The ACTN4 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population that disrupts the ACTN4 gene in HEK293T cells. This loss-of-function model enables investigation of alpha-actinin-4’s roles in actin crosslinking, focal adhesion dynamics, and mechanotransduction.
The HEK293T host cell line, a derivative of HEK293 stably expressing SV40 large T-antigen, is an adherent epithelial line widely used for protein expression, viral packaging, and kidney cell biology studies. Its robust growth, high transfectability, and well-characterized signaling pathways provide an ideal platform for CRISPR-mediated gene disruption and functional assays.
ACTN4 is an actin-binding protein that crosslinks filaments into bundles and connects them to focal adhesions through interactions with vinculin and integrin ??1. It functions downstream of TGF-??, mechanical stress, Rho GTPases, and PI3K/AKT signaling, and acts as a scaffold influencing the Hippo pathway by regulating YAP/TAZ nuclear translocation. In the knockout model, disruption of ACTN4 leads to impaired actin stress fiber formation, reduced vinculin recruitment to adhesions, and altered transcriptional activity of YAP/TAZ, manifesting as decreased expression of CTGF and CYR61. These changes compromise focal adhesion maturation, cell adhesion, and migration.
The HEK293T background, with its renal epithelial origin and adherent growth, is particularly relevant for investigating kidney podocyte biology and cancer cell motility. ACTN4 knockout in this context recapitulates features of focal segmental glomerulosclerosis (FSGS) and podocytopathies linked to ACTN4 mutations, including disrupted cytoskeletal architecture and adhesion defects. Additionally, the model aids in studying metastatic mechanisms where actin remodeling and altered mechanotransduction drive invasion.
Experimental applications encompass high-resolution immunofluorescence for actin and vinculin localization, quantitative migration and invasion assays, and biochemical characterization of focal adhesion complexes by co-immunoprecipitation and western blotting. The model further enables YAP/TAZ-centered studies using luciferase reporters and RT-qPCR for target genes CTGF and CYR61, as well as flow cytometric analysis of integrin ??1 expression. Phospho-signaling profiling of PI3K/AKT and Rho GTPase pathways can reveal upstream regulatory nodes. These polyclonal knockout cells are also suitable for screening small molecules that rescue cytoskeletal defects in FSGS or inhibit metastatic migration. For additional details, please contact Ascent Research.
