Abstract
Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos.
Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos.
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Acknowledgements
The authors are thankful to Chizuko Tsurumi and Benoit Kanzler for making the H2b-GFP-transgenic mouse strain, without which this project would not have been possible. The strain is publicly available from The Mutant Mouse Regional Resource Center 8U42OD010924-13. We thank our coauthors of the original publication [8], Telma Esteves, Franchesca Houghton, Marcin Siatkowski, Martin Pfeiffer and Georg Fuellen for valuable input and the Max Planck Institute for Molecular Biomedicine, Münster, in particular Hans Schöler, for infrastructural support.
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Balbach, S.T., Boiani, M. (2015). Live Embryo Imaging to Follow Cell Cycle and Chromosomes Stability After Nuclear Transfer. In: Beaujean, N., Jammes, H., Jouneau, A. (eds) Nuclear Reprogramming. Methods in Molecular Biology, vol 1222. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1594-1_11
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DOI: https://doi.org/10.1007/978-1-4939-1594-1_11
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