Generation of Chimeras by Aggregation of Embryonic Stem Cells with Diploid or Tetraploid Mouse Embryos

  • Jérôme Artus
  • Anna-Katerina HadjantonakisEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 693)


From the hybrid creatures of the Greek and Egyptian mythologies, the concept of the chimera has evolved and, in modern day biology, refers to an organism comprises of at least two populations of genetically distinct cells. Mouse chimeras have proven an invaluable tool for the generation of genetically modified strains. In addition, chimeras have been extensively used in developmental biology as a powerful tool to analyze the phenotype of specific mutations, to attribute function to gene products and to address the question of cell autonomy versus noncell autonomy of gene function. This chapter describes a simple and economical technique used to generate mouse chimeras by embryo aggregation. Multiple aggregation combinations are described each of which can be tailored to answer particular biological questions.

Key words

Mouse Embryo Chimera Aggregation Blastocyst Morula Development ES cells Diploid Tetraploid Developmental potential Lineage contribution 


  1. 1.
    Collins, F. S., Rossant, J., and Wurst, W. (2007) A mouse for all reasons, Cell 128, 9–13.PubMedCrossRefGoogle Scholar
  2. 2.
    Mintz, B. (1962) Formation of genetically mosaic mouse embryos, Am Zool 2, 432.Google Scholar
  3. 3.
    Tarkowski, A. K. (1961) Mouse chimaeras developed from fused eggs, Nature 190, 857–860.PubMedCrossRefGoogle Scholar
  4. 4.
    Gardner, R. L. (1968) Mouse chimeras obtained by the injection of cells into the blastocyst, Nature 220, 596–597.PubMedCrossRefGoogle Scholar
  5. 5.
    Arnold, S. J., and Robertson, E. J. (2009) Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo, Nat Rev Mol Cell Biol 10, 91–103.PubMedCrossRefGoogle Scholar
  6. 6.
    Dietrich, J. E., and Hiiragi, T. (2008) Stochastic processes during mouse blastocyst patterning, Cells Tissues Organs 188, 46–51.PubMedCrossRefGoogle Scholar
  7. 7.
    Rossant, J., and Tam, P. P. (2009) Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse, Development 136, 701–713.PubMedCrossRefGoogle Scholar
  8. 8.
    Yamanaka, Y., Ralston, A., Stephenson, R. O., and Rossant, J. (2006) Cell and molecular regulation of the mouse blastocyst, Dev Dyn 235, 2301–2314.PubMedCrossRefGoogle Scholar
  9. 9.
    Kwon, G. S., Viotti, M., and Hadjantonakis, A. K. (2008) The endoderm of the mouse embryo arises by dynamic widespread intercalation of embryonic and extraembryonic lineages, Dev Cell 15, 509–520.PubMedCrossRefGoogle Scholar
  10. 10.
    Evans, M. J., and Kaufman, M. H. (1981) Establishment in culture of pluripotential cells from mouse embryos, Nature 292, 154–156.PubMedCrossRefGoogle Scholar
  11. 11.
    Martin, G. R. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells, Proc Natl Acad Sci USA 78, 7634–7638.PubMedCrossRefGoogle Scholar
  12. 12.
    Smith, A. G., Heath, J. K., Donaldson, D. D., Wong, G. G., Moreau, J., Stahl, M., and Rogers, D. (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides, Nature 336, 688–690.PubMedCrossRefGoogle Scholar
  13. 13.
    Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A., Stewart, C. L., Gearing, D. P., Wagner, E. F., Metcalf, D., Nicola, N. A., and Gough, N. M. (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells, Nature 336, 684–687.PubMedCrossRefGoogle Scholar
  14. 14.
