Summary
Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.
Abstract
Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Panté, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus.
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.
Protocol
Discussion
Microinjection of Xenopus oocytes combined with thin-sectioning EM is a highly effective tool for studying nucleocytoplasmic transport. This system has been used to map distinct steps of import through the NPC, for example interaction of a nuclear import substrate with structural components of the NPC such as the cytoplasmic filaments and nuclear basket (reviewed by Panté, 2006). It has also been used to study the nuclear import of nuclear-replicating viruses (Panté and Kann, 2002; Rabe et al.<…
Acknowledgements
We thank David Theilmann (Pacific Agri-Food Research Centre, Summerland, British Columbia) for providing the baculovirus AcMNPV and for helpful discussion.
This work was supported by grants from the Canada Foundation for Innovation (CFI), the Canadian Institutes of Health Research (CIHR), the Michael Smith Foundation for Health Research (MSFHR), and the Natural Sciences and Engineering Research Council of Canada (NSERC).
References
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