双顺反子表达技术在线虫神经元钙成像中的应用(英文)  

A Novel Bicistronic Expression Strategy for Neuronal Calcium Imaging in C.elegans

作　　者：张海宁[1] 黄文明[1] 杨松[1] 徐涛[2] 

机构地区：[1]华中科技大学生命科学与技术学院分子生物物理教育部重点实验室,武汉430074 [2]中国科学院生物物理研究所生物大分子国家重点实验室,北京100101

出　　处：《生物化学与生物物理进展》2014年第5期515-519,共5页Progress In Biochemistry and Biophysics

基　　金：supported by a grant from The National Natural ScienceFoundation of China(30871225)~~

摘　　要：在线虫中,钙成像技术已被广泛用于检测不同神经元的活性.然而,对于准确记录爬行中的活体线虫神经元钙信号仍然存在许多挑战,其中一个困难即来自于标记目标神经元。在同一个目标神经元中共同表达基因编码的钙指示蛋白和常量参考值荧光蛋白常常具有无法共表达的不确定性.另外,光谱的串扰影响存在于目前最常用的绿色钙指示蛋白系列G-CaMP与其参考值荧光蛋白DsRed系列之间,光谱的串扰有时会给信号记录带来假阳性结果.综上所述,本文首次提出应用双顺反子表达技术用于同一神经元的双蛋白标记,这不仅提高了共表达效率,更简化了线虫神经元标记的工作量.同时,本文还首次采用mKate2,一种与G-CaMP没有串扰的红色荧光蛋白作为参考量.以上改进已在感觉神经元ASH中得到验证.希望本文提出的方法能给线虫神经回路的研究提供一个更为方便、有效的途径.Calcium imaging has been widely used to monitor the activity of various neurons in C. elegans. However, it is a challenge to determine the calcium transient in a freely moving worm for two reasons. One reason is the challenge in ensuring the co-expression of the genetically encoded calcium indicator and reference fluorescent protein in the same target neurons. Another reason is the common problem with spectrum cross-talk between the fluorescent pair used most often： G-CaMPs （calcium indicator） and DsReds （the reference fluorescent protein）. Spectrum cross-talk occasionally introduces artifacts in the calcium transient measurements. Herein, we developed a novel bicistronic expression strategy to ensure co-expression efficiency and simplify the labeling for the target neurons. Moreover, we presented a new fluorescent protein pair for calcium imaging, mKate2 and G-CaMPs, which have less spectrum cross-talk. We successfully tested our new labeling strategy in the C. elegans ASH sensory neuron. We anticipate that this improved technique will facilitate neural circuit studies in C. elegans.

关 键 词：双顺反子 钙成像 秀丽隐杆线虫 

分 类 号：Q633[生物学—生物物理学] Q336