Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres.
Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres.
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DOI：
10.3791/53617
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发表时间：
2016-02-29
期刊：
影响因子：
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通讯作者：
Nicoli S
中科院分区：
文献类型：
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作者：
Lopez-Ramirez MA;Calvo CF;Ristori E;Thomas JL;Nicoli S
斑马鱼是一种极为重要的模式生物，有助于理解脊椎动物神经发生和大脑再生所涉及的细胞与分子机制。然而，由于缺乏可靠的分离和培养成年神经干/祖细胞的方案，对斑马鱼成体神经发生潜在分子机制的深入分析一直受到限制。在此，我们提供一种可重复的方法，利用源自成年斑马鱼全脑或其端脑、中脑顶盖及小脑区域的神经球分析来研究成体神经发生。该方案首先对成年斑马鱼大脑进行显微解剖，然后进行单细胞解离并分离具有自我更新能力的多能神经干/祖细胞。整个过程需时八天。此外，我们还阐述了如何操控斑马鱼神经球中的基因表达，这对于研究斑马鱼成年神经干/祖细胞增殖和分化过程中特定信号通路的作用尤为有用。
The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.