Lombardo Verónica A, Sporbert Anje, Abdelilah-Seyfried Salim
Max Delbrück Center for Molecular Medicine.
J Vis Exp. 2012 Sep 28(67):4350. doi: 10.3791/4350.
Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede, KikGR, Dendra and EosFP, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed β-elimination and subsequent extension of a π-conjugated system. PCFPs and their monomeric variants are useful tools for tracking cells and studying protein dynamics, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish, chicken and mouse for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.
胚胎发生是一个动态过程，最好通过使用能够记录体内发育变化的技术来进行研究。事实证明，使用基因编码荧光蛋白是阐明完整生物体中发生的动态形态发生过程的一种有价值的策略。在过去十年中，光激活和光转换荧光蛋白的发展为研究标记蛋白离散亚群的命运开辟了可能性。与光激活蛋白不同，光转换荧光蛋白（PCFP）在光转换之前很容易在其天然发射状态下进行追踪和成像，这使得通过光学检查更容易识别和选择区域。由于His-Tyr-Gly三肽序列，PCFP如Kaede、KikGR、Dendra和EosFP在暴露于紫外线或蓝光时可从绿色转变为红色，该序列形成绿色发色团，可通过光催化的β-消除和随后的π共轭体系扩展而光转换为红色发色团。PCFP及其单体变体分别是追踪细胞和研究蛋白质动力学的有用工具。近年来，PCFP已在不同的动物模型中表达，如斑马鱼、鸡和小鼠，用于细胞命运追踪。在这里，我们报告了一种在活斑马鱼胚胎中对PCFP进行细胞特异性光转换并在后期发育阶段进一步追踪光转换蛋白的方案。这种方法能够以组织特异性的方式研究斑马鱼动物模型中形态发生背后的细胞生物学事件。
