Inducing hZSCAN4 expression in hES cells

SEES3 hES cells^{22 (link)} were obtained from the Center for Regenerative Medicine, National Research Institute for Child Health and Development, Japan. The cells were cultured in StemFit AK-02 medium (Ajinomoto) on iMatrix-511 (Nippi)-coated plates. The medium was changed daily, and cells were routinely split every 4–5 days. ES cells were transfected with synthetic mRNA encoding DUX4, as previously described,^{23–25 (link)} to induce hZSCAN4 expression. Briefly, DUX4 cDNA^{11 (link)} was subcloned into a plasmid containing a T7 promoter. DUX4 mRNA was synthesized following a previously described in vitro transcription protocol.^{26 (link)} The synthesized RNA was then transfected with Lipofectamine MessengerMAX (Invitrogen) according to the instructions.