Generating Biallelic KO Pig Fibroblasts

Pig fetal fibroblasts (PFFs) were prepared as previously described [29 (link)]. Prior to transfection, the PFFs were thawed and cultured in medium (10% FBS and 1% PS) until sub-confluence was reached. Approximately 7 × 10⁵ PFFs in 700 μL PBS mixed with 10.5 μg of the TALEN plasmid pairs were transfected by electroporation at 250 V for a single 20-ms pulse (Gene Pulser Xcell Microbial System, Bio-Rad, USA) in a 4-mm gap cuvette. Then, the cells were seeded in 5 mL of fresh DMEM containing 10% FBS in a T25 culture flask following a 48-h incubation at 37 °C. The cells were then trypsinsed, and the extremely dilute culture method was used to cultivate the cells and obtain single cell colonies. After 12–14 days, the colonies were assessed via polymerase chain reaction (PCR) (upstream primer, 5′-ACTGCTCTCTGCCCTTGTCTT-3′; downstream primer, 5′-AGAGTGTGATGGGAAGGATGAG-3′), and the amplified fragments were used for genotyping, including restriction endonuclease analysis and sequencing. Finally, we selected positive fibroblasts cell lines with a biallelic KO as nuclear donors for SCNT.