SHPRH Knockout HAP1 Cell Line

The choice depends on whether you are studying SHPRH's role as a ubiquitin ligase mediating PCNA polyubiquitination during DNA damage tolerance or its functions in genome stability maintenance. The Knockout line is the standard tool for asking whether SHPRH is required for K63-linked polyubiquitination of PCNA at lysine 164 — SHPRH and HLTF redundantly catalyze this modification, which switches translesion synthesis from error-prone to template-switching error-free pathway. Overexpression is useful for testing SHPRH activity or for studying its tumor suppressor functions. For DNA damage tolerance research, the EDITGENE SHPRH Knockout in HAP1 is a clean genetic background for studying PCNA-K164 polyubiquitination biology. HLTF expression analysis is essential given functional redundancy — combined SHPRH/HLTF analyses provide comprehensive characterization of K63-polyubiquitin-dependent template switching. Rescue with wild-type or RING-domain-mutant (E3-ligase-dead) SHPRH enables structure-function studies.

Primary applications: • PCNA polyubiquitination: ubiquitin Western blot for K63-polyubiquitinated PCNA following DNA damage (UV, methyl methanesulfonate) — assess SHPRH-dependent modification. • Template switching activity: replication fork progression assays following damage induction to assess damage tolerance pathway integrity. • HLTF paralog studies: HLTF expression analysis and combined SHPRH/HLTF knockdown to dissect redundant K63-polyubiquitination roles. • Genome stability: micronucleus assays, sister chromatid exchange, and damage-induced mutagenesis given SHPRH's reported tumor suppressor functions. EDITGENE recommends this model for researchers investigating DNA damage tolerance, PCNA polyubiquitination, and SHPRH-mediated genome stability.

Yes. SHPRH rescue experiments require attention to multi-domain architecture: • Construct design: SHPRH is large (~1683 amino acids); use codon-modified sequences with C-terminal tags (FLAG, HA). The N-terminal H15 (linker histone H1.5) domain, central SF2 helicase domain, RING domain (E3 ligase), and C-terminal regions should all be preserved. • RING-dead rescue: RING domain mutations abolish E3 ligase activity and are the standard specificity control for K63-polyubiquitin ligase function. • Helicase-dead rescue: ATP-binding mutations in the SF2 domain enable separation of ATPase from E3 ligase functions. • Functional readout: rescue should restore K63-polyubiquitinated PCNA following DNA damage and template switching activity. 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.