This polyclonal knockout cell product is a CRISPR/Cas9-edited population of HEK293T cells in which the ATP5MK gene has been disrupted. As a polyclonal pool, the cells contain a heterogeneous mixture of loss-of-function mutations, eliminating the need for single-cell cloning while providing a robust model for studying gene function. The targeted disruption of ATP5MK enables researchers to investigate the role of this mitochondrial ATP synthase subunit in cellular energy metabolism and mitochondrial physiology.
The host cell line, HEK293T, is a widely used human embryonic kidney epithelial cell derivative that stably expresses the SV40 large T antigen. This feature permits episomal replication of plasmids bearing the SV40 origin, making these cells highly efficient for viral packaging, recombinant protein production, and transient transfection experiments. Despite their tumor-derived origin, HEK293T cells maintain functional mitochondria and a balance between glycolytic and oxidative metabolism, offering a tractable system for mitochondrial studies.
ATP5MK encodes a critical component of the F0 sector of the mitochondrial ATP synthase (Complex V), where it participates in proton translocation across the inner mitochondrial membrane to drive ATP synthesis. The gene is transcriptionally regulated by key metabolic regulators including PGC-1??, NRF1, and TFAM, and its expression is influenced by cellular energy status through the AMP/ATP ratio. ATP5MK interacts with other F0 subunits such as ATP6 and ATP8 as well as assembly factors to form a functional proton channel. Downstream, ATP synthase activity governs mitochondrial membrane potential and reactive oxygen species production, and its impairment is linked to deficiencies in oxidative phosphorylation and Complex V assembly.
In the HEK293T background, knockout of ATP5MK creates a relevant model for dissecting the contributions of ATP synthase to cellular bioenergetics. Because these cells rely on both glycolysis and oxidative phosphorylation, disruption of the F0 complex leads to compensatory metabolic shifts that can be experimentally monitored. This model recapitulates features of mitochondrial Complex V deficiency, Leigh syndrome, and other metabolic disorders, thereby enabling mechanistic studies into the molecular pathogenesis of mitochondrial diseases.
This knockout cell pool is suitable for a broad range of research applications, including mitochondrial biology, oxidative phosphorylation analysis, and drug screening for metabolic disorders. Researchers can employ functional assays such as Seahorse metabolic flux analysis, ATP luminescence measurements, and mitochondrial membrane potential assessment using dyes like JC-1 or TMRM. Molecular characterization can be performed via western blotting for ATP synthase subunits and RT-qPCR for mitochondrial gene expression. For additional information about this product, please contact Ascent Research.
