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In vitro culture of mouse blastocysts beyond the implantation stages

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Abstract

The implanting mouse blastocyst invades the uterine stroma and undergoes a dramatic transformation into an egg cylinder. The morphogenetic and signaling events during this transition are largely unexplored, as the uterine tissues engulf the embryo. Here we describe a protocol supporting the development of the mouse embryo beyond the blastocyst stage in vitro. We established two types of medium to be applied sequentially, and we used a substrate permitting high-resolution imaging of the transition from blastocyst to egg cylinder. We developed two variants of this protocol: the first starts with intact early blastocysts that upon zona removal can attach to the substrate and develop into egg cylinders after 5 d, and the second starts with late blastocysts that upon dissection of the mural trophectoderm form egg cylinders in only 3 d. This method allows observation of a previously hidden period of development, and it provides a platform for novel research into peri-implantation embryogenesis and beyond.

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Figure 1: In vitro culture of E3.5 blastocyst to the egg cylinder stage.
Figure 2: In vitro culture of E4.5 blastocyst to the egg cylinder stage.
Figure 3: Characterization of egg cylinders developed from blastocyst-stage embryos in vitro.

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Acknowledgements

We are grateful to the Wellcome Trust for the Senior Research Fellowship to M.Z.-G. that funded this work. We also thank the European Molecular Biology Organization (EMBO) for supporting M.B.

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Author notes

  1. Ivan Bedzhov, Chuen Yan Leung and Monika Bialecka: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physiology, Development and Neuroscience, Downing Site, University of Cambridge, Cambridge, UK

    Ivan Bedzhov, Chuen Yan Leung, Monika Bialecka & Magdalena Zernicka-Goetz

  2. Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK

    Ivan Bedzhov, Chuen Yan Leung, Monika Bialecka & Magdalena Zernicka-Goetz

Authors
  1. Ivan Bedzhov
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  2. Chuen Yan Leung
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  3. Monika Bialecka
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  4. Magdalena Zernicka-Goetz
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Contributions

I.B., C.Y.L., M.B. and M.Z.-G. designed the protocol, carried out the work, analyzed the results and prepared the manuscript.

Corresponding author

Correspondence to Magdalena Zernicka-Goetz.

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The authors declare no competing financial interests.

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Bedzhov, I., Leung, C., Bialecka, M. et al. In vitro culture of mouse blastocysts beyond the implantation stages. Nat Protoc 9, 2732–2739 (2014). https://doi.org/10.1038/nprot.2014.186

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