The ATP2B4 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout population derived from the HAP1 cell line, providing a loss-of-function model for the ATP2B4 gene. Disruption of ATP2B4 eliminates the plasma membrane calcium ATPase 4 (PMCA4), enabling functional studies of calcium homeostasis in a near-haploid background. The polyclonal composition avoids single-cell cloning artifacts and offers a heterogeneous knockout pool suitable for pooled analyses and straightforward expansion.
HAP1 cells are a human male near-haploid chronic myeloid leukemia (CML) cell line originating from KBM-7. Their near-haploid karyotype simplifies genetic perturbation, as disruption of a single allele yields unambiguous phenotypes. Retaining leukemic features, HAP1 cells serve as a robust platform for investigating oncogenic signaling, drug resistance, and functional genomics. This background is extensively used in CRISPR screens and mechanistic studies due to its genetic tractability.
ATP2B4 encodes PMCA4, a high-affinity Ca2+-ATPase that actively extrudes cytosolic calcium. The pump is activated by Ca2+/calmodulin and regulated by cAMP/PKA, PKC, and PIP2, while scaffolding interactions with CASK, NOS1, DLG1, and MAGI1 localize its activity to specific membrane domains. PMCA4 modulates downstream effectors including calcineurin, NFAT, CaMKII, and nitric oxide synthase. Knockout elevates intracellular calcium, driving calcineurin-mediated NFAT dephosphorylation and altering nitric oxide production, with consequent impacts on transcription of NFAT target genes including cell cycle regulators and modulation of CaMKII-dependent phospho-signaling.
In HAP1 CML cells, ATP2B4 knockout disrupts calcium-dependent pathways controlling proliferation, apoptosis, and migration. The near-haploid state amplifies phenotypic effects, facilitating studies of calcium signaling in leukemia progression and chemoresistance. This model is valuable for dissecting PMCA4??s role in MAPK and calcineurin/NFAT crosstalk, and for validating targets in myeloid malignancies. Furthermore, this knockout model facilitates investigation of crosstalk between calcium and cAMP signaling pathways that govern leukemic cell fate decisions.
Applications include calcium imaging with Fluo-4 AM, Western blotting for PMCA4 deficiency, RT-qPCR for ATP2B4 mRNA validation, NFAT reporter assays, nitric oxide detection, and phospho-signaling profiling of CaMKII and NFAT. The knockout cells are suited for functional genomics screens, drug sensitivity testing, apoptosis assays (Annexin V), and proliferation analyses (MTS). Co-immunoprecipitation can probe altered PMCA4 interactions. For further details, please contact Ascent Research.
