Summary
This chapter describes the tools and the experimental route of targeted manipulation by aggregation in the mouse using targeted embryonic stem cells (ES cells). Instead of injecting ES cells into the blastocoel of a diploid blastocyst-stage embryo (3.5 dpc) ES cells can be brought together with diploid morula-stage embryos (2.5 dpc). The zona pellucida of the embryo needs to be removed and one or two embryos (sandwich aggregation) are put together with ES cells into an indentation well of a cell culture grade dish overnight for aggregation. This can be performed manually using a stereomicroscope and does not require any special training or expensive instrumentation.
The next day, the embryo would have developed into a blastocyst in vitro and can be transferred to a pseudopregnant female mouse (see Chapter 15).
The use of tetraploid embryos generated by electrofusion will lead to entirely ES cell-derived fetuses.
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References
Tarkowsky, A. K. (1961) Mouse chimaeras from fused eggs. Nature 190, 857–860.
Thomas, K. R., and Capecchi M. R. (1990) Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature 346, 847–850.
Wood, S. A., Allen, N. D., Rossant, J., Auerbach, A., and Nagy, A. (1993) Noninjection methods for the production of chimeric stem cell–embryo chimeras. Nature 365, 87–89.
Wang, Z., and Jaenisch, R. (2004) At most three ES cells contribute to the somatic lineages of chimeric mice and of mice produced by ES-tetraploid complementation. Dev. Biol 275, 192–201.
Acknowledgments
Dr. Alfonso Gutierrez-Adan, Departamento de Reproduccion Animal y Conservacion de Recursos Zoogeneticos, INIA, Ctra de la Coruna Km 5,9, Spain, agutierr@inia.es)
Ronald Naumann, Transgenic Core Facility, MPI of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany, naumann@mpi-cbg.de
Kristina Vintersten, Mount Sinai Hospital, Samuel Lunenfeld Research Institute, 600 University Ave, Toronto, Canada, vintersten@mshri.on.ca
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Plück, A., Klasen, C. (2009). Generation of Chimeras by Morula Aggregation. In: Cartwright, E. (eds) Transgenesis Techniques. Methods in Molecular Biology, vol 561. Humana Press. https://doi.org/10.1007/978-1-60327-019-9_14
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DOI: https://doi.org/10.1007/978-1-60327-019-9_14
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