FIG4 Knockout HAP1 Cell Line

The choice depends on whether you are studying FIG4 (factor-induced gene 4, SAC3)'s role as a PI(3,5)P2 5-phosphatase or modeling Charcot-Marie-Tooth disease type 4J (CMT4J), Yunis-Varón syndrome, and amyotrophic lateral sclerosis. The Knockout line is the standard tool for asking whether FIG4 is required for these processes — FIG4 is a phosphoinositide phosphatase that dephosphorylates PI(3,5)P2 to PI(3)P; paradoxically, FIG4 loss reduces (rather than increases) PI(3,5)P2 levels because FIG4 also positively regulates the PI(3,5)P2-synthesizing kinase PIKfyve via scaffolding within the PAS complex (PIKfyve, FIG4, VAC14). Overexpression is useful for studying FIG4 gain-of-function effects. For lysosomal biology research, the EDITGENE FIG4 Knockout in HAP1 enables study of PI(3,5)P2 biology and PIKfyve pathway. FIG4 biallelic loss-of-function mutations cause Charcot-Marie-Tooth disease type 4J (autosomal recessive peripheral neuropathy) and Yunis-Varón syndrome; FIG4 has been characterized as an ALS risk gene. Rescue with wild-type or phosphatase-dead FIG4 enables structure-function studies. The knockout is valuable for studying endolysosomal biology, autophagy, and PIKfyve inhibitor (apilimod) specificity.

Primary applications: • PI(3,5)P2 levels: phosphoinositide HPLC or biosensor (ML1Nx2-mCherry) imaging analysis given the paradoxical FIG4-PIKfyve scaffolding effect. • Endolysosomal morphology: lysosomal enlargement (a hallmark of PI(3,5)P2 deficiency) by LAMP1 imaging. • Autophagy regulation: LC3-II accumulation and autophagic flux given PI(3,5)P2's role in autophagy. • CMT4J modeling: rescue with patient-derived FIG4 mutations for genotype-function studies of Charcot-Marie-Tooth disease type 4J. • Apilimod sensitivity: critical genetic control for apilimod (PIKfyve inhibitor) — FIG4 loss should phenocopy PIKfyve inhibition. EDITGENE recommends this model for researchers investigating endolysosomal biology, CMT4J disease mechanisms, and PIKfyve pathway research.

Yes. FIG4 rescue experiments require attention to PAS complex scaffolding: • Construct design: use a codon-modified FIG4 sequence with a small C-terminal tag (FLAG, HA). FIG4 has SAC domain phosphatase region and C-terminal VAC14-interaction region — preserve all elements. • Phosphatase-dead rescue: SAC domain catalytic residue mutations abolish phosphatase activity but retain scaffolding function — invaluable for separating catalytic from scaffolding roles. • VAC14-binding-deficient rescue: C-terminal mutations disrupt PAS complex assembly. • CMT4J mutation rescue: patient-derived FIG4 mutations (I41T common in CMT4J, others) enable disease genotype-function studies. • Functional readout: rescue should restore endolysosomal morphology (no enlarged vacuoles) and PI(3,5)P2 levels. 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.