Visualizing Cytoplasmic Spindle and F-Actin

The cytoplasmic spindle formation and F-actin meshwork were investigated by live imaging of F-actin with mRNA encoding b5-tubulin–GFP [11 (link), 12 (link)] and UtrCH–eGFP [11 (link), 13 (link)] as previously described. Templates of in vitro transcription from constructed plasmids were obtained by PCR with F and R primers. PCR products were diluted in RNAse-free water. cRNA transcripts were synthesized in vitro with T7 RNA polymerase mMESSAGE mMACHINE T7 kit (Ambion, Life. Co, Calsbad, CA, USA). Poly(A) tail was added to the sequence end by polymerase tailing kit (PAP5104, Lucigen, Beijing, China). The RNA solutions were then stored at − 80 °C in a final concentration of 400 μg/mL until further use. Approximately 50 pl of RNA solution was injected into each GV oocyte.