ANKRD28 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal population in which the gene encoding ankyrin repeat domain 28 (ANKRD28) has been disrupted to generate a loss-of-function model. This polyclonal knockout cell pool, generated from the widely used HEK293T host line, facilitates the study of ANKRD28-dependent processes without the use of clonal selection, thereby preserving population-level heterogeneity. The engineered disruption abolishes functional ANKRD28 expression, enabling downstream investigations into protein phosphatase 1 (PP1) targeting and cytoskeletal regulation.
HEK293T cells are human embryonic kidney epithelial cells transformed with sheared adenovirus 5 DNA and stably expressing the SV40 large T antigen. This antigen permits episomal replication of plasmids containing the SV40 origin of replication, making the line highly amenable to transient transfection, protein overexpression, and viral vector production. Their robust growth characteristics and ease of manipulation have established HEK293T as a standard host for functional genomics, signal transduction studies, and high-throughput screening applications.
ANKRD28 encodes a regulatory subunit of serine/threonine protein phosphatase 1 (PP1) that directs catalytic activity toward substrates involved in actin cytoskeleton organization, cell adhesion, and mitotic progression. Mechanistically, ANKRD28 scaffolds PPP1CA to macromolecular complexes at focal adhesions and stress fibers, where it dephosphorylates targets such as myosin light chain, cofilin, and retinoblastoma protein. Disruption of ANKRD28 leads to hyperphosphorylation of these substrates, resulting in impaired cytoskeletal dynamics, defective cell adhesion and migration, and aberrant mitotic progression. ANKRD28 also interacts with death-associated protein kinase 1 (DAPK1) to modulate apoptosis, integrating signals from the cell cycle machinery and E2F transcription factors.
Knockout of ANKRD28 in HEK293T cells provides a physiologically relevant model to dissect PP1-directed regulation within an epithelial background. Given the role of HEK293T in cell adhesion and migration studies, this knockout line enables precise examination of how ANKRD28-dependent dephosphorylation controls focal adhesion turnover, stress fiber assembly, and motility. Additionally, the expression of SV40 large T antigen may intersect with PP1/Rb pathways, offering a unique context to explore cell cycle control and transformation. The model is therefore valuable for both basic cell biology and translational oncology research.
This knockout cell pool is suitable for a range of advanced applications, including elucidation of PP1 targeting mechanisms in actin cytoskeletal remodeling, investigation of cancer cell migration and invasion via transwell and wound healing assays, and dissection of the ANKRD28?CDAPK1 complex in apoptosis through Annexin V staining and caspase activation profiling. Users may perform co-immunoprecipitation with PPP1CA to assess phosphatase complex integrity, quantify phospho-myosin light chain by Western blotting, visualize focal adhesion dynamics by immunofluorescence for vinculin, and conduct phosphoproteomic mass spectrometry to identify downstream phosphorylation events. High-content screening for PP1 modulators is also feasible. For further technical information or to request custom knockout services, please contact Ascent Research.
