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Derivation of mouse embryonic stem cells

Nature Protocols volume 1, pages 2082–2087 (2006)Cite this article

Abstract

Here we describe a simple and efficient protocol for derivation of germline chimera-competent mouse embryonic stem cells (mESCs) from embryonic day 3.5 (E3.5) blastocysts. The protocol involves the use of early-passage mouse embryonic fibroblast feeders (MEF) and the alternation of fetal bovine serum– and serum replacement (SR)–containing media. As compared to other available protocols for mESCs derivation, our protocol differs in the combination of commercial availability of all reagents, technical simplicity and high efficiency. mESC lines are derived with approximately 50% efficiency (50 independent mESC lines derived from 96 blastocysts). We believe that this protocol could be a good starting point for (i) setting up the derivation of mESC lines in a laboratory and (ii) incorporating further steps to improve efficiency or adapt the protocol to other applications. The whole process (from blastocyst extraction to the freezing of mESC line) usually takes between 15 and 20 d.

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Figure 1: Individual steps of mESC derivation.
Figure 2: Derivation of germline-competent chimeras from newly derived mESCs.

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Acknowledgements

We would like to thank J. Wilbertz and his team from Karolinska Center for Transgene Technologies for ESC microinjections, C. Tello, J. Söderlund and L. Bryjová for excellent technical assistance, P. Sacchetti for critical reading of the manuscript, C. Collstam and C. Olsson for extraordinary animal maintenance, and members of the Arenas laboratory for assistance and stimulating discussions. Financial support was obtained from the Swedish Foundation for Strategic Research, Swedish Royal Academy of Sciences, Knut and Alice Wallenberg Foundation, European Commission (EuroStemCell), Swedish MRC, Karolinska Institutet and Lars Hiertas Minnesfond. S.B. is the recipient of a postdoctoral fellowship from the Ministerio de Educación y Ciencia (Spain).

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  1. Department of Medical Biochemistry and Biophysics, Laboratory of Molecular Neurobiology, Karolinska Institutet, Stockholm, S-171 77, Sweden

    Vítězslav Bryja, Sonia Bonilla & Ernest Arenas

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  1. Vítězslav Bryja
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  2. Sonia Bonilla
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  3. Ernest Arenas
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Correspondence to Ernest Arenas.

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Bryja, V., Bonilla, S. & Arenas, E. Derivation of mouse embryonic stem cells. Nat Protoc 1, 2082–2087 (2006). https://doi.org/10.1038/nprot.2006.355

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