"Color Timer" mice: visualization of neuronal differentiation with fluorescent proteins.
"Color Timer" mice: visualization of neuronal differentiation with fluorescent proteins.
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DOI：
10.1186/1756-6606-3-5
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发表时间：
2010-02-02
期刊：
影响因子：
3.6
通讯作者：
Okano H
中科院分区：
文献类型：
--
作者：
Kanki H;Shimabukuro MK;Miyawaki A;Okano H
神经干细胞（neural stem cells, NSCs）分化为神经元祖细胞并最终分化为神经元的分子机制正逐渐被揭示。然而，由于缺乏实时测定单个神经细胞分化阶段的方便手段，一直阻碍着阐明其机制的进展。为了能够轻松地识别神经细胞的分化阶段，我们一直在尝试建立一个小鼠系统，该系统将利用小鼠遗传学和不断扩大的荧光蛋白库的组合，使基于荧光颜色之间的转换的神经元分化进程可视化。在这项研究中，我们报告了这种鼠标系统的初始版本，我们称之为“颜色计时器”。我们首先培育了转基因（Tg; nestin/KOr Tg）小鼠，其荧光蛋白Kusabira-Orange （KOr）的产生受nestin基因第2内含子增强子内的基因调控元件控制，该基因是NSCs的良好标记，因此NSCs在激发时发出橙色荧光。然后，我们通过免疫组织化学和免疫细胞化学分析证实，KOr荧光密切反映了Nestin蛋白的存在。我们还通过神经球形成实验证实，KOr荧光的强度与细胞的“干性”相关，并且可以很容易地识别出成年nestin/KOr Tg小鼠大脑中两个神经发生区域（即海马齿状回和侧脑室室下区）中的NSCs。然后，我们将巢蛋白/KOr小鼠与双皮质素增强的绿色荧光蛋白Tg小鼠杂交，其未成熟的神经元在激发时发出绿色荧光，并且可以通过荧光颜色从橙色到绿色的转变来可视化nsc向神经元分化的过程。这种双色初始版本的“颜色定时器”鼠标系统将为神经发生研究提供一个强大的新工具。
The molecular mechanisms governing the differentiation of neural stem cells (NSCs) into neuronal progenitor cells and finally into neurons are gradually being revealed. The lack of convenient means for real-time determination of the stages of differentiation of individual neural cells, however, has been hindering progress in elucidating the mechanisms. In order to be able to easily identify the stages of differentiation of neural cells, we have been attempting to establish a mouse system that would allow progression of neuronal differentiation to be visualized based on transitions between fluorescence colors by using a combination of mouse genetics and the ever-expanding repertoire of fluorescent proteins. In this study we report the initial version of such a mouse system, which we call "Color Timer." We first generated transgenic (Tg; nestin/KOr Tg) mice in which production of the fluorescent protein Kusabira-Orange (KOr) is controlled by the gene regulatory elements within the 2nd intronic enhancer of the nestin gene, which is a good marker for NSCs, so that NSCs would emit orange fluorescence upon excitation. We then confirmed by immunohistochemical and immunocytochemical analyses that the KOr fluorescence closely reflected the presence of the Nestin protein. We also confirmed by a neurosphere formation assay that the intensity of the KOr fluorescence correlated with "stemness" of the cell and it was possible to readily identify NSCs in the two neurogenic regions, namely the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricle, in the brain of adult nestin/KOr Tg mice by the orange fluorescence they emitted. We then crossed nestin/KOr mice with doublecortin-enhanced Green Fluorescent Protein Tg mice, whose immature neurons emit green fluorescence upon excitation, and it was possible to visualize the progress of NSC-to-neuron differentiation by the transition between fluorescence colors from orange to green. This two-color initial version of the "Color Timer" mouse system will provide a powerful new tool for neurogenesis research.
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