USP7 Knockout HAP1 Cell Line

The choice depends on the experimental question. The Knockout line is appropriate for asking whether USP7 is required for its established functions — particularly MDM2/p53 axis regulation, FOXO3 stability, and PTEN deubiquitination. Overexpression is useful for testing whether elevated USP7 activity is sufficient to drive substrate stabilization, or for studying USP7 in cancer contexts where it is frequently dysregulated. For USP7 research, the EDITGENE Knockout line in HAP1 is highly informative — USP7 is a major drug target with multiple inhibitors in clinical development (P5091, FT827, etc.), and HAP1 KO provides a critical genetic specificity control for testing on-target activity of these compounds. Rescue with wild-type or catalytically-dead (C223A) USP7 is the standard approach for confirming substrate dependence on USP7 deubiquitinase activity.

Primary applications: • p53/MDM2 axis: protein stability and ubiquitination analysis of p53 and MDM2 following USP7 loss; comparison with USP7 inhibitor treatment to assess on-target activity. • Substrate stability assays: cycloheximide chase experiments for USP7 substrates (FOXO3, PTEN, ICP0, others) to measure deubiquitinase-dependent protein stability. • USP7 inhibitor specificity: critical genetic control for testing P5091, FT827, and other USP7 inhibitors in clinical development. • Cancer phenotype assays: proliferation, apoptosis, and DNA damage response in the context of USP7 loss. EDITGENE recommends this model for researchers investigating USP7 biology, p53 regulation, and USP7 inhibitor pharmacology.

Yes. USP7 rescue experiments have a well-established framework given USP7's status as a major drug target: • Construct design: use a codon-modified USP7 sequence with a small N- or C-terminal tag (FLAG, HA). Both positions are tolerated. • Catalytically-dead rescue: the C223A mutation abolishes deubiquitinase activity and is the standard specificity control for assigning observed effects to USP7 catalytic function. • USP7 inhibitor specificity controls: rescue with inhibitor-resistant USP7 variants (where available) is particularly valuable for confirming on-target activity of P5091, FT827, and related compounds. • Functional readout: rescue should restore p53 stability, MDM2 ubiquitination, and substrate-specific deubiquitination of FOXO3, PTEN, and other reported USP7 substrates. 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.