Generation of PERV-resistant Porcine Fibroblasts

Immortalized porcine fibroblasts [22 (link)] were cultured in DMEM (Gibco, Waltham, MA, USA) supplemented with 1% penicillin/streptomycin (Gibco), 10% fetal bovine serum (Gibco), 100 mM β-mercaptoethanol (Sigma, St Louis, MO, USA), and 1% nonessential amino acids (Gibco) at 37 °C with 5% CO₂. We transfected 3 × 10⁵ cells using the Neon Nucleofection system (Invitrogen, Carlsbad, CA, USA). Briefly, 500 ng of the PB-CMV-Target-AID-PERV(pol-gag) vector and 500 ng of the transposase vector (pCy43, provided by the Sanger Institute) were transfected at 1400 V for 20 ms, with a pulse number of 2. After 2 d of transfection, the fibroblasts were treated with 2 µg/mL neomycin (Sigma) for 10 d for antibiotic selection. After selection, single cells were subcultured in each well of 96-well plates and then expanded. Cell colonies from each single cell were analyzed by PCR to confirm the integration of the vector. The primer sequences are listed in Table 2.