The INPP5F Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from the well-characterized HEK293T human embryonic kidney cell line. These cells harbor a disrupted INPP5F gene, established via CRISPR/Cas9-mediated gene disruption, providing a loss-of-function model for investigating the role of this phosphoinositide 5-phosphatase in receptor trafficking and signal transduction. The polyclonal format offers a heterogeneous population with a range of editing outcomes, reflecting natural biological variability and enabling the study of INPP5F function at the population level.
The parental HEK293T cell line is a widely used model in cell and molecular biology, originating from human embryonic kidney epithelial cells transformed with sheared adenovirus 5 DNA. These cells stably express the SV40 large T-antigen, which promotes episomal replication of plasmids containing the SV40 origin, leading to high-level transient protein expression. Their high transfection efficiency, rapid growth, and robust protein production make HEK293T cells an ideal host for signaling studies, lentivirus production, and functional genomics applications.
INPP5F encodes a 5-phosphatase that specifically hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) at the 5-position to generate phosphatidylinositol 4-phosphate (PIP) on endosomal membranes. This enzymatic activity is critical for the uncoating of clathrin-coated vesicles, thereby facilitating the recycling of internalized receptors such as the epidermal growth factor receptor (EGFR). INPP5F interacts with components of the endocytic machinery, including the AP-2 adaptor complex, clathrin, and EPS15, and is regulated by upstream stimuli like EGF and insulin. By modulating PIP2 levels, INPP5F controls EGFR and insulin receptor trafficking, actin cytoskeleton dynamics, and downstream signal attenuation through pathways including PI3K/AKT. Loss of INPP5F disrupts this regulation, leading to prolonged growth factor signaling and potentially enhanced proliferative responses.
In the HEK293T background, disruption of INPP5F provides a powerful tool to dissect the interplay between phosphoinositide metabolism and membrane trafficking. The high transfection efficiency of these cells allows for straightforward introduction of reporters, mutant constructs, or fluorescently tagged proteins, facilitating real-time visualization of endosomal dynamics. The knockout model enables systematic analysis of altered EGFR internalization kinetics, sustained AKT phosphorylation, and changes in cell proliferation, offering a relevant cellular context for exploring oncogenic signaling mechanisms.
These polyclonal knockout cells are suited for a wide range of research applications, including cancer biology, endocytosis studies, receptor trafficking analysis, drug target validation, and functional genomics. Representative assays compatible with this model include Western blotting, RT-qPCR, immunofluorescence, flow cytometry, EGFR internalization assays, phospho-AKT analysis, transferrin recycling assays, and cell proliferation assays. Researchers can leverage this knockout system to interrogate the specific role of INPP5F in clathrin-mediated endocytosis and to screen for modulators of growth factor receptor signaling. For additional technical information or to discuss your experimental requirements, please contact Ascent Research.
