The CCDC25 Knockout HEK293T Polyclonal Cells product comprises a heterogeneous population of HEK293T cells in which the CCDC25 gene has been disrupted using CRISPR/Cas9-mediated genome editing. This polyclonal knockout pool provides a loss-of-function model for studying CCDC25-dependent signaling without the need for single-cell cloning. The gene-edited cells are suitable for investigating the role of CCDC25 as a transmembrane receptor for neutrophil extracellular trap DNA (NET-DNA) and its contribution to cell migration and metastatic progression.
The parental HEK293T cell line is a widely employed human embryonic kidney epithelial cell line known for its high transfection efficiency and constitutive expression of the SV40 large T-antigen. This genetic background facilitates episomal replication of plasmids containing the SV40 origin, making HEK293T cells a preferred host for recombinant protein production, lentivirus packaging, and various functional assays. The epithelial origin and robust growth characteristics also render them a suitable model for studying cell migration and adhesion processes relevant to cancer biology.
CCDC25 encodes a transmembrane protein that functions as a receptor for NET-DNA, a component of neutrophil extracellular traps. Upon ligand binding, CCDC25 activates intracellular signaling through integrin-linked kinase (ILK) and the adaptor protein ??-parvin. This triggers downstream activation of the small GTPase Rac1, leading to actin polymerization and cytoskeletal reorganization that drives cell motility. Thus, CCDC25 acts as a critical sensor linking extracellular NET signals to the cellular migration machinery, with the ILK?C??-parvin?CRac1 axis serving as a central signaling module.
In the HEK293T background, disruption of CCDC25 abrogates the NET-DNA-induced migratory response, providing a clean system to dissect CCDC25-dependent and -independent pathways. The epithelial nature of HEK293T cells, combined with their ease of manipulation, allows for straightforward interrogation of the CCDC25?CILK?C??-parvin?CRac1 cascade. This knockout model is particularly valuable for addressing whether CCDC25 cooperates with other migratory receptors or signaling hubs endogenously expressed in kidney epithelial cells. Additionally, the polyclonal format ensures representation of diverse editing events, mitigating clonal artifacts and better reflecting population-level responses.
Researchers can employ these polyclonal knockout cells in a range of experimental workflows. Comparative Western blotting and phospho-ILK detection between wild-type and knockout cultures can reveal baseline and NET-stimulated activation states of the ILK pathway. RT-qPCR profiling enables investigation of transcriptional changes downstream of CCDC25 signaling. Transwell migration assays directly assess the requirement for CCDC25 in NET-DNA-driven cell motility, while immunofluorescence allows visualization of cytoskeletal dynamics and ??-parvin localization. Flow cytometry with labeled NET-DNA can confirm loss of receptor binding. These assays support studies aimed at elucidating NETosis-mediated metastasis, screening CCDC25 inhibitors, and evaluating anti-metastatic strategies. For additional technical details, protocols, and inquiry about custom services, please contact Ascent Research.
