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Regulation of DNA Synthesis at the First Cell Cycle in the Sea Urchin In Vivo

  • Jolanta Kisielewska
  • Michael Whitaker
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1128)

Abstract

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 words

Fluorescent proteins PCNA GFP/RFP Microinjection Confocal/2P imaging Male and female pronuclei 

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Jolanta Kisielewska
    • 1
  • Michael Whitaker
    • 1
  1. 1.Institute of Cell and Molecular Biosciences, Faculty of Medical Sciences, The Medical SchoolNewcastle UniversityNewcastle upon TyneUK

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