The ADAM10 Knockout HAP1 Polyclonal Cells constitute a CRISPR/Cas9-edited polyclonal knockout population designed to disrupt the expression of the ADAM10 gene in the HAP1 human near-haploid cell line. This product provides a pooled loss-of-function model, avoiding the artefacts associated with single-cell cloning, and enables the examination of ADAM10-dependent proteolytic shedding in a simplified genomic context. The cells are well-suited for high-throughput screens and mechanistic studies that benefit from a uniform genetic background and robust loss of ADAM10 function across the population.
HAP1 cells are derived from the KBM-7 chronic myeloid leukemia line and possess a near-haploid genome, except for a disomic region on chromosome 8. This near-haploidy means that disruption of a single allele is sufficient to eliminate gene function, facilitating clean phenotypic readouts. The cells retain intact signaling pathways, including Notch, EGFR, and other cascades, making them a powerful host for investigating protein functions without the complexities of diploidy. Their adherent growth and rapid doubling time further enhance their utility in functional genomics and drug discovery.
ADAM10 is a membrane-anchored metalloproteinase that serves as a primary sheddase for a diverse array of transmembrane substrates. It cleaves Notch receptors to release the Notch intracellular domain (NICD), which translocates to the nucleus and activates transcription of Hes/Hey family target genes. ADAM10 also processes amyloid precursor protein (APP) via the non-amyloidogenic pathway, generating the neuroprotective sAPP?? fragment, and mediates the shedding of E-cadherin and EGFR ligands such as HB-EGF. Its activity is regulated by upstream signals including protein kinase C and calcium influx, with tetraspanins Tspan5 and Tspan14 acting as cofactors. Downstream, NICD and sAPP?? are key effectors, while EGFR ligand release triggers ERK and Akt signaling. Interacting factors include ADAM17, calmodulin, and TIMPs.
In the HAP1 context, ADAM10 disruption abolishes ligand-induced Notch signaling, leading to reduced expression of Hes/Hey genes, and shifts APP processing towards the amyloidogenic pathway, reducing sAPP?? levels. The shedding of adhesion molecules and growth factors is also impaired, impacting cell migration and downstream signaling. This model recapitulates critical aspects of ADAM10 deficiency seen in neurodegenerative diseases and cancer, offering a tractable system to dissect these pathways without confounding genetic redundancy. The polyclonal nature ensures that the population represents a range of knockout alleles, enhancing robustness in pooled assays.
This knockout product is instrumental for research applications including Alzheimer??s disease studies, where APP processing can be monitored by Western blotting for sAPP?? and C83 fragments or by ELISA. In cancer research, the cells enable evaluation of EGFR ligand shedding and migration/invasion through transwell assays, alongside phospho-signaling analysis of ERK and Akt. Drug screening for ADAM10 inhibitors can employ Notch luciferase reporters or RT-qPCR of target genes. Co-immunoprecipitation can probe altered substrate interactions, and flow cytometry can assess surface receptor shedding. For further information, please contact Ascent Research.
