The ATPAF2 Knockout HAP1 Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population with targeted disruption of the ATPAF2 gene in the HAP1 near-haploid cell line. This product provides a loss-of-function model for investigating mitochondrial Complex V biogenesis and oxidative phosphorylation. The polyclonal pool offers a heterogeneous genetic background, facilitating robust phenotype analysis without the artifacts of clonal selection.
The HAP1 cell line, derived from the chronic myelogenous leukemia KBM-7 line, is a male, near-haploid human cell model that carries the BCR-ABL fusion oncogene. Its haploid karyotype simplifies CRISPR/Cas9-mediated knockout by requiring modification of a single allele, enhancing editing efficiency. HAP1 cells retain critical metabolic pathways and are well-suited for functional genomic screens, particularly for essential mitochondrial genes where complete knockout phenotypes can be directly assessed.
ATPAF2 encodes a specific assembly factor for the F1 catalytic core of mitochondrial F1Fo-ATP synthase. It interacts directly with the ATP5A1 and ATP5B subunits to facilitate proper ATP synthase complex formation. Transcription of ATPAF2 is regulated by PGC-1??, NRF1, and TFAM. Knockout of ATPAF2 disrupts Complex V assembly, resulting in loss of ATP synthase activity, decreased ATP production, and collapse of mitochondrial membrane potential, recapitulating the bioenergetic failure seen in mitochondrial disorders.
In the near-haploid HAP1 background, ATPAF2 knockout yields a complete loss of gene function without allelic compensation, leading to pronounced oxidative phosphorylation defects. The BCR-ABL oncogene-driven metabolic adaptation may further sensitize cells to mitochondrial dysfunction, making this model useful for exploring the intersection of cancer cell metabolism and mitochondrial pathology. This system enables precise dissection of ATP synthase assembly defects in a genetically tractable human cell context.
Applications include functional studies of mitochondrial Complex V assembly, modeling of nuclear-encoded mitochondrial disorders such as Leigh syndrome, and drug screening for ATP synthase modulators. Compatible assays include western blotting for ATP synthase subunits, bioluminescent ATP measurements, JC-1/TMRM-based mitochondrial membrane potential analysis, and Seahorse oxygen consumption measurements. Complementation rescue with ATPAF2 and immunofluorescence for mitochondrial markers further validate phenotypes. For technical inquiries, contact Ascent Research.
