A11105 01

Human embryonal NTERA-2 cells (NT2/D1, a kind gift from Peter Andrews, University of Sheffield) were grown in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) high glucose with 2 mM L-Glutamine, 10% fetal calf serum (FCS), and 1% Penicillin/Streptomycin (P/S). They were split in half by cell scraping. 293T cells were grown in standard DMEM + 10% FCS and P/S. J1 ESCs (129S4/SvJae) or their derived triple knockout (TKO) cells, which are knockout for Dnmt3a, Dnmt3b, and Dnmt1 (from M. Okano) were used where stated, or as an independent mESC line, ES3 ESCs (Rowe et al. 2010 (link)). All mESCs were cultured on gelatin-coated plates (0.2%), either in standard media as previously described (Rowe et al. 2013a (link)), or in media containing two small-molecule inhibitors of MEK and GSK3 plus LIF (2i + LIF), as described (Ying et al. 2008 (link); Ficz et al. 2013 (link)). Cells were split every other day using accutase (Gibco, A11105-01) and cultured in 2i media for 7 d before transduction.

Blastocysts were seeded on Mitomycin C-treated mouse embryonic fibroblasts on 0.1% gelatin-coated plates (ES-006-B, Millipore, USA) with PKC inhibitor (5 μM Gӧ6983, 133053-19-7, Selleck, USA) in basic culture medium supplemented with Dulbecco’s modified Eagle’s medium (DMEM; 10829018, Gibco, USA) containing 15% knockout serum replacement (10828028, Gibco), 1% penicillin/streptomycin (SV30010, HyClone, USA), 2 mM glutamine (35050061, Gibco), 1 mM sodium pyruvate (11360088, Gibco), and 0.1 mM 2-mercaptoethanol (ES-007-E, Millipore). After culture for 7 days, outgrowths were collected and digested into single-cell suspensions with accutase (A1110501, Gibco) and re-seeded in new plates coated with feeder cells. mES passaging was performed by incubating colonies with accutase, followed by plating at a density of 1 × 10³ cells/cm² into a new 24-well plate coated with new feeder cells at 3- to 4-day intervals. Collected mES were frozen in a cryopreservation medium with 90% fetal bovine serum (FBS; SH30070.03, HyClone) and 10% dimethyl sulfoxide (D5879) and stored in liquid nitrogen.