The technology includes Pink1 knockout HeLa cells that were generated using CRISPR technology. Pink1 is the key master gene to trigger degradation of mitochondria, mitophagy, and is implicated in familial Parkinson Disease. Knocking out Pink1 allows us to study the roles of Pink1 in many aspects of mitophagy and to display Pink1-dependent or independent activity.
To create the HeLa cells, two CRISPR gRNAs targeting exon 1 and exon 7 of the Pink1 genome were used for transfection with Cas9 and GFP-C1 reporter. Cells were sorted 2 days after transfection and plated out in 96-well plates. Ten days later, single colonies were transferred to 24-well plates and then genomic DNA of each colony was isolated for genotyping. The positive clones were then confirmed by Western blot.
Pink1 is constantly degraded in normal condition and it has been difficult to raise a good and specific Pink1 antibody. The Pink1 knockout cell line can be the gold standard to verify if the antibody is specific.