAD) of misfolded glycoproteins. The polyclonal format provides a heterogeneous pool of knockout cells, avoiding clonal selection and offering robust population-level responses for studying ER quality control mechanisms.
The Huh-7 cell line is a well-differentiated, adherent hepatocellular carcinoma model with retained hepatocyte functions, including active protein secretion and metabolic activity. Permissive to hepatitis C virus (HCV) replication, Huh-7 cells are widely used in liver cancer and virology research. Their epithelial origin and intact endoplasmic reticulum (ER) structure make them well-suited for examining the ERAD pathway and unfolded protein response (UPR) in a hepatic context.
EDEM3 is an ??1,2-mannosidase that trims the terminal mannose from Man8GlcNAc2 on misfolded glycoproteins, generating a Man7GlcNAc2 glycan code recognized by OS9. This marks substrates for retrotranslocation via the HRD1-SEL1L complex and proteasomal degradation by VCP/p97. EDEM3 is transcriptionally activated by XBP1s downstream of IRE1 and ATF6 during ER stress. It cooperates with EDEM1, EDEM2, and the lectin OS9 to process clients such as the Z variant of alpha1-antitrypsin. Loss of EDEM3 disrupts ERAD, leading to accumulation of misfolded proteins and UPR activation.
In Huh-7 cells, EDEM3 knockout exacerbates ER stress inherent to hepatocellular carcinoma, increasing sensitivity to proteasome inhibitors and ER stressors. This model is valuable for studying the contribution of ERAD defects to liver cancer progression and metabolic disorders. Additionally, because HCV replication relies on ER membranes, EDEM3 disruption may illuminate viral-host interactions that depend on glycoprotein quality control. The knockout cells thus provide a platform to investigate the intersection of ER proteostasis, liver disease, and viral pathogenesis.
Typical applications include western blotting for EDEM3, BiP, and CHOP; RT-qPCR for XBP1s; cycloheximide chase assays to monitor substrate turnover; immunofluorescence for ER morphology; and flow cytometry for apoptosis or viability after tunicamycin treatment. Proteasome activity assays can assess ERAD functionality. These polyclonal EDEM3 knockout cells are a robust tool for ER quality control research, drug screening, and mechanistic studies. For further details, contact Ascent Research.
