Regulation of DNA Synthesis at the First Cell Cycle in the Sea Urchin In Vivo
Using fluorescent and non-fluorescent recombinant proteins has proved to be a very successful technique for following postfertilization events, in both male and female pronuclei during the first cell cycle of sea urchin in vivo. Proteins and dyes are introduced by microinjection into the unfertilized egg, and their function can be monitored by fluorescence or confocal/two-photon (2P) and transmitted light microscopy after insemination. Here, we describe expression and purification of GFP/RFP-tagged proteins involved in regulation of DNA replication. We also explain the techniques used to introduce recombinant proteins and fluorescent tubulin into sea urchin eggs and embryos.
Key wordsFluorescent proteins PCNA GFP/RFP Microinjection Confocal/2P imaging Male and female pronuclei