EFNA5 Knockout NCI-H1299 Polyclonal Cells are a heterogeneous population of NCI-H1299 cells engineered with CRISPR/Cas9-mediated gene disruption at the EFNA5 locus. This polyclonal knockout pool provides a loss-of-function model in which ephrin-A5 expression is ablated across a diverse cell population, enabling robust analysis of ephrin-A5-dependent phenotypes without the clonal selection bias associated with single-cell-derived knockouts. The use of a polyclonal format is particularly suited for studying population-level behaviors such as collective cell migration and tumor heterogeneity.
The parental NCI-H1299 cell line is derived from a lymph node metastasis of a human lung adenocarcinoma and serves as a well-characterized model for non-small cell lung carcinoma (NSCLC). These cells exhibit an adherent epithelial morphology and carry a homozygous partial deletion of the TP53 gene, resulting in p53 protein deficiency. The p53-null background contributes to genomic instability and a highly aggressive, metastatic phenotype, making NCI-H1299 an ideal host for investigating molecular drivers of NSCLC invasion and therapeutic resistance.
EFNA5 encodes ephrin-A5, a glycosylphosphatidylinositol (GPI)-anchored ligand that activates EphA receptor tyrosine kinases (EphA1?CEphA8) through direct cell-cell contact. Ephrin-A5/EphA binding triggers bidirectional signaling: forward via the receptor kinase domain and reverse through Src family kinases. Downstream, ephrin-A5 modulates focal adhesion kinase (FAK), Rho GTPases (RhoA, Rac1), ERK1/2, and AKT, thereby controlling cytoskeletal dynamics, cell adhesion, and migration. Its expression is regulated by HIF1A, Wnt/??-catenin, and inflammatory cytokines (TNF??, IL-1??), linking ephrin-A5 to microenvironmental cues.
In NCI-H1299 cells, EFNA5 knockout abrogates ephrin-A5-mediated juxtacrine signaling, disrupting the balance between cell?Ccell repulsion and adhesion that governs collective tumor cell movement. Loss of ephrin-A5 attenuates activation of Src/FAK and ERK1/2-AKT cascades, impairing lamellipodial protrusion and invasive behavior. This model enables direct interrogation of ephrin-A5??s contribution to NSCLC metastasis, including matrix degradation by MMPs and tumor angiogenesis, which is influenced by ephrin-A5/EphA crosstalk in the tumor stroma.
This polyclonal knockout tool is suitable for scratch wound healing, Transwell migration, and Matrigel invasion assays. Western blotting for ephrin-A5, phospho-FAK (Tyr397), and phospho-ERK1/2 (Thr202/Tyr204) confirms loss of protein and downstream signaling; RT-qPCR verifies transcript depletion. Immunofluorescence can assess adhesion dynamics, while co-immunoprecipitation reveals disrupted EphA receptor interactions. Xenograft tumorigenicity assays demonstrate the impact on in vivo growth and metastasis. By providing a ready-to-use polyclonal knockout population, this product accelerates research into ephrin-A5 as a therapeutic target in NSCLC. For further technical details, please contact Ascent Research.
