Pmx reprogramming vectors

The pMX reprogramming vectors were purchased from Addgene (Cat. 17964, 17965, 17966, and 17967, respectively). The retroviral vectors were produced by transfection of these vectors (15 μg each) into PLAT-A cells in 150 μl of iN-fect in vitro transfection reagent (iNtRON, Seongnam, Korea). The first virus-containing supernatant of the transfected cells was collected 24 h after transfection and replaced with fresh medium, which was collected 24 h later as the second virus-containing supernatant. Each virus-containing supernatant was filtered through a 0.45 μm syringe membrane filter. The pMX-DsRed vector was used to evaluate viral infection efficiency.

The cDNAs from chicken POUV (POU5F3), SOX2, SOX3, KLF4, c-MYC, NANOG, and LIN28 were cloned, sequenced, inserted into the pGAE lentivirus or pPB transposon backbones, and deposited to Addgene. All constructs were generated using the Gibson Assembly Mix (NEB) and sequenced to validate the cDNA insertion. Viral ready-to-use stocks were purchased for the polycistronic pLentG-mKOSM lentiviral vector expressing mouse genes (Cell Biolabs) and for the Sendai viral vectors expressing the human genes (Cytotune Kit, Life Technologies). pMX reprogramming vectors were purchased from Addgene (nos. 13366, 13367, 13375, and 13370).