The DIAPH1 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the near-haploid human HAP1 cell line, designed for loss-of-function analysis of DIAPH1 (mDia1). This model facilitates studies of actin cytoskeleton dynamics, Rho GTPase signaling, and transcriptional regulation. The polyclonal format avoids clonal selection, providing heterogeneous gene disruptions for rapid functional assessment.
HAP1 is a male near-haploid cell line originating from KBM-7 chronic myeloid leukemia cells, retained for its haploid karyotype that simplifies gene targeting. CRISPR/Cas9 disruption of a single allele suffices to eliminate gene function. HAP1 cells are widely used in genetic screens and pathway analyses due to their simplified genome and robust proliferation, particularly advantageous for cytoskeletal and adhesion studies where diploidy can mask phenotypes.
DIAPH1 (mDia1) is a formin protein that functions as a key Rho GTPase effector, catalyzing actin nucleation and elongation downstream of RhoA, Rac1, and Cdc42. Activated RhoA recruits mDia1 to the plasma membrane, where it interacts with profilin?CG-actin to assemble unbranched actin filaments that form stress fibers, filopodia, and lamellipodia. mDia1 also stabilizes microtubules through APC and CLIP-170 and regulates SRF/MRTF-dependent transcription by modulating actin monomer levels, controlling genes such as FOS and ACTA2. Additional interactions with IQGAP1 and DIP/WASF2, and upstream modulation by EGFR and PIP2, integrate extracellular signals with cytoskeletal and transcriptional outputs. Knockout of DIAPH1 therefore decouples adhesion and migration from gene expression programs.
In HAP1 cells, DIAPH1 knockout provides a clean genetic background to study Rho?CmDia1?CSRF signaling without allelic redundancy. This model is relevant to autosomal dominant deafness (DFNA1), cancer metastasis, Sezary syndrome, and myeloproliferative disorders. The haploid state enhances phenotypic sensitivity in migration, adhesion, and transcriptional reporter assays, making it suitable for high-content screening of compounds or genetic modulators affecting actin cytoskeleton and SRF pathways.
Typical applications include phalloidin staining and confocal microscopy to visualize F-actin organization, transwell migration/invasion assays to assess cell motility, and western blotting to detect SRF target proteins (FOS, ACTA2). Co-immunoprecipitation studies reveal DIAPH1 protein complexes, and Rhotekin pull-down assays measure RhoA activation. Time-lapse actin imaging and haploid genetic screens further exploit this polyclonal knockout population. For additional details, please contact Ascent Research.
