The CAST Knockout HAP1 Polyclonal Cells product provides researchers with a population of HAP1 cells subjected to CRISPR/Cas9-mediated disruption of the CAST gene, generating a mixed population of loss-of-function mutants. This polyclonal knockout model avoids single-cell clonal selection, preserving natural heterogeneity while introducing targeted gene disruption across the cell pool.
The HAP1 cell line is a human near-haploid line originally derived from the KBM-7 chronic myeloid leukemia (CML) cell line. Its haploid karyotype makes it exceptionally useful for genetic loss-of-function screens, as single-allele disruption often suffices to produce a phenotype. HAP1 cells retain many features of hematopoietic cells and provide a physiologically relevant platform for investigating signaling pathways in a leukemic background.
The CAST gene encodes calpastatin, the endogenous inhibitor of the calpain proteases CAPN1 (calpain-1) and CAPN2 (calpain-2). Calpastatin activity is regulated by upstream signals including NF-??B, c-Jun, and intracellular calcium levels. When active, calpastatin binds and inhibits calpains, preventing the cleavage of downstream substrates such as spectrin, talin, PKC, Bid, and Bax. This inhibition is critical for controlling calpain-mediated processes like apoptosis, cell migration, and cytoskeletal remodeling.
Disruption of CAST in HAP1 cells eliminates calpastatin expression, removing the primary brake on calpain activity. Consequently, unregulated calpain proteolysis may lead to enhanced degradation of key substrates, altering apoptosis thresholds, cell adhesion dynamics, and signal transduction. This model is particularly relevant for studying neurodegenerative conditions such as Alzheimer disease, in which calpain dysregulation contributes to pathology, as well as ischemia-reperfusion injury and muscular dystrophy. The haploid background simplifies genetic studies, allowing cleaner interpretation of the effects of CAST loss.
This polyclonal knockout cell population is well-suited for a range of downstream applications including western blotting to assess calpastatin and calpain substrate levels, fluorometric or colorimetric calpain activity assays, scratch wound-healing or transwell migration assays, and flow cytometric apoptosis assays with Annexin V staining. Researchers can also use the cells in calcium imaging studies to examine how CAST loss influences calcium-dependent signaling. Additionally, the cells serve as a platform for screening small-molecule calpain inhibitors. For further information, please contact Ascent Research.
