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In vitro culture of mouse blastocysts beyond the implantation stages


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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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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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.

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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).

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