The DYSF Knockout HAP1 Polyclonal Cells are a pooled population of HAP1 cells that have been subjected to CRISPR/Cas9-mediated disruption of the DYSF gene, generating a heterogeneous mix of loss-of-function alleles. This polyclonal knockout format provides a robust cellular model for studying dysferlin biology without clone-specific artifacts, enabling functional analyses of membrane repair and associated pathways.
The HAP1 cell line is a near-haploid human cell line derived from KBM-7 chronic myeloid leukemia cells. These adherent, fibroblast-like cells possess a stable haploid karyotype for most chromosomes, making them exceptionally useful for genetic perturbation studies. The reduced genetic complexity facilitates straightforward genotype-phenotype correlations, and the cell line’s rapid growth and ease of culture support high-throughput applications.
Dysferlin, encoded by DYSF, is a transmembrane protein critical for calcium-dependent plasma membrane repair. Upon membrane injury, dysferlin mediates vesicle fusion at damage sites, a process essential for maintaining myofiber integrity. Its activity is transcriptionally regulated by myogenic factors such as MYOD1, MEF2C, and MYOG. Dysferlin interacts with caveolin-3 (CAV3), annexins A1 and A2 (ANXA1, ANXA2), AHNAK, TRIM72 (also known as MG53), and the protease CAPN3 to orchestrate membrane resealing. Downstream effects include annexin recruitment and vesicle trafficking, ultimately preserving sarcolemmal integrity. Disruption of dysferlin impairs this repair mechanism, leading to progressive muscle degeneration characteristic of dysferlinopathies.
In the HAP1 context, DYSF knockout cells provide a simplified, genetically tractable platform to dissect the molecular requirements for membrane repair. The near-haploid nature of HAP1 ensures that single-copy gene disruption yields a complete loss of function, facilitating clean phenotypic readouts. These cells can be used to study defective membrane sealing, calcium dysregulation, and the interplay between dysferlin and its binding partners without the confounding complexity of diploid muscle cells. Moreover, the leukemic origin of HAP1 allows for exploration of dysferlin’s potential roles beyond muscle, including in hematopoietic cells.
Researchers can employ these knockout cells in a variety of assays, such as laser-induced membrane wounding, FM1-43 dye uptake to assess repair kinetics, calcium imaging to monitor injury-induced fluxes, and co-immunoprecipitation to map protein interactions. Western blotting and immunofluorescence can confirm loss of dysferlin expression. This model is particularly suited for studying the pathophysiology of limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal myopathy with anterior tibial onset, as well as for screening compounds that rescue membrane repair defects. For additional information, please contact Ascent Research.
