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.
Highlights
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 visualized in the Xenopus oocyte
Transfer the injected oocytes to a small (35-mm diameter) Petri dish filled with modified Barth’s saline (MBS), and incubate at room temperature for the desired amount of time
Summary
Place the tube on a shaker platform and rock it gently (at 100 RPM) for one hour This time varies with different lots of collagenase. 4. Using a dissecting microscope, select mature stage VI oocytes for microinjection. Select mature stage VI oocytes for microinjection These oocytes are large, with good contrast between the black animal hemisphere and the creamy-colored vegetal hemisphere. 5. Transfer the mature stage VI oocytes into a microwell plate (Nunc, 10 μl well volume). Transfer the mature stage VI oocytes into a microwell plate (Nunc, 10 μl well volume) This should be done carefully, using a 200-μl pipettor with a pipette tip that has been cut at the end to allow undisrupted suction of the oocytes
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