The ANKFY1 Knockout HEK293T Polyclonal Cells product provides a polyclonal population of HEK293T cells that have undergone CRISPR/Cas9-mediated gene disruption of ANKFY1. This loss-of-function model is generated without single-cell cloning, yielding a heterogeneous knockout pool that mitigates artifacts associated with clonal selection. The polyclonal format enables robust, population-level analysis of ANKFY1-dependent processes and is particularly suited for experiments requiring consistent phenotypic penetrance in endosomal biology, signal transduction, and cell migration research.
HEK293T is a human embryonic kidney epithelial cell line immortalized by expression of the SV40 large T antigen, a modification that confers high transfectability and supports episomal plasmid amplification. These adherent cells retain a functional endocytic pathway and are extensively characterized in studies of vesicular trafficking, receptor signaling, and viral production. Their epithelial nature and genetic tractability make them an optimal host for investigating macropinocytosis and endosomal maturation in a controlled cell culture environment.
ANKFY1 is a phosphoinositide-binding protein that localizes to early endosomes through its FYVE domain, which specifically recognizes phosphatidylinositol 3-phosphate (PI3P) generated by the lipid kinase PI3K/Vps34. Positioned downstream of growth factor receptors such as EGFR, ANKFY1 cooperates with the tethering factor EEA1 and the small GTPase Rab5 to regulate endosomal progression. It further promotes the recruitment of Rab7 and the ESCRT-0 component Hrs, thereby ushering cargo into multivesicular bodies for degradation. This functional linkage makes ANKFY1 a central regulator of the endosomal maturation checkpoint that controls EGFR downregulation and macropinocytic flux.
Disruption of ANKFY1 in HEK293T cells creates a relevant model to dissect the PI3P?CRab5?CRab7 signaling axis and its contribution to membrane trafficking. Because the host line is of epithelial origin, this knockout pool is particularly valuable for studying how endosomal dysfunction influences cell migration and invasion??processes in which integrin and growth factor receptor turnover are critical. The expected delay in early-to-late endosome transition offers a direct window into altered EGFR degradation kinetics and macropinocytic capacity, phenotypes that are implicated in cancer progression and, potentially, in neurodegenerative conditions linked to endosomal trafficking defects.
Common applications for these polyclonal knockout cells include western blotting of EGFR turnover, quantitative RT?qPCR for ANKFY1 mRNA, immunofluorescence localization of early (EEA1) and late (LAMP1) endosomal markers, and flow?based macropinocytosis assays using fluorescent dextran. The cells are also suited for co?immunoprecipitation studies with Rab proteins, confocal colocalization analysis, and wound?healing or Transwell invasion assays. Moreover, this polyclonal platform can be employed in high?content screening efforts to identify modulators of endocytosis or to evaluate macropinocytosis?dependent drug delivery vectors. For additional product information and ordering support, please contact Ascent Research.
