Plasmids and in vitro transcription of cRNA

The following constructs were used: pCS2+ GFP-Myosin10(MYO10) (a gift from Nicolas Plachta, University of Pennsylvania, PA, USA), pspe3-GFP-UtrCH (Azoury et al., 2008) and pRN3-Histone(H2B)-RFP (Tsurumi et al., 2004). Plasmids were linearized with proper restriction enzymes and subsequently in vitro transcribed using the T3 or SP6 mMESSAGE mMACHINE (Ambion, AM1348 and 1340) transcription kits. The resulting cRNAs were purified using the RNeasy Mini Kit (Qiagen, 74104) and were centrifuged for 90 min at 4°C before being microinjected into the cytoplasm of prophase I-arrested oocytes.