This CRISPR/Cas9-edited polyclonal knockout cell population disrupts the EFNA1 gene in the 786-O human clear cell renal carcinoma line. The polyclonal product comprises a heterogeneous pool of edited cells, enabling loss-of-function studies without clonal bias.
786-O is a widely used model for clear cell renal cell carcinoma (ccRCC), harboring a constitutive VHL mutation that stabilizes HIF-1??. This leads to upregulation of VEGF and other angiogenic factors, promoting tumorigenicity. The cells exhibit adherent epithelial morphology and form tumors in immunocompromised mice, making them a standard system for studying VHL-HIF pathway contributions to renal cancer.
EFNA1 encodes ephrin-A1, a glycosylphosphatidylinositol (GPI)-anchored ligand that binds EphA receptor tyrosine kinases, primarily EphA2 and EphA4. Upon cell-cell contact, ephrin-A1 engagement triggers bidirectional signaling: forward signaling through EphA receptors activates SRC and FAK kinases, stimulating PI3K/AKT and MAPK/ERK1/2 (MAPK1/3) pathways, while reverse signaling recruits GRB2 and NCK1 adaptor proteins to modulate RHOA-driven cytoskeletal rearrangements. EFNA1 expression is transcriptionally regulated by TNF-??, VEGF, HIF-1??, TGF-??, and NF-??B, linking inflammatory, hypoxic, and growth factor stimuli to changes in cell adhesion, repulsion, and migration.
In the VHL-mutant 786-O background, constitutive HIF-1?? activity promotes VEGF and ephrin-A1 overexpression, establishing an autocrine/paracrine loop that enhances EphA2 forward signaling and tumor angiogenesis. Knockout of EFNA1 disrupts this loop, permitting dissection of ligand-dependent versus ligand-independent EphA2 functions. Additionally, loss of ephrin-A1 reverse signaling allows investigation of its role in RHOA-mediated cytoskeletal dynamics and invasive behavior. This model thus provides a controlled system to elucidate how hypoxia-driven ephrin signaling contributes to ccRCC progression and metastasis.
This polyclonal knockout cell population is suitable for diverse functional studies, including Transwell migration and invasion assays to quantify motility, HUVEC tube formation cocultures to evaluate angiogenic potential, and phospho-EphA2 immunoblotting to assess receptor activation. Downstream signaling can be probed via western blotting for AKT1 and MAPK1/3 phosphorylation, while RT-qPCR or RNA-seq can profile transcriptional changes. Validation of EFNA1 ablation is achieved by western blotting and immunofluorescence, which may also reveal EphA2 redistribution. For further information, please contact Ascent Research.
