Cell tracking using a photoconvertible fluorescent protein
Cell tracking using a photoconvertible fluorescent protein
复制标题
DOI：
10.1038/nprot.2006.96
复制
发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Omura, Tomomi
中科院分区：
文献类型：
--
作者：
Hatta, Kohei;Tsujii, Hitomi;Omura, Tomomi
跟踪细胞的命运、形状和迁移是研究多细胞生物发育的重要组成部分。在这里，我们报告了一个协议，使用的蛋白质Kaede，这是荧光绿色合成后，但可以光转换为红色的紫色或紫外线。我们已经使用Kaede沿着与共聚焦激光扫描显微镜跟踪标记的细胞在斑马鱼胚胎中的感兴趣的模式。这种技术允许细胞运动的可视化和神经元形状的跟踪。我们提供了通过mRNA注射表达、通过DNA注射嵌合表达以及用UAS-Gal 4系统创建永久性转基因鱼以可视化形态发生过程（如神经形成、基板形成和后脑中早期连合轴突的导航）的说明性实例。该程序可以适用于其他光转换和可逆的荧光分子，包括KikGR和Dronpa;这些分子可以与双光子共聚焦显微镜结合使用，以特异性地突出组织中埋藏的细胞。进行包括通过注射mRNA或DNA瞬时表达Kaede、光转化和成像的方案所需的总时间为2 - 8 d。
The tracking of cell fate, shape and migration is an essential component in the study of the development of multicellular organisms. Here we report a protocol that uses the protein Kaede, which is fluorescent green after synthesis but can be photoconverted red by violet or UV light. We have used Kaede along with confocal laser scanning microscopy to track labeled cells in a pattern of interest in zebrafish embryos. This technique allows the visualization of cell movements and the tracing of neuronal shapes. We provide illustrative examples of expression by mRNA injection, mosaic expression by DNA injection, and the creation of permanent transgenic fish with the UAS- Gal4 system to visualize morphogenetic processes such as neurulation, placode formation and navigation of early commissural axons in the hindbrain. The procedure can be adapted to other photoconvertible and reversible fluorescent molecules, including KikGR and Dronpa; these molecules can be used in combination with two- photon confocal microscopy to specifically highlight cells buried in tissues. The total time needed to carry out the protocol involving transient expression of Kaede by injection of mRNA or DNA, photoconversion and imaging is 2 - 8 d.