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首页> 外文期刊>The European Journal of Neuroscience >Purification of Purkinje cells by fluorescence-activated cell sorting from transgenic mice that express green fluorescent protein.
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Purification of Purkinje cells by fluorescence-activated cell sorting from transgenic mice that express green fluorescent protein.

机译：通过表达绿色荧光蛋白的转基因小鼠的荧光激活细胞分选纯化Purkinje细胞。

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The cerebellar Purkinje cell has been the focus of numerous studies involving the analysis of development and information processing in the nervous system. Purkinje cells represent less than 0.1% of the total cell content of the cerebellum. To facilitate studies of molecules that are expressed in such a small proportion of neurons, we have established procedures for the purification of these cells. Transgenic mice were developed in which the expression of green fluorescent protein (GFP) was controlled by the L7 promoter. In adult cerebellum, GFP fluorescence was only detected in Purkinje cells, where it filled dendrites, soma and axons. GFP fluorescence was detected in Purkinje cells as early as embryonic day 17 and increased during development in vivo and in dissociated cerebellar culture. Mirroring endogenous L7 expression, high levels of GFP were observed in retinal rod bipolar cells. Lower levels of GFP were seen in olfactory periglomerular cells, neurons in the interpeduncular nucleus, and superior colliculus neurons. Cerebella from transgenic mice were dissociated by mild enzymatic treatment and Purkinje cells were isolated by fluorescence-activated cell sorting (FACS). By selecting optimal parameters, a fraction of viable Purkinje cells that was 94% pure was obtained. These results indicate that FACS is a powerful tool for isolating Purkinje cells from postnatal L7-GFP transgenic mice. GFP-positive neurons will also be useful in the real-time observation of dendritic morphogenesis and axonal outgrowth during development, or after neuronal activity in vitro.
机译：小脑浦肯野细胞一直是众多研究的重点，涉及神经系统发育和信息处理的分析。浦肯野细胞占小脑总细胞含量的不到0.1％。为了促进研究在如此小的神经元中表达的分子，我们建立了纯化这些细胞的程序。已开发出转基因小鼠，其中绿色荧光蛋白（GFP）的表达受L7启动子控制。在成年小脑中，仅在Purkinje细胞中检测到GFP荧光，其中充满了树突，体细胞和轴突。早在胚胎第17天就在Purkinje细胞中检测到GFP荧光，并在体内发育和离体小脑培养中增加了GFP荧光。镜像内源性L7表达，在视网膜视杆双极细胞中观察到高水平的GFP。嗅觉性肾小球细胞，足突间核中的神经元和上丘神经元中的GFP水平较低。通过温和的酶处理将转基因小鼠的小脑解离，并通过荧光激活细胞分选术（FACS）分离Purkinje细胞。通过选择最佳参数，获得了纯度为94％的存活的Purkinje细胞部分。这些结果表明，FACS是从产后L7-GFP转基因小鼠中分离Purkinje细胞的强大工具。 GFP阳性神经元还将用于实时观察发育过程中或体外神经元活动后的树突形态发生和轴突生长。

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