Overexpression of Myc-Tfap2a in Oocytes

Oocytes were microinjected as previously described [77 (link)] using an Narishige microinjector (Narishige Inc., Sea Cliff, NY, USA) with an Olympus IX70 microscope (Olympus, Center Valley, PA, USA) equipped with an Eppendorf TM FemtoJet TM 4i microinjector (Eppendorf, Hauppauge, NY, USA) and completed within 30 min. For the overexpression experiments, 10 pL Myc-Tfap2a mRNA solution (1 μg/μL) was injected into the cytoplasm of denuded GV oocytes. The same amount of Myc mRNA (1 μg/μL) was injected as a control group. Following microinjection, the oocytes were arrested at the GV stage in α-MEM containing 10% (v/v) fetal bovine serum (Gibco-Invitrogen, Karlsruhe, Germany) and 200 nM IBMX for 12 h. Then, the oocytes were washed and cultured in an IBMX-free α-MEM medium (containing 10% (v/v) fetal bovine serum) under a humidified atmosphere of 5% CO₂ at 37 °C for 14 h.