    Ying, Q. L., Nichols, J., Chambers, I., and Smith, A. (2003) BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3, Cell 115, 281–292.PubMedCrossRefGoogle Scholar
  15. 15.
    Ying, Q. L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. (2008) The ground state of embryonic stem cell self-renewal, Nature 453, 519–523.PubMedCrossRefGoogle Scholar
  16. 16.
    Beddington, R. S., and Robertson, E. J. (1989) An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo, Development 105, 733–737.PubMedGoogle Scholar
  17. 17.
    Bradley, A., Evans, M., Kaufman, M. H., and Robertson, E. (1984) Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines, Nature 309, 255–256.PubMedCrossRefGoogle Scholar
  18. 18.
    Nagy, A., Sass, M., and Markkula, M. (1989) Systematic non-uniform distribution of parthenogenetic cells in adult mouse chimaeras, Development 106, 321–324.PubMedGoogle Scholar
  19. 19.
    Kunath, T., Arnaud, D., Uy, G. D., Okamoto, I., Chureau, C., Yamanaka, Y., Heard, E., Gardner, R. L., Avner, P., and Rossant, J. (2005) Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts, Development 132, 1649–1661.PubMedCrossRefGoogle Scholar
  20. 20.
    Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., and Rossant, J. (1998) Promotion of trophoblast stem cell proliferation by FGF4, Science 282, 2072–2075.PubMedCrossRefGoogle Scholar
  21. 21.
    Eakin, G. S., and Behringer, R. R. (2003) Tetraploid development in the mouse, Dev Dyn 228, 751–766.PubMedCrossRefGoogle Scholar
  22. 22.
    Snow, M. H. (1973) Tetraploid mouse embryos produced by cytochalasin B during cleavage, Nature 244, 513–515.PubMedCrossRefGoogle Scholar
  23. 23.
    Tarkowski, A. K., Witkowska, A., and Opas, J. (1977) Development of cytochalasin in B-induced tetraploid and diploid/tetraploid mosaic mouse embryos, J Embryol Exp Morphol 41, 47–64.PubMedGoogle Scholar
  24. 24.
    Eakin, G. S., Hadjantonakis, A. K., Papaioannou, V. E., and Behringer, R. R. (2005) Developmental potential and behavior of tetraploid cells in the mouse embryo, Dev Biol 288, 150–159.PubMedCrossRefGoogle Scholar
  25. 25.
    Mackay, G. E., and West, J. D. (2005) Fate of tetraploid cells in 4n ↔ 2n chimeric mouse blastocysts, Mech Dev 122, 1266–1281.PubMedCrossRefGoogle Scholar
  26. 26.
    Cross, J. C. (2001) Factors affecting the developmental potential of cloned mammalian embryos, Proc Natl Acad Sci U S A 98, 5949–5951.PubMedCrossRefGoogle Scholar
  27. 27.
    Eggan, K., Akutsu, H., Loring, J., Jackson-Grusby, L., Klemm, M., Rideout, W. M., 3rd, Yanagimachi, R., and Jaenisch, R. (2001) Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation, Proc Natl Acad Sci U S A 98, 6209–6214.PubMedCrossRefGoogle Scholar
  28. 28.
    Wakayama, T., Rodriguez, I., Perry, A. C., Yanagimachi, R., and Mombaerts, P. (1999) Mice cloned from embryonic stem cells, Proc Natl Acad Sci U S A 96, 14984–14989.PubMedCrossRefGoogle Scholar
  29. 29.
    Joyner, A. L. (2001) Gene targeting: A practical approach, second edition, The Practical Approach Series.Google Scholar
  30. 30.
    Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. (2003) Manipulating the mouse embryo. A laboratory manual, third edition, Cold Spring Harbor Press.Google Scholar
  31. 31.
    Hadjantonakis, A. K., Dickinson, M. E., Fraser, S. E., and Papaioannou, V. E. (2003) Technicolour transgenics: imaging tools for functional genomics in the mouse, Nat Rev Genet 4, 613–625.PubMedCrossRefGoogle Scholar
  32. 32.
