Ooga Masatoshi, Funaya Satoshi, Aoki Fugaku, Wakayama Teruhiko
Faculty of Life and Environmental Sciences, Department of Biotechnology, University of Yamanashi; Advanced Biotechnology Center, University of Yamanashi;
Department of Integrated Bioscience, Graduate School of Frontier Sciences, University of Tokyo.
J Vis Exp. 2018 Jun 12(136):57068. doi: 10.3791/57068.
Live imaging is a powerful tool that allows for the analysis of molecular events during ontogenesis. Recently, chromatin looseness or openness has been shown to be involved in the cellular differentiation potential of pluripotent embryonic stem cells. It was previously reported that compared with embryonic stem cells, zygotes harbor an extremely loosened chromatin structure, suggesting its association with their totipotency. However, until now, it has not been addressed whether this extremely loosened/open chromatin structure is important for embryonic developmental potential. In the present study, to examine this hypothesis, an experimental system in which zygotes that were analyzed by fluorescence recovery after photo-bleaching can develop to term without any significant damage was developed. Importantly, this experimental system needs only a thermos-plate heater in addition to a confocal laser scanning microscope. The findings of this study suggest that fluorescence recovery after photo-bleaching analysis (FRAP) analysis can be used to investigate whether the molecular events in zygotic chromatin are important for full-term development.
实时成像技术是一种强大的工具，可用于分析个体发育过程中的分子事件。最近，染色质的松散或开放状态已被证明与多能胚胎干细胞的细胞分化潜能有关。此前有报道称，与胚胎干细胞相比，受精卵具有极其松散的染色质结构，这表明其与全能性有关。然而，到目前为止，尚未探讨这种极其松散/开放的染色质结构对胚胎发育潜能是否重要。在本研究中，为了验证这一假设，开发了一种实验系统，在该系统中，经光漂白后荧光恢复分析的受精卵能够发育至足月，且无任何明显损伤。重要的是，该实验系统除了共聚焦激光扫描显微镜外，仅需要一个热板加热器。本研究结果表明，光漂白后荧光恢复分析（FRAP）可用于研究合子染色质中的分子事件对足月发育是否重要。
