Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level
Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level
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DOI：
10.1073/pnas.97.13.7260
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发表时间：
2000-06-20
影响因子：
11.1
通讯作者：
Brûlet, P
中科院分区：
文献类型：
--
作者：
Baubet, V;Le Mouellic, H;Brûlet, P
实时监测钙通量有助于了解中枢神经系统的发育、可塑性和功能。在水母中，化学发光的钙结合蛋白与绿色荧光蛋白相关，在Ca2+刺激下发出绿色生物发光信号。我们决定利用这种化学发光共振在两个分子之间传递能量。通过融合绿色荧光蛋白和绿光蛋白，构建了钙敏感的生物发光报告基因，从而产生更多的光。这些融合蛋白的化学发光和荧光活性已在哺乳动物细胞中进行了评估。细胞质Ca2+增加成像在单细胞水平与冷却强化电荷耦合器件相机。这种双功能报告基因应该允许在转基因动物的神经网络和特定亚细胞区室中研究钙的活性。
Monitoring calcium fluxes in real time could help to understand the development, the plasticity, and the functioning of the central nervous system. In jellyfish, the chemiluminescent calcium binding aequorin protein is associated with the green fluorescent protein and a green bioluminescent signal is emitted upon Ca2+ stimulation. We decided to use this chemiluminescence resonance energy transfer between the two molecules. Calcium-sensitive bioluminescent reporter genes have been constructed by fusing green fluorescent protein and aequorin, resulting in much more light being emitted. Chemiluminescent and fluorescent activities of these fusion proteins have been assessed in mammalian cells. Cytosolic Ca2+ increases were imaged at the single-cell level with a cooled intensified charge-coupled device camera. This bifunctional reporter gene should allow the investigation of calcium activities in neuronal networks and in specific subcellular compartments in transgenic animals.