The ARHGAP35 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed to disrupt the human ARHGAP35 gene, encoding p190-A RhoGAP, a critical GTPase-activating protein for Rho family GTPases. This polyclonal pool, derived from the near-haploid HAP1 cell line, provides a loss-of-function model for studying ARHGAP35-dependent processes, including cytoskeletal regulation, adhesion, and migration. The CRISPR/Cas9-mediated gene disruption introduces targeted modifications across the ARHGAP35 locus, generating a mixed population of cells with loss-of-function mutations, suitable for functional genomics and screening applications without clonal bias.
HAP1 cells originate from the KBM-7 chronic myeloid leukemia cell line, which is naturally near-haploid, thus facilitating straightforward gene knockout studies by minimizing genetic redundancy. Their haploid genome enables efficient mutagenesis and phenotypic screening, making them a preferred host for arrayed and pooled CRISPR screens. In this polyclonal format, the ARHGAP35 knockout cells retain the utility of the HAP1 background while offering a heterogeneous population that can be used directly for biochemical, imaging, and signaling investigations, avoiding clonal expansion and monoclonal artifacts.
ARHGAP35 acts as a negative regulator of Rho-mediated signaling by accelerating GTP hydrolysis on RhoA, Rac1, and Cdc42, thereby promoting inactivation of these small GTPases. Its activity is modulated by upstream signals from integrins, EGFR, PDGFR, and Src family kinases like Fyn, as well as focal adhesion kinase (FAK). Activated ARHGAP35 interacts with p120-RasGAP (RASA1), FAK, cortactin, and filamin at adhesions, leading to actin depolymerization, reduced stress fibers, and decreased adhesion turnover. This makes ARHGAP35 a key integrator of growth factor and adhesion signals controlling cytoskeletal dynamics.
In HAP1 cells, ARHGAP35 disruption enables dissection of its role in Rho GTPase regulation in a myeloid leukemia context, known for altered adhesion and migration. The knockout enhances constitutive RhoA, Rac1, and Cdc42 activity, leading to increased actin polymerization and stress fiber assembly, providing a tractable model for studying cytoskeletal reorganisation and its impact on cancer cell behaviours. This system is particularly informative for exploring how ARHGAP35 loss contributes to pathologies such as glioblastoma, breast cancer, and leukemia, where dysregulation of Rho signaling promotes invasion and metastasis.
These polyclonal cells support applications including RhoA activation assays (e.g., G-LISA), western blotting for phospho-signaling readouts, immunofluorescence imaging of focal adhesions and actin structures, and real-time cell migration assays. They enable functional genomics screens for synthetic lethal partners and drug targets in Rho pathways, along with validation of ROCK or LIMK inhibitors. By providing a mixed population of knockout genotypes, these cells enable robust, high-throughput analyses that better represent the genetic heterogeneity encountered in tumors. For more details, please contact Ascent Research.
