The EDN3 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population for studying the EDN3 gene, encoding Endothelin-3, a peptide ligand vital for vasoconstriction, neural crest development, and melanocyte differentiation. This knockout model uses CRISPR/Cas9-mediated gene disruption in HAP1 haploid human cells, yielding a mixed edited population. The polyclonal format offers a versatile tool for investigating EDN3-dependent signaling without clonal selection, supporting functional screening and pathway analysis.
HAP1 is a near-haploid human cell line derived from KBM-7, isolated from a chronic myeloid leukemia (CML) patient. With only one allele per gene, HAP1 is ideal for CRISPR knockout screens and genetic perturbation. The hematopoietic lineage provides a relevant background for exploring endothelin signaling in leukemia and blood cancers.
EDN3 is a secreted ligand that binds the G protein-coupled receptor EDNRB, activating G??q/11 and stimulating phospholipase C to produce IP3 and DAG. IP3 mobilizes calcium, while DAG activates protein kinase C. Downstream, ERK1/2 (MAPK3/MAPK1) and AKT1 are phosphorylated, regulating transcription factors MITF and CREB1. Upstream regulators include PAX3, SOX10, TGFB1, BMP4, and WNT signals; activation requires cleavage by furin and ECE1. These pathways drive neural crest migration, enteric neurogenesis, and melanocyte specification.
In HAP1 cells, EDN3 knockout disrupts endothelin signaling potentially influencing hematopoietic cell behavior, given the CML origin. The model aids study of EDN3 in cancer proliferation, survival, and migration, especially melanoma where EDNRB and MITF are key. Haploidy ensures clean phenotypes without allelic compensation, facilitating pooled screens and dose-response studies. This unique platform also enables investigation of crosstalk between endothelin and BCR-ABL pathways.
These polyclonal knockout cells are suitable for Western blotting for EDN3, EDNRB, and phospho-ERK; RT-qPCR for MITF and EDNRB; immunofluorescence for melanocyte markers; flow cytometry for EDNRB expression; calcium flux assays; and migration/invasion studies. Drug sensitivity assays with endothelin receptor antagonists (e.g., bosentan) can be performed. The haploid background also supports genetic modifier screens. For further details or to discuss your specific research needs, please contact Ascent Research.
