Espinosa-Medina Isabel
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Methods Mol Biol. 2025;2886:327-353. doi: 10.1007/978-1-0716-4310-5_17.
During development, cells undergo a sequence of specification events to form functional tissues and organs. To investigate complex tissue development, it is crucial to visualize how cell lineages emerge and to be able to manipulate regulatory factors with temporal control. We recently developed TEMPO (Temporal Encoding and Manipulation in a Predefined Order), a genetic tool to label with different colors and genetically manipulate consecutive cell generations in vertebrates. TEMPO relies on CRISPR to activate a cascade of fluorescent proteins which can be imaged in vivo. Here, we explain the steps to design, generate, and express TEMPO constructs in zebrafish and mice.
在发育过程中，细胞经历一系列特化事件以形成功能组织和器官。为了研究复杂的组织发育，可视化细胞谱系如何出现并能够在时间控制下操纵调控因子至关重要。我们最近开发了TEMPO（按预定义顺序进行时间编码和操纵），这是一种用于在脊椎动物中用不同颜色标记并对连续细胞世代进行基因操纵的遗传工具。TEMPO依靠CRISPR激活一系列荧光蛋白，这些蛋白可以在体内成像。在这里，我们解释在斑马鱼和小鼠中设计、生成和表达TEMPO构建体的步骤。
