ITCH Knockout HAP1 Cell Line

The choice depends on whether you are studying ITCH's role as a HECT family E3 ubiquitin ligase or its functions in immune regulation and substrate-specific protein degradation. The Knockout line is the standard tool for asking whether ITCH is required for these processes — ITCH (atrophin-interacting protein 4, AIP4) is a HECT-family E3 ligase that ubiquitinates diverse substrates including JunB, Notch1, p73, c-FLIP, and CXCR4, regulating immune cell activation, NOTCH signaling, and apoptosis. ITCH loss causes immune dysregulation in the itchy mouse and in human ITCH-deficiency syndrome. Overexpression is useful for studying ITCH gain-of-function effects. For immune regulation research, the EDITGENE ITCH Knockout in HAP1 enables study of ITCH biology — ITCH loss-of-function causes autoinflammation, hepatic disease, and lung disease in patients (syndromic multisystem autoimmune disease). Rescue with wild-type or catalytically-dead (C830A in the HECT catalytic cysteine) ITCH is the standard specificity control. The knockout is valuable for studying ITCH substrate ubiquitination biology and emerging ITCH-targeted immune drug development.

Primary applications: • Substrate ubiquitination: JunB, Notch1, p73, c-FLIP, CXCR4 ubiquitination levels analysis in ITCH-null cells. • Notch signaling regulation: Notch1 protein stability and target gene (HES1, HEY1) expression given ITCH-mediated Notch turnover. • Immune dysregulation studies: in immune-relevant contexts, characterization of ITCH-deficiency immune phenotypes mimicking ITCH-deficiency syndrome. • Substrate discovery: proteomics in ITCH-null cells to identify candidate ITCH-dependent ubiquitination targets. EDITGENE recommends this model for researchers investigating HECT family E3 ligase biology and ITCH-mediated immune regulation.

Yes. ITCH rescue experiments require attention to HECT E3 ligase architecture: • Construct design: use a codon-modified ITCH sequence with a small C-terminal tag (FLAG, HA). ITCH has N-terminal C2 domain (membrane binding), four WW domains (substrate recognition via PPxY motifs), and C-terminal HECT catalytic domain — preserve all elements. • Catalytically-dead rescue: C830A mutation in the HECT catalytic cysteine abolishes ubiquitin ligase activity and is the standard specificity control. • WW-domain-mutant rescue: WW domain mutations disrupt PPxY-motif substrate binding without affecting catalytic activity. • Functional readout: rescue should restore ITCH-dependent substrate ubiquitination (JunB, Notch1, p73, c-FLIP) and downstream protein stability. HAP1-specific considerations: • Diploidization: HAP1 cells gradually diploidize during extended culture — confirm ploidy by flow cytometry at the time of phenotypic assay. • Integration site sensitivity: position effects on transgene expression are more pronounced in near-haploid backgrounds; generating multiple independent rescue clones is strongly recommended. • Transduction efficiency: HAP1 transduces with lentivirus at moderate efficiency — increase MOI compared to standard immortalized lines.