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Production of cloned mice by somatic cellnuclear transfer

Nature Protocols volume 1, pages 125–138 (2006)Cite this article

Abstract

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), the success rate for producing live offspring by cloning remains <5%. Nevertheless, the techniques have potential as important tools for future research in basic biology. We have been able to develop a stable NT method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although manipulation of the piezo unit is complex, once mastered it is of great help not only in NT experiments but also in almost all other forms of micromanipulation. In addition to this technique, embryonic stem (ES) cell lines established from somatic cell nuclei by NT can be generated relatively easily from a variety of mouse genotypes and cell types. Such NT-ES cells can be used not only for experimental models of human therapeutic cloning but also as a backup of the donor cell's genome. Our most recent protocols for mouse cloning, as described here, will allow the production of cloned mice in ≥3 months.

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Figure 2: Piezo unit and manipulation chamber.
Figure 1: Injection pipette construction.
Figure 3: Oocyte enucleation.
Figure 4: Donor nucleus collection.
Figure 5: Injection.
Figure 6: Aggregation of cloned embryos.
Figure 7: The mouse cloning procedure.

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References

  1. McGrath, J. & Solter, D. Inability of mouse blastomere nuclei transferred to enucleated zygotes to support development in vitro. Science 226, 1317–1319 (1984).

    Article  CAS  PubMed  Google Scholar 

  2. Willadsen, S.M. Nuclear transplantation in sheep embryos. Nature 320, 63–65 (1986).

    Article  CAS  PubMed  Google Scholar 

  3. Tsunoda, Y. et al. Full-term development of mouse blastomere nuclei transplanted into enucleated two-cell embryos. J. Exp. Zool. 242, 147–151 (1987).

    Article  CAS  PubMed  Google Scholar 

  4. Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. & Campbell, K.H. Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–813 (1997).

    Article  CAS  PubMed  Google Scholar 

  5. Kono, T., Tsunoda, Y. & Nakahara, T. Production of identical twin and triplet mice by nuclear transplantation. J. Exp. Zool. 257, 214–219 (1991).

    Article  CAS  PubMed  Google Scholar 

  6. Tsunoda, Y. & Kato, Y. Full-term development after transfer of nuclei from 4-cell and compacted morula stage embryos to enucleated oocytes in the mouse. J. Exp. Zool. 278, 250–254 (1997).

    Article  CAS  PubMed  Google Scholar 

  7. Tsunoda, Y. & Kato, Y. Not only inner cell mass cell nuclei but also trophectoderm nuclei of mouse blastocysts have a developmental totipotency. J. Reprod. Fertil. 113, 181–184 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Tsunoda, Y. & Kato, Y. A manual for nuclear transfer is missing. in Nuclear Transfer Technology in Transgenic Animal Technology, A Laboratory Handbook, 2nd edn. (ed. Pinkert, C.A.) 195–231 (Academic Press, Amsterdam, 2002).

    Chapter  Google Scholar 

  9. Kimura, Y. & Yanagimachi, R. Intracytoplasmic sperm injection in the mouse. Biol. Reprod. 52, 709–720 (1995).

    Article  CAS  PubMed  Google Scholar 

  10. 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  PubMed  Google Scholar 

  11. Kawase, Y. et al. Application of the piezo-micromanipulator for injection of embryoic stem cells into mouse blastocysts. Contemp. Top. Lab. Anim. Sci. 40, 31–4 (2001).

    CAS  PubMed  Google Scholar 

  12. Wakayama, T., Perry, A.C., Zuccotti, M., Johnson, K.R. & Yanagimachi, R. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–374 (1998).

    Article  CAS  PubMed  Google Scholar 

  13. Wakayama, T. & Yanagimachi, R. Cloning of male mice from adult tail-tip cells. Nat. Genet. 22, 127–128 (1999).

    Article  CAS  PubMed  Google Scholar 

  14. Ogura, A. et al. Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells. Biol. Reprod. 62, 1579–1584 (2000).

    Article  CAS  PubMed  Google Scholar 

  15. Wakayama, T. & Yanagimachi, R. Mouse cloning with nucleus donor cells of different age and type. Mol. Reprod. Dev. 58, 376–383 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. Ono, Y., Shimozawa, N., Ito, M. & Kono, T. Cloned mice from fetal fibroblast cells arrested at metaphase by a serial nuclear transfer. Biol. Reprod. 64, 44–50 (2001).

    Article  CAS  PubMed  Google Scholar 

  17. Wakayama, T., Rodriguez, I., Perry, A.C., Yanagimachi, R. & Mombaerts, P. Mice cloned from embryonic stem cells. Proc. Natl. Acad. Sci. USA 96, 14984–14989 (1999).

    Article  CAS  PubMed  Google Scholar 

  18. Inoue, K. et al. Generation of cloned mice by direct nuclear transfer from natural killer T cells. Curr. Biol. 15, 1114–1118 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Miki, H. et al. Birth of mice produced by germ cell nuclear transfer. Genesis 41, 81–86 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Inoue, K. et al. Inefficient reprogramming of the hematopoietic stem cell genome following nuclear transfer. J. Cell Sci. 119, 1985–1991 (2006).

    Article  CAS  PubMed  Google Scholar 

  21. Yamazaki, Y. et al. Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer. Proc. Natl. Acad. Sci. USA 98, 14022–14026 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Inoue, K. et al. Effects of donor cell type and genotype on the efficiency of mouse somatic cell cloning. Biol. Reprod. 69, 1394–1400 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Boiani, M., Eckardt, S., Scholer, H.R. & McLaughlin, K.J. Oct4 distribution and level in mouse clones: consequences for pluripotency. Genes Dev. 16, 1209–1219 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Tanaka, S. et al. Placentomegaly in cloned mouse concepti caused by expansion of the spongiotrophoblast layer. Biol. Reprod. 65, 1813–1821 (2001).

    Article  CAS  PubMed  Google Scholar 

  25. Tamashiro, K.L. et al. Cloned mice have an obese phenotype not transmitted to their offspring. Nat. Med. 8, 262–267 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Ogonuki, N. et al. Early death of mice cloned from somatic cells. Nat. Genet. 30, 253–254 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Munsie, M.J. et al. Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei. Curr. Biol. 10, 989–992 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Wakayama, T. et. al. Equivalency of nuclear transfer-derived embryonic stem cells to those derived from fertilized mouse blastocysts. Stem Cells, published online 2006. http://stemcells.alphamedpress.org/cgi/reprint/2005-0537v1.pdf

  29. Wakayama, S. et al. Mice cloned by nuclear transfer from somatic and ntES cells derived from the same individuals. J. