The EDNRB Knockout HAP1 Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout cell population designed for targeted disruption of the EDNRB gene, encoding the endothelin receptor type B. This loss-of-function model is generated in the HAP1 host cell line using CRISPR/Cas9 technology to introduce gene disruption across a polyclonal pool, providing a heterogeneous knockout background suitable for pooled functional genomics and signaling studies. The product is provided as a live cell population, enabling researchers to immediately deploy isogenic EDNRB-deficient cells in downstream assays without single-cell cloning steps, thereby preserving population-level diversity while eliminating wild-type receptor activity.
The HAP1 parental cell line is a near-haploid, chronic myeloid leukemia (CML)-derived cell line of male origin, originally isolated from a patient with CML. HAP1 cells exhibit a stable near-haploid karyotype, which simplifies genetic manipulation and phenotypic interpretation by reducing gene redundancy. As an adherent myeloid progenitor cell line, HAP1 retains many features of hematopoietic cells while offering the practical advantages of robust proliferation and straightforward culture. The near-haploid genome makes HAP1 an ideal host for knockout studies, as a single targeting event is often sufficient to eliminate gene function, and the reduced genetic complexity facilitates clear genotype-phenotype correlations in signaling and drug response experiments.
EDNRB functions as a G protein-coupled receptor (GPCR) that is specifically activated by the endothelin family of peptide ligands??EDN1, EDN2, and EDN3. Upon ligand binding, EDNRB predominantly couples to the heterotrimeric G proteins G??q/11 (encoded by GNAQ and GNA11) and G??i (GNAI), initiating bifurcating intracellular cascades. Signaling through G??q/11 activates phospholipase C beta (PLC??), which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 triggers calcium release from the endoplasmic reticulum, while DAG activates protein kinase C (PKC). Downstream, PKC phosphorylates and activates the RAF1-MAP2K1-MAPK1/3 (ERK1/2) kinase module, promoting the activity of transcription factors such as FOS, JUN, and MYC. Concurrently, G??i-mediated inhibition of adenylyl cyclase reduces cellular cAMP levels, dampening protein kinase A (PKA) signaling. EDNRB also engages the PI3K/AKT pathway, supporting cell survival and proliferation. In addition, the receptor interacts with regulatory proteins including beta-arrestins (ARRB1, ARRB2) and caveolin-1 (CAV1), which modulate receptor desensitization, internalization, and signal compartmentalization.
The ablation of EDNRB in the HAP1 near-haploid background creates a powerful model for dissecting endothelin-mediated signaling networks in a genetically clean system. Loss of EDNRB function abrogates both the G??q/11-Ca2+-PKC-ERK axis and the G??i-cAMP-PKA module, as well as attenuates PI3K/AKT activation, thereby eliminating the receptor??s contributions to cell proliferation, migration, and transcriptional responses. This is particularly relevant for studying neural crest biology, as EDNRB is essential for enteric nervous system development and its dysfunction underlies Hirschsprung disease and Waardenburg syndrome. In addition, the model is valuable for cancer research, especially melanoma, where EDNRB can promote tumor cell proliferation and migration. The near-haploid setting also aids synthetic lethality screens and drug-gene interaction studies aimed at identifying vulnerabilities in EDNRB-null contexts.
This polyclonal knockout cell population is suited for a broad range of experimental applications, including functional genomics, GPCR signal transduction analysis, and drug discovery. Researchers can use these cells in calcium flux assays to verify the absence of endothelin-induced calcium mobilization, or assess ERK phosphorylation via Western blotting or imaging to confirm disrupted MAPK cascade activity. Migration and invasion assays (e.g., transwell or scratch wound) can quantify the receptor??s role in cell motility, while cAMP assays reveal the impact on G??i-dependent adenylate cyclase regulation. Transcriptomic profiling by RNA-seq enables unbiased assessment of EDNRB-dependent gene networks, and drug sensitivity testing with endothelin receptor antagonists (e.g., bosentan) or downstream kinase inhibitors supports translational pharmacology studies. For further details or to request a quote, please contact Ascent Research.
