Cell type-selective expression of green fluorescent protein and the calcium indicating protein, yellow cameleon, in rat cortical primary cultures
Cell type-selective expression of green fluorescent protein and the calcium indicating protein, yellow cameleon, in rat cortical primary cultures
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DOI：
10.1016/s0006-8993(02)03518-7
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发表时间：
2002-11
期刊：
影响因子：
2.9
通讯作者：
R. Tsuchiya;F. Yoshiki;Y. Kudo;M. Morita
中科院分区：
文献类型：
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作者：
R. Tsuchiya;F. Yoshiki;Y. Kudo;M. Morita
建立了一种细胞类型特异性的绿色荧光蛋白（GFP）表达系统，用于脑细胞的荧光标记。采用脂质介导的转染（脂质转染），允许建立用于分析单个细胞的方便有效的系统。为了实现细胞类型特异性标记，构建了包含大鼠神经元特异性烯醇化酶（NSE）基因启动子、人胶质纤维酸性蛋白（GFAP）基因启动子、人延伸因子（EF-1α）基因启动子和人巨细胞病毒（CMV）立即早期启动子的GFP表达载体，并检测了它们的特异性。含有CMV或GFAP启动子的载体主要导致星形胶质细胞中的GFP表达，而含有EF 1-α或NSE启动子的载体主要导致神经元中的GFP表达。该标记系统被应用于活神经元的形态学分析和细胞类型选择性钙成像。共聚焦显微镜显示单个GFP表达神经元具有突起，其长度超过500 μm，并具有棘状突起。在同一系统中表达了钙指示GFP变体黄色cameleon（YC 2.1），并进行了细胞类型选择性钙成像。在药理学刺激，YC 2.1表达神经元响应去极化刺激，但不代谢型谷氨酸受体激动剂，反式-（1 S，3R）-1-氨基-1，3-环戊烷二羧酸（tACPD），而星形胶质细胞只tACPD。
A cell type-specific green fluorescent protein (GFP) expression system in rat cortical primary cultures has been developed for the fluorescence labeling of brain cells. Lipid-mediated transfection (lipofection) was employed, allowing the establishment of a convenient efficient system for the analysis of individual cells. To achieve cell type-specific labeling, GFP expression vectors containing the rat neuron-specific enolase (NSE) gene promoter, human glial fibril acidic protein (GFAP) gene promoter, human elongation factor (EF-1α) gene promoter, or human cytomegalovirus (CMV) immediate early promoter were constructed, and their specificities examined. Vectors containing the CMV or GFAP promoter resulted primarily in GFP expression in astrocytes, while those containing the EF1-α or NSE promoter resulted primarily in GFP expression in neurons. This labeling system was applied to the morphological analysis of living neurons and to cell type-selective calcium imaging. Confocal microscopy revealed that individual GFP-expressing neurons had processes, which were longer than 500 μm and bore spine-like protrusions. A calcium-indicating GFP variant, yellow cameleon (YC2.1), was expressed in the same system, and cell type-selective calcium imaging performed. On pharmacological stimulation, YC2.1-expressing neurons responded to depolarizing stimuli, but not to the metabotropic glutamate receptor agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (tACPD), while astrocytes responded only to tACPD.