Abstract
Meiotic division is a dynamic process that exhibits active interactive behaviors amongst different intracellular structures and components for spindle assembly and chromosome segregation. Understanding the mechanisms of meiotic spindle assembly and chromosome segregation therefore requires a quantitative analysis of spatiotemporal relationships among different structures and components. In this chapter, we describe a method for triple-color live imaging of meiotic division in mouse oocytes. This approach combines the microinjection of RNAs encoding proteins tagged with green and red fluorescent proteins and the visualization of microtubules with the fluorogenic far-red probe SiR-Tubulin. This method enables the simultaneous spatiotemporal mapping of three different components of the spindle and chromosomes, which opens the way to quantitative analysis of their interactive behaviors.
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Acknowledgment
We thank Dr. Jan Ellenberg for a macro for automated confocal microscopy, Dr. Melina Schuh for the Cep192 construct, and the RIKEN Kobe imaging and animal facilities for support. This work was supported by the National Sustainability Programme of the Czech Ministry of Education, Youth and Sports [project number LO1609] to P.S., and by the research grants JSPS KAKENHI 26650072/16H01226/16H06161, JSPS Bilateral Joint Research projects with P.S., and RIKEN CDB intramural grant to T.S.K.
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Courtois, A., Solc, P., Kitajima, T.S. (2018). Triple-Color Live Imaging of Mouse Oocytes. In: Verlhac, MH., Terret, ME. (eds) Mouse Oocyte Development. Methods in Molecular Biology, vol 1818. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8603-3_10
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DOI: https://doi.org/10.1007/978-1-4939-8603-3_10
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