Green Rebecca A, Audhya Anjon, Pozniakovsky Andrei, Dammermann Alexander, Pemble Hayley, Monen Joost, Portier Nathan, Hyman Anthony, Desai Arshad, Oegema Karen
Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093, USA.
Methods Cell Biol. 2008;85:179-218. doi: 10.1016/S0091-679X(08)85009-1.
The Caenorhabditis elegans gonad and early embryo have recently emerged as an attractive metazoan model system for studying cell and developmental biology. The success of this system is attributable to the stereotypical architecture and reproducible cell divisions of the gonad/early embryo, coupled with penetrant RNAi-mediated protein depletion. These features have facilitated the development of visual assays with high spatiotemporal resolution to monitor specific subcellular processes. Assay development has relied heavily on the emergence of methods to circumvent germline silencing to allow the expression of transgenes encoding fluorescent fusion proteins. In this chapter, we discuss methods for the expression and imaging of fluorescent proteins in the C. elegans germline, including the design of transgenes for optimal expression, the generation of transgenic worm lines by ballistic bombardment, the construction of multimarker lines by mating, and methods for live imaging of the gonad and early embryo.
秀丽隐杆线虫的性腺和早期胚胎最近已成为用于研究细胞和发育生物学的一种有吸引力的后生动物模型系统。该系统的成功归因于性腺/早期胚胎的定型结构和可重复的细胞分裂，以及可渗透的RNAi介导的蛋白质消耗。这些特征促进了具有高时空分辨率的视觉检测方法的发展，以监测特定的亚细胞过程。检测方法的开发在很大程度上依赖于能够规避种系沉默以允许表达编码荧光融合蛋白的转基因的方法的出现。在本章中，我们讨论了秀丽隐杆线虫种系中荧光蛋白的表达和成像方法，包括用于优化表达的转基因设计、通过基因枪轰击产生转基因虫系、通过交配构建多标记系以及性腺和早期胚胎的实时成像方法。
