Generation of Miniature Pig iPSCs from Fibroblasts

For the generation of miniature pig iPSCs, 10⁶ fibroblasts at P3 were transfected with 4 μg of episomal plasmid pMaster12 (Addgene # 58527)^{17 (link)} using Nucleofector™ II with the A-024 program (Amaxa, Walkersville, MD, USA) and the fibroblast-specific Nucleofector kit (Lonza, Hayward, CA, USA). Transfected cells were plated on a feeder layer of γ-ray-treated MEFs in a 6-well plate containing modified E8 medium (E8 medium supplemented with 5 ng/ml activin A, 1.5 µM CHIR99021, 2.5 µM IWR-1, and 10 ng/ml LIF). The medium was replaced with fresh modified E8 medium and G418 (400 µg/ml) the next day and then maintained for 5 days before switching back to modified E8 medium. After 3 weeks, cell colonies were picked under a microscope. Each colony was individually transferred to a well of 24-well plates containing MEF feeder layers with modified E8 medium for expansion and passaging afterward. The SIGMAFAST BCIP/NBT kit (Sigma–Aldrich, Burlington, MA, USA) was used to stain iPSCs for ALP activity following the manufacturer’s instructions. The reprogramming efficiency of fibroblasts was determined by counting the number of iPSC colonies positive for ALP staining per million cells in culture 21 days after transfection.