The ATP2B2 Knockout HAP1 Polyclonal Cells represent a CRISPR/Cas9-edited polyclonal knockout cell population designed to disrupt the ATP2B2 gene, which encodes the plasma membrane calcium-transporting ATPase 2 (PMCA2). This product provides a heterogeneous pool of HAP1 cells carrying targeted genetic ablations within the ATP2B2 locus, yielding a robust loss-of-function model without clonal isolation. The polyclonal format preserves genetic diversity across the population, making it suitable for pooled screening approaches and functional studies where monoclonality is not required. The gene disruption is achieved through CRISPR/Cas9-mediated editing, generating a stable knockout background for downstream applications.
HAP1 cells are a near-haploid human cell line derived from the male chronic myeloid leukemia (CML) cell line KBM-7. This adherent, fibroblast-like cell line maintains a predominantly haploid karyotype, which simplifies genetic manipulation and facilitates knockout generation by requiring disruption of a single allele. The near-haploid state enhances the fidelity of CRISPR/Cas9 editing and reduces complications from heterozygous mutations, making HAP1 an ideal platform for functional genomics, cancer modeling, and pathway dissection. The cell line??s CML origin provides a relevant context for studying hematopoietic signaling, while its adherent morphology supports imaging-based assays and high-content screening.
ATP2B2 encodes PMCA2, a high-affinity calcium pump responsible for extruding cytosolic Ca2+ to the extracellular space, thereby maintaining low resting intracellular calcium levels. The pump is tightly regulated by calmodulin, which binds to the C-terminal autoinhibitory domain upon Ca2+ elevation, relieving inhibition and stimulating transport activity. Additional regulatory inputs include phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase A (PKA), and protein kinase C (PKC), which modulate pump kinetics and membrane targeting. Downstream, PMCA2 activity limits the activation of calcineurin, a Ca2+/calmodulin-dependent phosphatase that dephosphorylates nuclear factor of activated T-cells (NFAT), promoting its nuclear translocation and transcriptional activity. PMCA2 also influences CaMKII autophosphorylation, CREB phosphorylation, and expression of the calcineurin regulator RCAN1. Interacting partners such as PDZ domain proteins (e.g., NHERF2 and MAGUKs) scaffold PMCA2 at signaling microdomains, while Homer proteins and ??-synuclein further fine-tune its localization and function within the broader calcium signaling network, which includes TRPC channels, ORAI1, and SERCA pumps.
Disruption of ATP2B2 in HAP1 cells impairs calcium efflux, resulting in elevated basal and stimulated cytosolic Ca2+ concentrations. This dysregulation potently activates Ca2+-dependent effectors, notably calcineurin/NFAT and CaMKII signaling cascades, while altering the balance of pro-survival and apoptotic pathways. The near-haploid genetic background of HAP1 cells eliminates confounding heterozygosity, allowing a clear interpretation of the knockout??s impact on calcium homeostasis and downstream transcriptional programs. This model is particularly valuable for investigating calcium dynamics in a leukemic context, as many hematopoietic malignancies exhibit altered PMCA expression and Ca2+ signaling. Moreover, the model recapitulates molecular features relevant to ATP2B2-associated disorders, including autosomal dominant deafness and neurological conditions, providing a tractable in vitro system for mechanistic studies.
This polyclonal knockout population is well-suited for a range of applications in calcium signaling research, disease modeling, and functional genomics. Researchers can employ intracellular calcium imaging with Fluo-4 AM or genetically encoded indicators to monitor real-time Ca2+ fluxes and assess store-operated calcium entry. Western blotting with PMCA2-specific antibodies and RT-qPCR confirm loss of expression, while flow cytometry enables quantification of calcium responses in mixed populations. The model supports hearing loss research by enabling analysis of hair cell-like calcium handling, as well as studies of synaptic function and neurotoxicity linked to autism spectrum disorders and spinocerebellar ataxia. In drug discovery, the cells can serve as a screening platform for modulators of PMCA activity or calcium signaling pathways. For further information, please contact Ascent Research.
