The ASAP2 Knockout HT29 Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal population of HT29 cells with targeted disruption of the ASAP2 gene. This gene-edited product provides a powerful loss-of-function model for investigating Arf GTPase-activating protein 1 (ASAP2)-dependent processes in a colorectal cancer background, avoiding clonal artefacts and enabling robust functional studies.
The HT29 cell line is a widely employed human colorectal adenocarcinoma model derived from a female patient, characterized by a BRAF V600E mutation, epithelial morphology, and tumorigenic capacity. It serves as a relevant system for examining colorectal cancer biology, including signaling pathways driving proliferation, survival, and metastasis, as well as for therapeutic compound screening.
ASAP2 (also known as DDEF2 or PAG3) is a multidomain protein that functions as an Arf GTPase-activating protein (GAP) and scaffolds key signaling complexes at focal adhesions. It is activated downstream of integrin engagement and growth factor stimulation, including EGF, through PI3K-AKT signaling and small GTPases. ASAP2 accelerates GTP hydrolysis on Arf1 and Arf6, thereby regulating membrane trafficking, actin cytoskeleton reorganization, and focal adhesion dynamics. It interacts directly with c-Src, FAK, paxillin, cortactin, and SH3-containing adaptor proteins, positioning it at the nexus of adhesion, migration, and invasion signaling. The mechanistic interplay involves Arf GTPase cycles, actin remodeling, and focal adhesion turnover, with downstream impact on Rho family GTPases and integrin-mediated adhesion.
In HT29 colorectal cancer cells, disruption of ASAP2 impairs Arf-dependent regulation of actin architecture and membrane dynamics, leading to defects in cell migration, invasion, and focal adhesion disassembly. This model enables dissection of ASAP2??s role in integrin signaling, focal adhesion kinase (FAK) activation, and Src family kinase pathways that promote tumor cell motility. The polyclonal knockout population, lacking single-cell cloning artifacts, provides a physiologically relevant representation of gene function loss, facilitating the study of metastatic processes and the identification of critical nodes in colorectal cancer progression.
Researchers can apply this tool in a variety of experimental settings: Western blotting and RT-qPCR to confirm protein and transcript loss, transwell migration and wound healing assays to quantify cell motility defects, and immunofluorescence to visualize focal adhesion morphology and composition. It is also suited for co-immunoprecipitation studies to map protein interaction networks, phospho-FAK and phospho-Src analyses to probe signaling rewiring, and drug screens aimed at identifying compounds that counteract ASAP2-driven invasion. Additionally, combining this knockout with inhibitors of the PI3K-AKT pathway or integrin antagonists can uncover synthetic vulnerabilities. For further details, please contact Ascent Research.
