Miller D M, Desai N S, Hardin D C, Piston D W, Patterson G H, Fleenor J, Xu S, Fire A
Vanderbilt University Medical Center, Nashville, TN, USA.
Biotechniques. 1999 May;26(5):914-8, 920-1. doi: 10.2144/99265rr01.
We describe the use of modified versions of the Aequora victoria green fluorescent protein (GFP) to simultaneously follow the expression and distribution of two different proteins in the nematode, Caenorhabditis elegans. A cyan-colored GFP derivative, designated CFP, contains amino acid (aa) substitutions Y66W, N146I, M153T and V163A relative to the original GFP sequence and is similar to the previously reported "W7" form. A yellow-shifted GFP derivative, designated YFP, contains aa substitutions S65G, V68A, S72A and T203Y and is similar to the previously described "I0C" variant. Coding regions for CFP and YFP were constructed in the context of a high-activity C. elegans expression system. Previously characterized promoters and localization signals have been used to express CFP and YFP in C. elegans. Filter sets designed to distinguish YFP and CFP fluorescence spectra allowed visualization of the two distinct forms of GFP in neurons and in muscle cells. A series of expression vectors carrying CFP and YFP have been constructed and are being made available to the scientific community.
我们描述了利用维多利亚水母绿色荧光蛋白（GFP）的修饰版本，在线虫秀丽隐杆线虫中同时追踪两种不同蛋白质的表达和分布情况。一种蓝色的GFP衍生物，命名为CFP，相对于原始GFP序列含有氨基酸（aa）替换Y66W、N146I、M153T和V163A，并且与先前报道的“W7”形式相似。一种向黄色偏移的GFP衍生物，命名为YFP，含有aa替换S65G、V68A、S72A和T203Y，并且与先前描述的“I0C”变体相似。CFP和YFP的编码区是在高活性秀丽隐杆线虫表达系统的背景下构建的。先前已表征的启动子和定位信号已用于在秀丽隐杆线虫中表达CFP和YFP。设计用于区分YFP和CFP荧光光谱的滤光片组使得能够在神经元和肌肉细胞中观察到两种不同形式的GFP。已经构建了一系列携带CFP和YFP的表达载体，并将提供给科学界。
