Name: microinjection of xenopus laevis oocytes
Uploaded: 2009-02-23T00:00:00
Description: Watch this Scientific Journal Video about Microinjection of Xenopus Laevis Oocytes at JoVE.com

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).

Part 1: Preparation of Xenopus oocytes for microinjection
<ol>
	<li>Place a small piece (about 2 cm) of Xenopus laevis ovary into a 50-ml conical tube containing 20 ml of collagenase solution (5 mg/ml collagenase in calcium-free modified Barth&#8217;s saline: 88 mM NaCl, 1 mM KCl, 0.82 mM MgSO4, 10 mM Hepes, pH 7.5). Collagenase is used to remove the follicle cells which surround the oocytes.</li>
	<li>Place the tube on a shaker platform and rock it gently (at 100 RPM) for one hour. This ti...

<ol>
	<li>Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+intact+Xenopus+oocytes+in+nucleocytoplasmic+transport+studies.">Use of intact Xenopus oocytes in nucleocytoplasmic transport studies.</a> Methods Mol. Biol. 322, 301-314 (2006).</li><li>Pant&#233;, N., Kann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+pore+complex+is+able+to+transport+macromolecules+with+diameters+of+about+39+nm.">Nuclear pore complex is able to transport macromolecules with diameters of about 39 nm.</a> Mol. Biol. Cell. 13 (2), 425-434 (2002).</li><li>Rabe, B., Vlachou, A., Pant&#233;, N., Helenius, N., Kann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+import+of+hepatitis+B+virus+capsids+and+release+of+the+viral+genome.">Nuclear import of hepatitis B virus capsids and release of the viral genome.</a> Proc. Natl. Acad. Sci. U.S.A. 100 (17), 9849-9854 (2003).</li><li>Cohen, S., Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pushing+the+Envelope:+Microinjection+of+MVM+into+Xenopus+oocytes+causes+damage+to+the+nuclear+envelope.">Pushing the Envelope: Microinjection of MVM into Xenopus oocytes causes damage to the nuclear envelope.</a> J. Gen. Virol. 86 (12), 3243-3252 (2005).</li><li>Pant&#233;, N., Jarmolowski, A., Izaurralde, E., Sauder, U., Baschong, W., Mattaj, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+nuclear+export+of+different+classes+of+RNA+by+electron+microscopy.">Visualizing nuclear export of different classes of RNA by electron microscopy.</a> RNA. 3, 498-513 (1997).</li></ol>

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 Pant&eacute;, 2006). It has also been used to study the nuclear import of nuclear-replicating viruses (Pant&eacute; and Kann, 2002; Rabe et al.<...

microinjection of xenopus laevis oocytes

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 Pant&eacute;, 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.

Watch this Scientific Journal Video about Microinjection of Xenopus Laevis Oocytes at JoVE.com

Microinjection of Xenopus Laevis Oocytes

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.

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

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