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Production of chimeras by aggregation of embryonic stem cells with diploid or tetraploid mouse embryos

Nature Protocols volume 1, pages 1145–1153 (2006)Cite this article

Abstract

The production of mouse chimeras is a common step in the establishment of genetically modified animal strains. Chimeras also provide a powerful experimental tool for following cell behavior during both prenatal and postnatal development. This protocol outlines a simple and economical technique for the production of large numbers of mouse chimeras using traditional diploid morula↔diploid embryonic stem (ES) cell aggregations. Additional steps are included to describe the procedures necessary to produce specialized tetraploid chimeras using tetraploid morula↔diploid ES cell aggregations. This increasingly popular form of chimera produces embryos of nearly complete ES cell derivation that can be used to speed transgenic production or ask developmental questions. Using this protocol, mouse chimeras can be generated and transferred to pseudopregnant surrogate mothers in a 5-d period.

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Figure 1: Timeline and integration of the protocols.
Figure 2: Generalized scheme for the production of aggregation chimeras.
Figure 3: Protocol for generating tetraploid embryos by electrofusion.
Figure 4: Morphology of embryos throughout the procedure.

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References

  1. Bradley, A., Evans, M., Kaufman, M.H. & Robertson, E. Formation of germ-line chimeras from embryo-derived teratocarcinoma cell lines. Nature 309, 255–256 (1984).

    Article  CAS  Google Scholar 

  2. Thompson, S., Clarke, A.R., Pow, A.M., Hooper, M.L. & Melton, D.W. Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells. Cell 56, 313–321 (1989).

    Article  CAS  Google Scholar 

  3. Tam, P.P. & Rossant, J. Mouse embryonic chimeras: tools for studying mammalian development. Development 130, 6155–6163 (2003).

    Article  CAS  Google Scholar 

  4. Kunath, T. et al. Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts. Development 132, 1649–1661 (2005).

    Article  CAS  Google Scholar 

  5. Tanaka, S., Kunath, T., Hadjantonakis, A.K., Nagy, A. & Rossant, J. Promotion of trophoblast stem cell proliferation by FGF4. Science 282, 2072–2075 (1998).

    Article  CAS  Google Scholar 

  6. Nagy, A., Gertsenstein, M., Vinterstein, K. & Behringer, R.R. Manipulating the mouse embryo: a laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2003).

    Google Scholar 

  7. Papaioannou, V.E. & Behringer, R.R. Mouse Phenotypes: A handbook of mutation analysis (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2005).

    Google Scholar 

  8. Wakayama, T. et al. Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science 292, 740–743 (2001).

    Article  CAS  Google Scholar 

  9. Wood, S.A. et al. Simple and efficient production of embryonic stem cell-embryo chimeras by coculture. Proc. Natl. Acad. Sci. USA 90, 4582–4585 (1993).

    Article  CAS  Google Scholar 

  10. Papaioannou, V. & Johnson, R. Production of chimeras by blastocyst and morula injection of targeted ES cells. in Gene Targeting: A Practical Approach 2nd edn. (ed. Joyner, A.L.) 133–175 (Oxford Univ. Press, Oxford, 2000).

    Google Scholar 

  11. Nagy, A. & Rossant, J. Production and analysis of ES cell aggregation chimeras. in Gene Targeting: A Practical Approach 2nd edn. (ed. Joyner, A.L.) 177–206 (Oxford Univ. Press, Oxford, 2000).

    Google Scholar 

  12. Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. & Roder, J.C. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 8424–8428 (1993).

    Article  CAS  Google Scholar 

  13. Li, X. et al. The genetic heterozygosity and fitness of tetraploid embryos and embryonic stem cells are crucial parameters influencing survival of mice derived from embryonic stem cells by tetraploid embryo aggregation. Reproduction 130, 53–59 (2005).

    Article  CAS  Google Scholar 

  14. Eggan, K. et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc. Natl. Acad. Sci. USA 98, 6209–6214 (2001).

    Article  CAS  Google Scholar 

  15. Beddington, R.S. & Robertson, E.J. An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. Development 105, 733–737 (1989).

    CAS  PubMed  Google Scholar 

  16. Nagy, A. et al. Embryonic stem cells alone are able to support fetal development in the mouse. Development 110, 815–821 (1990).

    CAS  PubMed  Google Scholar 

  17. Eakin, G.S. & Behringer, R.R. Tetraploid development in the mouse. Dev. Dyn. 228, 751–766 (2003).

    Article  Google Scholar 

  18. Eakin, G.S., Hadjantonakis, A.K., Papaioannou, V.E. & Behringer, R.R. Developmental potential and behavior of tetraploid cells in the mouse embryo. Dev. Biol. 288, 150–159 (2005).

    Article  CAS  Google Scholar 

  19. Mackay, G.E. & West, J.D. Fate of tetraploid cells in 4n↔2n chimeric mouse blastocysts. Mech. Dev. 122, 1266–1281 (2005).

    Article  CAS  Google Scholar 

  20. Iwasaki, S., Campbell, K.H., Galli, C. & Akiyama, K. Production of live calves derived from embryonic stem-like cells aggregated with tetraploid embryos. Biol. Reprod. 62, 470–475 (2000).

