Cell transfection

For the overexpression vector (carrier framework pCDNA3.0), 1.5×10⁵/well chick embryo fibroblasts (CEFs)/ESCs/PGCs were seeded into a 24-well plate. CEFs/ESCs/PGCs were seeded in 24-well plates (Corning) at 1×10⁵ cells/well. When cell density became 70%, cells were transfected with FuGENE^® HD (Promega) at a 3:1 ratio of reagent (volume, μl) to plasmid (mass, ng). It should be noted that in ESCs and PGCs, transfection should be conducted continuously three times (at 1 day intervals).

For interference vectors (lentiviral vector), 1×10⁵/well CEFs/ESCs/PGCs were seeded into a 24-well plate. When cell density became 70%, the lentiviral vector was used to infect CEFs/ESCs/PGCs under 10 multiplicity of infection and 5 ng/ml polybrene (Santa Cruz Biotechnology, sc-134220).