Mek inhibitor pd0325901

To overexpress Nanog or Klf5 in Fgf5-P2A-Venus BAC Tg mEpiSCs, we used piggyBac transposon and a transposase system. The pPB-CAG-Flox-Nanog/Klf5-dsRedT4-iresHygroR plasmid was generated by combination of the PB-CAG backbone and pPyCAG-Flox-Nanog/Klf5-dsRedT4-iresHygroR, both of which were kindly provided by Dr. Hitoshi Niwa (Kumamoto University, Japan). Plasmids were then co-transfected with piggyBac transposase into the Tg mEpiSCs as previously described [16 (link)]. After 7 days of selection with 250 μg/ml Hygromycin B (InvivoGen, San Diego, CA, USA), colonies were picked for stable Klf5 and Nanog-overexpressing Tg mEpiSC lines.
For reprogramming experiments, 2–4 × 10⁴ cells were seeded onto fibronectin-coated 6-well plates in EpiSC culture conditions. After 24 h, the medium was switched to 2i/LIF conditions; the 2i inhibitors included 1 μM Mek inhibitor PD0325901 (Wako Pure Chemical Industries) and 3 μM Gsk3 inhibitor CHIR99021 (Wako Pure Chemical Industries). After 7 days, immunofluorescence analysis was performed. To check the characteristics of the resulting miPSCs, several miPSC colonies were picked up, expanded, and then cultured in 2i/LIF conditions for further experiments.

Female mT/mG mice were superovulated by intraperitoneal injections of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) at an interval of 48 h. hCG-injected female mice were mated with Oct4dPE-CreER^T2 male mice, and the copulatory plug was confirmed the next morning. Blastocysts were collected from the uterus 3 days later. Collected blastocysts were plated on mitomycin-treated mouse embryonic fibroblast feeder cells in 2i-LIF medium (ESGRO Complete Basal medium; Millipore, Germany) containing leukemia inhibitory factor (LIF) (Wako, Tokyo, Japan), 0.4 µM MEK inhibitor PD0325901 (Wako, Tokyo, Japan), 3 µM GSK3 inhibitor CHIR99021 (Wako, Tokyo, Japan) and Penicillin-Streptomycin (Invitrogen). Outgrowths from blastocysts were disaggregated and passaged to new culture wells containing feeder cells in 2i-LIF medium. Once ES cell colonies emerged, these cells were expanded for genotyping and frozen. ES cells were treated with Proteinase K (Roche) and genotyped using the following primers: [WT Rosa locus; Rosa-F (5′-ctc tgc tgc ctc ctg gct tct-3′) and Rosa-R (5′-cga ggc gga tca caa gca ata-3′), mT/mG locus; Rosa-F and mT/mG R (5′-tca atg ggc ggg ggt cgt t-3′), and CreER^T2; CreER^T2-F (5′-gaa gca act cat cga ttg att tac gg-3′) and CreER^T2-R(5′-tga agg gtc tgg tag gat cat act c-3′)]. We named the established ES cells TGOC referring to Rosa-mTmG/Oct-dPE-CreERT.