    Article  CAS  Google Scholar 

  21. Tarkowski, A.K., Witkowska, A. & Opas, J. Development of cytochalasin in B-induced tetraploid and diploid/tetraploid mosaic mouse embryos. J. Embryol. Exp. Morphol. 41, 47–64 (1977).

    CAS  PubMed  Google Scholar 

  22. Ueda, O., Jishage, K., Kamada, N., Uchida, S. & Suzuki, H. Production of mice entirely derived from embryonic stem (ES) cell with many passages by coculture of ES cells with cytochalasin B induced tetraploid embryos. Exp. Anim. 44, 205–210 (1995).

    Article  CAS  Google Scholar 

  23. Kubiak, J.Z. & Tarkowski, A.K. Electrofusion of mouse blastomeres. Exp. Cell Res. 157, 561–566 (1985).

    Article  CAS  Google Scholar 

  24. McLaughlin, K.J. Production of tetraploid embryos by electrofusion. Methods Enzymol. 225, 919–930 (1993).

    Article  CAS  Google Scholar 

  25. Mintz, B. Allophenic mice of mutiple embryo origin. in Methods in Mammalian Embryology (ed. Daniel, J.C. Jr.) 186–214 (W.H. Freeman and Company, San Francisco, 1971).

    Google Scholar 

  26. Kishigami, S. et al. Production of cloned mice by somatic cell nuclear transfer. Nat. Protocols 1, 125–138 (2006).

    Article  CAS  Google Scholar 

  27. Singer, O., Tiscornia, G., Ikawa, M. & Verma, I.M. Rapid generation of knockdown transgenic mice by silencing lentiviral vectors. Nat. Protocols 1, 1–7 (2006).

    Article  Google Scholar 

  28. Ware, L.M. & Axelrad, A.A. Inherited resistance to N- and B-tropic murine leukemia viruses in vitro: evidence that congenic mouse strains SIM and SIM.R differ at the Fv-1 locus. Virology 50, 339–348 (1972).

    Article  CAS  Google Scholar 

  29. Evans, M.J. & Kaufman, M.H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).

    Article  CAS  Google Scholar 

  30. Hadjantonakis, A.K., Gertsenstein, M., Ikawa, M., Okabe, M. & Nagy, A. Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech. Dev. 76, 79–90 (1998).

    Article  CAS  Google Scholar 

  31. Mullen, R.J. & Whitten, W.K. Relationship of genotype and degree of chimerism in coat color to sex ratios and gametogenesis in chimeric mice. J. Exp. Zool. 178, 165–176 (1971).

    Article  CAS  Google Scholar 

  32. Eggan, K. & Jaenisch, R. Differentiation of F1 embryonic stem cells into viable male and female mice by tetraploid embryo complementation. Methods Enzymol. 365, 25–39 (2003).

    Article  Google Scholar 

  33. Eggan, K. et al. Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation. Nat. Biotechnol. 20, 455–459 (2002).

    Article  CAS  Google Scholar 

  34. Schwenk, F. et al. Hybrid embryonic stem cell-derived tetraploid mice show apparently normal morphological, physiological, and neurological characteristics. Mol. Cell. Biol. 23, 3982–3989 (2003).

    Article  CAS  Google Scholar 

  35. Hadjantonakis, A.K., Dickinson, M.E., Fraser, S.E. & Papaioannou, V.E. Technicolour transgenics: imaging tools for functional genomics in the mouse. Nat. Rev. Genet. 4, 613–625 (2003).

    Article  CAS  Google Scholar 

  36. Nijs, M., Camus, M. & Van Steirteghem, A.C. Evaluation of different biopsy methods of blastomeres from 2-cell mouse embryos. Hum. Reprod. 3, 999–1003 (1988).

    Article  CAS  Google Scholar 

  37. Hartshorn, C., Rice, J.E. & Wangh, L.J. Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling. Mol. Reprod. Dev. 64, 41–51 (2003).

    Article  CAS  Google Scholar 

  38. Wang, Z. & Jaenisch, R. 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 (2004).

    Article  CAS  Google Scholar 

  39. James, R.M., Kaufman, M.H., Webb, S. & West, J.D. Electrofusion of mouse embryos results in uniform tetraploidy and not tetraploid/diploid mosaicism. Genet. Res. 60, 185–194 (1992).

    Article  CAS  Google Scholar 

  40. Bronson, R.A. & McLaren, A. Transfer to the mouse oviduct of eggs with and without the zona pellucida. J. Reprod. Fertil. 22, 129–137 (1970).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank R.R. Behringer, E.H. Lacy and V.E. Papaioannou for advice, discussions and support.

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Authors and Affiliations

  1. Developmental Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, 10021, New York, USA

    Guy S Eakin & Anna-Katerina Hadjantonakis

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  1. Guy S Eakin
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  2. Anna-Katerina Hadjantonakis
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Corresponding author

Correspondence to Anna-Katerina Hadjantonakis.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Time-lapse movie showing induction of tetraploidy by electrofusion. (MOV 1057 kb)

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Eakin, G., Hadjantonakis, AK. Production of chimeras by aggregation of embryonic stem cells with diploid or tetraploid mouse embryos. Nat Protoc 1, 1145–1153 (2006). https://doi.org/10.1038/nprot.2006.173

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