Transfection efficiency was assessed by nucleofecting 5 µg of pmaxGFP vector into another set of cells to monitor GFP expression. For clonal isolation of modified hiPSCs after nucleofection, cultures were dissociated into single cells with Accutase and plated at 1 cell/well in 96-well plates coated with irradiated SNL feeders. Cloned hiPSCs were maintained in hES medium supplemented with 10 µM ROCKi for 1 week. Single colonies in 96-well plates were passaged into 48-well plates and expanded in 24-well plates. Cells were then harvested for isolation of genomic DNA with a GenElute Mammalian Genomic DNA Miniprep Kit (Sigma), following the manufacturer’s instructions. Genomic DNA (250 ng) was amplified by PCR with Phusion High Fidelity DNA Polymerase (Thermo Fisher Scientific) and 0.5 µM forward and reverse primers flanking the ZFN binding sequence on APOE to yield a 326-bp fragment. The primer sequences were 5´-CTCGGCCGCAGGGCGCTGATGGA-3´ (forward) and 5´-GCCCCGGCCTGGTACACTGCCA-3´ (reverse). The PCR products were sequenced to identify the ZFN-introduced apoE4 to apoE3 conversion. The converted apoE3 isogenic clones were further confirmed by genomic sequencing of the APOE allele covering the donor region and upstream and downstream regions. The hiPSC lines were karyotyped by Cell Line Genetics.
