Microinjection of Xenopus laevis oocytes.
Microinjection of Xenopus laevis oocytes.
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DOI：
10.3791/1106
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发表时间：
2009-02-23
期刊：
影响因子：
--
通讯作者：
Pante, Nelly
中科院分区：
文献类型：
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作者：
Cohen, Sarah;Au, Shelly;Pante, Nelly
非洲爪蟾卵母细胞显微注射后的薄层电镜（EM）是研究核细胞质转运的一个很好的系统。由于非洲爪蟾卵母细胞具有较大的细胞核和高密度的核孔复合物（NPCs），因此可以很容易地观察到核运输过程。通过使用这一系统，对核进出口机制有了更深入的了解（由Pante审查，2006年）。此外，我们还使用爪蟾卵母细胞显微注射来解剖几种病毒在宿主细胞核中复制的核输入途径。在这里，我们展示了带有核输入底物的非洲爪蟾卵母细胞的细胞质显微注射。我们还展示了通过薄切片电镜观察注射卵母细胞的制备，包括解剖、脱水和将卵母细胞包埋到环氧包埋树脂中。最后，我们提供了用加利福尼亚自体杆状病毒（AcMNPV）或小鼠细小病毒（MVM）衣壳微注射卵母细胞的代表性结果，并讨论了该技术的潜在应用。
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 Pante, 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.