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Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains

Nature Protocols volume 9, pages 559–574 (2014)Cite this article

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Abstract

Mouse embryonic stem cells (mESCs) are key tools for genetic engineering, development of stem cell–based therapies and basic research on pluripotency and early lineage commitment. However, successful derivation of germline-competent embryonic stem cell lines has, until recently, been limited to a small number of inbred mouse strains. Recently, there have been considerable advances in the field of embryonic stem cell biology, particularly in the area of pluripotency maintenance in the epiblast from which the mESCs are derived. Here we describe a protocol for efficient derivation of germline-competent mESCs from any mouse strain, including strains previously deemed nonpermissive. We provide a protocol that is generally applicable to most inbred strains, as well as a variant for nonpermissive strains. By using this protocol, mESCs can be derived in 3 weeks and fully characterized after an additional 12 weeks, at efficiencies as high as 90% and in any strain background.

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Figure 1: Overview of preimplantation development in mice.
Figure 2: NANOG immunolabeling in ICM outgrowths grown in the presence of 2i/LIF with or without serum or KOSR.
Figure 3: Overview of blastocyst-stage embryo collection.
Figure 4: Overview of ES cell derivation process.
Figure 5: Emergent mESC morphology.
Figure 6: mESC characterization by immunolabeling of pluripotency biomarkers.
Figure 7: Karyotypic analysis of ES cells.
Figure 8: Germline testing of mESC lines.

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Acknowledgements

This work was supported in part by the US National Institutes of Health (NIH), Office of Research Infrastructure Programs (grant nos. U42-OD011102, U42-OD010921). Work in A.-K.H.'s laboratory is supported by the Human Frontiers Sciences Program and the NIH (grant nos. RO1-HD052115 and RO1-DK084391). We are grateful for the excellent microinjection services provided by the Cell Biology and Microinjection core at The Jackson Laboratory.

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Authors and Affiliations

  1. Genetic Resource Science, The Jackson Laboratory, Bar Harbor, Maine, USA.,

    Anne Czechanski, Candice Byers, Ian Greenstein, Leah Rae Donahue & Laura G Reinholdt

  2. Developmental Biology Program, Sloan-Kettering Institute, New York, New York, USA

    Nadine Schrode & Anna-Katerina Hadjantonakis

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  1. Anne Czechanski
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Contributions

A.C. and L.G.R. developed the protocol and wrote the manuscript. C.B., I.G. and L.R.D. contributed to and/or supported the development of the protocol. N.S. and A.-K.H. contributed to and expanded the protocol and contributed to the writing manuscript.

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Correspondence to Laura G Reinholdt.

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The authors declare no competing financial interests.

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Czechanski, A., Byers, C., Greenstein, I. et al. Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains. Nat Protoc 9, 559–574 (2014). https://doi.org/10.1038/nprot.2014.030

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