The EHD4 Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population designed for the disruption of the EHD4 gene in the HEK293T host cell background. This product provides a loss-of-function model for investigating the cellular roles of EHD4 in endocytic recycling and membrane trafficking. As a polyclonal population, the cells offer a heterogeneous knockout model that maintains genetic diversity while enabling robust functional studies without the clonal selection artifacts that may arise in monoclonal lines. The CRISPR-mediated gene disruption targets the EHD4 locus, allowing researchers to dissect the contributions of this ATPase to vesicle transport and receptor dynamics in a well-characterized human cell system.
The HEK293T host cell line is a widely used derivative of the HEK293 human embryonic kidney epithelial cell line, stably expressing the SV40 large T antigen. This modification permits episomal replication of plasmids containing the SV40 origin of replication, resulting in high-level transient protein expression and efficient production of lentiviral and retroviral vectors. HEK293T cells are an established model for studying signal transduction, endocytosis, and membrane trafficking, owing to their robust growth, ease of transfection, and responsiveness to extracellular stimuli. Their epithelial origin and expression of key endocytic components make them particularly suitable for exploring the molecular mechanisms of receptor-mediated internalization and recycling pathways.
EHD4 (EH-domain-containing protein 4) is a member of the C-terminal Eps15 homology domain (EHD) ATPase family that functions as a membrane-remodeling factor on early and recycling endosomes. The protein oligomerizes upon membrane binding, generating curvature and tubulation to facilitate the fission of recycling vesicles. EHD4 is activated by membrane curvature sensing and binding to phosphoinositides such as PI(4,5)P2, and its ATPase activity is essential for its function. It acts downstream of receptor activation and cooperates with EHD1, Rab11, and Arf6 to sort and recycle internalized cargoes, including the transferrin receptor and integrin beta1, back to the plasma membrane. Additionally, EHD4 interacts with syndapin II and EHBP1, linking endosomal recycling to the actin cytoskeleton, and participates in ciliary membrane trafficking, influencing ciliogenesis.
In the HEK293T cellular context, disruption of EHD4 profoundly impacts endocytic recycling dynamics and plasma membrane composition. The loss of EHD4 is expected to delay the return of internalized receptors, leading to altered surface levels of key signaling molecules and potentially affecting downstream signaling cascades. This polyclonal knockout model allows researchers to examine the functional redundancy and distinct roles among EHD family members (EHD1?C4) and to dissect the interplay between EHD4 and its regulatory partners such as Rab11 and Arf6. Moreover, because HEK293T cells are widely used for viral production and protein expression, this knockout cell population provides a valuable tool for studying how endosomal trafficking influences these processes, with implications for understanding the limited association of EHD4 polymorphisms in complex diseases like schizophrenia and ciliopathies.
The EHD4 Knockout HEK293T Polyclonal Cells are suitable for a broad range of experimental applications, including western blotting to confirm EHD4 protein depletion, immunofluorescence microscopy to visualize endosomal morphology and receptor distribution, and co-immunoprecipitation to assess altered protein?Cprotein interactions within the EHD4 interactome. Functional assays such as transferrin recycling assays can quantitatively measure endocytic recycling rates, while flow cytometry enables detection of surface receptor levels (e.g., transferrin receptor, integrin beta1) to evaluate EHD4-dependent trafficking. These cells can also be employed in phenotypic rescue experiments by re-expressing wild-type or mutant EHD4, facilitating structure?Cfunction analyses of this membrane-remodeling ATPase. For further technical information and ordering details, contact Ascent Research.
