To generate isogenic apoE3/3-hiPSC lines, we dissociated the parental apoE4/4 hiPSCs with Accutase (Millipore) and placed 2×106 cells in nucleofection solution (Lonza) containing 4 µg of the APOE-ZFN mRNA pair together with 5 µg of apoE3 donor DNA. A specific pair of six-finger ZFNs engineered to target a region around the Arg-112 of apoE4 allele was designed and prepared by Sigma. The ZFNs bound (uppercase) and cut (lowercase) the following sequence on the APOE gene: CGGGCACGGCTGTCCAAGgagctGCAGGCGGCGCAGGCCCG. The mixture was transferred to a cuvette and nucleofected with Nucleofector II (Lonza) at Nucleofector setting A-23 and the Human Stem Cell Nucleofector Kit I, as reported29. Nucleofected cells were quickly transferred to 500 µl of prewarmed (37°C) hES cell medium, incubated at 37°C for 5 min, and plated at 1×106 cells per well in an irradiated SNL-coated six-well plate containing hES medium supplemented with 10 µM ROCK inhibitor (ROCKi) (Tocris). To maximize viability and efficiency, the entire procedure was completed within 20 min.
