Ward Brian M
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Methods Mol Biol. 2009;515:1-11. doi: 10.1007/978-1-59745-559-6_1.
Fluorescent protein (FP) fusions not only allow for the convenient visualization of a protein of -interest's subcellular localization but also permit the real-time monitoring of their subcellular trafficking. The subcellular fluorescent pattern of FP-fusions can also serve as a visual marker for various subcellular processes using either live or static microscopy. We have employed FP-fusions for the study of poxvirus morphogenesis. Fusion of FP with either a viral core protein or an extracellular virion-specific protein can serve as a visual read-out for normal poxvirus morphogenesis at the subcellular level. Recombinant viruses expressing a FP-fusion, in conjunction with the deletion of a gene involved in either morphogenesis or egress, usually display an aberrant FP pattern. Functional domains in the missing protein are then mapped by complementation in-trans followed by fluorescent microscopy for analysis of the FP pattern. The methods presented here describe how to infect and transfect cells for trans-complementation for the purpose of functional domain mapping. The imaging and analysis of these cells is described.
荧光蛋白（FP）融合不仅便于观察感兴趣蛋白质的亚细胞定位，还能实时监测其亚细胞转运。FP融合蛋白的亚细胞荧光模式也可作为使用活细胞或静态显微镜观察各种亚细胞过程的视觉标记。我们已利用FP融合来研究痘病毒的形态发生。FP与病毒核心蛋白或细胞外病毒粒子特异性蛋白的融合可作为亚细胞水平上正常痘病毒形态发生的视觉读数。表达FP融合蛋白的重组病毒，结合缺失参与形态发生或释放的基因，通常会呈现异常的FP模式。然后通过反式互补，随后用荧光显微镜分析FP模式，来定位缺失蛋白中的功能域。这里介绍的方法描述了为进行功能域定位而感染和转染细胞以进行反式互补的方法。还描述了对这些细胞的成像和分析。
