To evaluate possible genomic rearrangement and CNV of ATM in individual cells, we used a fluorescence in situ hybridization (FISH) probe specific for the ATM gene (chr11:108,089,374–108,241,929 in the hg19 genome). Results indicated that a majority of cells had an equal number of ATM-specific puncta as those specific for a chr11 centromere control FISH probe (Figure 3A, “Normal”). A much smaller percentage of cells had one ATM singlet punctum and a doublet puncta compared with two singlets for chr11 control (“SD”), indicating asynchronous DNA replication. ATM-deficient cells have difficulties responding to oxidative stress in cell culture, and they may have diminished control of asynchronous DNA replication due to deficiencies in p53 signaling (Nagler et al., 2010). Rarely, cells were characterized as aneuploid (“AP”) that likely reflected early stages of cell death. There were no significant differences in these percentages across genotype (ATM−/− versus ATM+/−) or iPSC line. This indicates that no deletion or duplication of the ATM gene was detected, even in a subset of cells and therefore there was no evidence for cell-to-cell variability in ATM gene copy number.
