Nowotschin Sonja, Garg Vidur, Piliszek Anna, Hadjantonakis Anna-Katerina
Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
Department of Experimental Embryology, Institute of Genetics and Animal Breeding, Polish Academy of Sciences, Jastrzębiec, Poland.
Methods Mol Biol. 2019;1920:163-182. doi: 10.1007/978-1-4939-9009-2_11.
Mouse genetic approaches when combined with live imaging tools are revolutionizing our current understanding of mammalian developmental biology. The availability and improvement of a wide variety of genetically encoded fluorescent proteins have provided indispensable tools to visualize cells and subcellular features in living organisms. It is now possible to generate genetically modified mouse lines expressing several spectrally distinct fluorescent proteins in a tissue-specific or -inducible manner. Such reporter-expressing lines make it possible to image dynamic cellular behaviors in the context of living embryos undergoing normal or aberrant development. As with all viviparous mammals, mouse embryos develop within the uterus, and so live imaging experiments require culture conditions that closely mimic the in vivo environment. Over the past decades, significant advances have been made in developing conditions for culturing both pre- and postimplantation-stage mouse embryos. In this chapter, we discuss routine methods for ex utero culture of preimplantation- and postimplantation-stage mouse embryos. In particular, we describe protocols for collecting mouse embryos of various stages, setting up culture conditions for their ex utero culture and imaging, and using laser scanning confocal microscopy to visualize live processes in mouse embryos expressing fluorescent reporters.
当与活体成像工具相结合时，小鼠遗传学方法正在彻底改变我们目前对哺乳动物发育生物学的理解。各种各样的基因编码荧光蛋白的可用性和改进为可视化活生物体中的细胞和亚细胞特征提供了不可或缺的工具。现在有可能生成以组织特异性或诱导性方式表达几种光谱不同的荧光蛋白的转基因小鼠品系。这种表达报告基因的品系使得在经历正常或异常发育的活胚胎背景下对动态细胞行为进行成像成为可能。与所有胎生哺乳动物一样，小鼠胚胎在子宫内发育，因此活体成像实验需要紧密模拟体内环境的培养条件。在过去几十年中，在开发用于培养植入前和植入后阶段小鼠胚胎的条件方面取得了重大进展。在本章中，我们讨论用于子宫外培养植入前和植入后阶段小鼠胚胎的常规方法。特别是，我们描述了收集各个阶段小鼠胚胎的方案，为其子宫外培养和成像设置培养条件，以及使用激光扫描共聚焦显微镜可视化表达荧光报告基因的小鼠胚胎中的活过程。
