The DS iPSC line was cultured in 10 μM of Rho-associated protein kinases (ROCK) inhibitor (Calbiochem; Y27632) 24 h before electroporation. Single cells (1×107) were harvested using TryPLE select (Invitrogen), resuspended in 1 x PBS and electroporated with a total of 55 μg DNA including five plasmids (XIST, DYRK1A ZFN1, DYRK1A ZFN2, rtTA/puro, and AAVS1 ZFN) with both 3:1 and 5:1 ratios of XIST: rtTA/puro. The electroporation conditions were 220v, and 750 μF (BioRad Gene Pulser II System). Cells were subsequently plated on puromycin-resistant DR4 MEF feeders (Open Biosystems, Cat#: MES3948) in hiPSC medium supplemented with ROCK inhibitor for the first 24 h. Over 300 colonies remained after 12 days of 0.4 μg/ml puromycin selection and 245 randomly chosen individual colonies across 36 pooled wells were examined by interphase DNA/RNA FISH for the presence and expression of XIST, correct targeting and retention of trisomy (since some subclones lacked XIST or showed just two DYRK1A DNA signals). Over 100 individual clones were isolated and characterized, and those of interest, containing targeted XIST on one of three DYRK1A loci, were frozen. Six single target clones with good pluripotent morphology, OCT4 positive staining, correct targeting to one trisomic chromosome, and good XIST RNA paint were expanded for further characterization. One double and one triple target line, two non-target clones, and one disomic clone were also isolated and frozen. Targeting and correct chromosome number (47) was confirmed by interphase and metaphase FISH and genome integrity by high resolution G-band karyotype and CGH array.