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Generating Chimeric Mice from Embryonic Stem Cells via Vial Coculturing or Hypertonic Microinjection

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Mouse Genetics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1194))

Abstract

The generation of a fertile embryonic stem cell (ESC)-derived or F0 (100 % coat color chimerism) mice is the final criterion in proving that the ESC is truly pluripotent. Many methods have been developed to produce chimeric mice. To date, the most popular methods for generating chimeric embryos is well sandwich aggregation between zona pellucida (ZP) removed (denuded) 2.5-day post-coitum (dpc) embryos and ESC clumps, or direct microinjection of ESCs into the cavity (blastocoel) of 3.5-dpc blastocysts. However, due to systemic limitations and the disadvantages of conventional microinjection, aggregation, and coculturing, two novel methods (vial coculturing and hypertonic microinjection) were developed in recent years at my laboratory.

Coculturing 2.5-dpc denuded embryos with ESCs in 1.7-mL vials for ~3 h generates chimeras that have significantly high levels of chimerism (including 100 % coat color chimerism) and germline transmission. This method has significantly fewer instrumental and technological limitations than existing methods, and is an efficient, simple, inexpensive, and reproducible method for “mass production” of chimeric embryos. For laboratories without a microinjection system, this is the method of choice for generating chimeric embryos. Microinjecting ESCs into a subzonal space of 2.5-dpc embryos can generate germline-transmitted chimeras including 100 % coat color chimerism. However, this method is adopted rarely due to the very small and tight space between ZP and blastomeres. Using a laser pulse or Piezo-driven instrument/device to help introduce ESCs into the subzonal space of 2.5-dpc embryos demonstrates the superior efficiency in generating ESC-derived (F0) chimeras. Unfortunately, due to the need for an expensive instrument/device and extra fine skill, not many studies have used either method. Recently, ESCs injected into the large subzonal space of 2.5-dpc embryos in an injection medium containing 0.2–0.3 M sucrose very efficiently generated viable, healthy, and fertile chimeric mice with 100 % coat color chimerism.

Both vial coculture and hypertonic microinjection methods are useful and effective alternatives for producing germline chimeric or F0 mice efficiently and reliably. Furthermore, both novel methods are also good for induced pluripotent stem cells (iPSCs) to generate chimeric embryos.

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Acknowledgements

I would like to thank the National Science Council of the Republic of China, Taiwan, for financially supporting my research under Contract Nos. NSC99-2324-B-059-001 and NSC100-2321-B-059-001-MY3. Drs. C.H. Chen, C.F. Tu, J.K. Juang, and S.F. Guo as well as Ms. H.R. Chang and T.L. Hsu at Animal Technology Institute Taiwan (ATIT) are commended for their critical comments, technical assistance, and routine maintenance in the laboratory and mouse facility.

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Authors and Affiliations

  1. Division of Biotechnology, Animal Technology Institute Taiwan, 23, Chunan (35053), Miaoli, Taiwan

    Kun-Hsiung Lee

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  1. Kun-Hsiung Lee
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Correspondence to Kun-Hsiung Lee .

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Editors and Affiliations

  1. Stem Cell Regulation and Animal Aging Section, Basic Research Laboratory,, National Cancer Institute, National Institutes of Health, Frederick, MD, USA

    Shree Ram Singh

  2. Genetically Engineered Mouse Modeling Core/Department of Molecular Virology, Immunology and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA

    Vincenzo Coppola

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Lee, KH. (2014). Generating Chimeric Mice from Embryonic Stem Cells via Vial Coculturing or Hypertonic Microinjection. In: Singh, S., Coppola, V. (eds) Mouse Genetics. Methods in Molecular Biology, vol 1194. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1215-5_5

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  • DOI: https://doi.org/10.1007/978-1-4939-1215-5_5

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