Neon microporator

The homologous recombination donor vector was composed of the MESP1 left arm, T2A fused with a membrane-bound tdTomato (mTomato), PGK promoter-driving puromycin resistance gene (PGK-Puro), MESP1 right arm, and MC-1 promoter-driving TK gene. H9 cells were electroporated with TALEN and donor vectors by Neon microporator (Invitrogen). After puromycin selection, targeted clones were picked and expanded for characterization. Detailed protocols (design of TALEN pair and synthesis of tandem arrays of TALE repeats) have been described elsewhere (manuscript submitted).

Fibroblasts were isolated from a 42-year-old female ADPKD patient who had the PKD1 frameshift mutation (c.7946_7947delCT) and cultured in DMEM supplemented with 10% FBS (Hyclone, Logan, UT, USA). Episomal vectors (Addgene #27077, #27078, #27080) were electroporated into fibroblasts (2 × 10⁵) using a NEON microporator (Invitrogen, Waltham, MA, USA) with 3 pulses at 1650 V for 10 min. Electroporated cells were cultured on a 6-well plate precoated with 8.7 µg/cm² Matrigel (Corning, Corning, NY, USA) for 3 days. On day 4, the media was switched to E8 medium (without TGF-β) for 2 more weeks. On week 4 of post electroporation, iPSC colonies were picked and cultured in a complete E8 medium containing DMEM/F12, L-ascorbic acid-2-phosphate magnesium (64 mg/L), FGF2 (100 µg/L), TGFβ1 (2 µg/L), insulin (19.4 mg/L), sodium selenium (14 µg/L), and transferrin (10.7 mg/L). Cells were routinely passaged and stocks were kept. For passaging, cells were treated with 0.5 mM EDTA for 6–8 min and iPSC clumps were gently collected and plated onto Matrigel-coated plates at a split ratio of 1:4. Rho-kinase inhibitor (Y-27632, Sigma, St. Louis, MO, USA) as an additive was found to be critical for cell survival following thawing or sub-culture. iPSCs were maintained in a CO₂ incubator at 37 °C with 5% CO₂.

A transcription activator-like effector nuclease (TALEN) pair was designed using online tool (http://boglabx.plp.iastate.edu/TALENT/). Tandem arrays of TALE repeats were synthesized by ViewSolid Biotech (http://www.v-solid.com) and joined to heterodimeric Fok I endonuclease. The homologous recombination donor vector consists of the following elements: the MESP1 left arm, T2A fused with a membrane-bound tdTomato (mTomato), PGK promoter driving puromycin resistance gene (PGK-Puro), MESP1 right arm and MC-1 promoter driving TK gene. H9 cells were electroporated with TALEN and donor vectors using Neon microporator (Invitrogen). After puromycin selection, individual undifferentiated colonies were picked and expanded for characterization. Detailed verification methods were described in Supplemental Methods.