    Nowotschin, S., Eakin, G. S., and Hadjantonakis, A. K. (2009) Live-imaging fluorescent proteins in mouse embryos: multi-dimensional, multi-spectral perspectives, Trends Biotechnol 27, 266–276.PubMedCrossRefGoogle Scholar
  33. 33.
    Tanaka, M., Gertsenstein, M., Rossant, J., and Nagy, A. (1997) Mash2 acts cell autonomously in mouse spongiotrophoblast development, Dev Biol 190, 55–65.PubMedCrossRefGoogle Scholar
  34. 34.
    Hadjantonakis, A. K., Macmaster, S., and Nagy, A. (2002) Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal, BMC Biotechnol 2, 11.PubMedCrossRefGoogle Scholar
  35. 35.
    Adams, R. H., Porras, A., Alonso, G., Jones, M., Vintersten, K., Panelli, S., Valladares, A., Perez, L., Klein, R., and Nebreda, A. R. (2000) Essential role of p38alpha MAP kinase in placental but not embryonic cardiovascular development, Mol Cell 6, 109–116.PubMedGoogle Scholar
  36. 36.
    Damert, A., Miquerol, L., Gertsenstein, M., Risau, W., and Nagy, A. (2002) Insufficient VEGFA activity in yolk sac endoderm compromises haematopoietic and endothelial differentiation, Development 129, 1881–1892.PubMedGoogle Scholar
  37. 37.
    Duncan, S. A., Nagy, A., and Chan, W. (1997) Murine gastrulation requires HNF-4 regulated gene expression in the visceral endoderm: tetraploid rescue of Hnf-4(−/−) embryos, Development 124, 279–287.PubMedGoogle Scholar
  38. 38.
    Guillemot, F., Nagy, A., Auerbach, A., Rossant, J., and Joyner, A. L. (1994) Essential role of Mash-2 in extraembryonic development, Nature 371, 333–336.PubMedCrossRefGoogle Scholar
  39. 39.
    Yamamoto, H., Flannery, M. L., Kupriyanov, S., Pearce, J., McKercher, S. R., Henkel, G. W., Maki, R. A., Werb, Z., and Oshima, R. G. (1998) Defective trophoblast function in mice with a targeted mutation of Ets2, Genes Dev 12, 1315–1326.PubMedCrossRefGoogle Scholar
  40. 40.
    Varlet, I., Collignon, J., and Robertson, E. J. (1997) Nodal expression in the primitive endoderm is required for specification of the anterior axis during mouse gastrulation, Development 124, 1033–1044.PubMedGoogle Scholar
  41. 41.
    Ciruna, B. G., Schwartz, L., Harpal, K., Yamaguchi, T. P., and Rossant, J. (1997) Chimeric analysis of fibroblast growth factor receptor-1 (Fgfr1) function: a role for FGFR1 in morphogenetic movement through the primitive streak, Development 124, 2829–2841.PubMedGoogle Scholar
  42. 42.
    de Bruin, A., Wu, L., Saavedra, H. I., Wilson, P., Yang, Y., Rosol, T. J., Weinstein, M., Robinson, M. L., and Leone, G. (2003) Rb function in extraembryonic lineages suppresses apoptosis in the CNS of Rb-deficient mice, Proc Natl Acad Sci USA 100, 6546–6551.PubMedCrossRefGoogle Scholar
  43. 43.
    Wu, L., de Bruin, A., Saavedra, H. I., Starovic, M., Trimboli, A., Yang, Y., Opavska, J., Wilson, P., Thompson, J. C., Ostrowski, M. C., Rosol, T. J., Woollett, L. A., Weinstein, M., Cross, J. C., Robinson, M. L., and Leone, G. (2003) Extra-embryonic function of Rb is essential for embryonic development and viability, Nature 421, 942–947.PubMedCrossRefGoogle Scholar
  44. 44.
    Mereau, A., Grey, L., Piquet-Pellorce, C., and Heath, J. K. (1993) Characterization of a binding protein for leukemia inhibitory factor localized in extracellular matrix, J Cell Biol 122, 713–719.PubMedCrossRefGoogle Scholar
  45. 45.
    McMahon, A. P., and Bradley, A. (1990) The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain, Cell 62, 1073–1085.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Developmental Biology ProgramSloan-Kettering InstituteNew YorkUSA

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