Pcag cre gfp vector

Patient-derived iPSC colonies were pretreated with 10 μM Y27632 for 2 h prior to electroporation. Cells were then washed once with dPBS and dissociated into single cells using TrypLE^TM Select (Gibco). iPSC cells (5 × 10⁵) were electroporated with 2 μg Cas9, 2 μg sgRNA expression vector, and 4 μg donor plasmids using a Neon^R electroporator (Invitrogen, Carlsbad, CA, USA) as previously described^{10 (link)}. Transfected cells were plated onto a Matrigel-coated plate with 10 μM Y27632 for 2 days. G418 (100 μg/mL) was added to the culture medium 2 days after electroporation. After 12–14 days of G418 selection, half of the surviving colonies were manually lifted and lysed for genotype as described previously^{9 (link)}. Correctly targeted colonies were dissociated into single cells and reseeded for expansion and further analysis. To generate single cell-derived correctly targeted iPSCs, we performed an additional three rounds of single colony passaging with G418 selection. After three rounds of single colony passaging and G418 selection, the correctly targeted cell lines underwent excision from the neomycin resistance cassette. We electroporated 2 μg pCAG-Cre:GFP vector (Addgene #13776) into 5 × 10⁵ iPSCs and performed clonal selection without a selection drug.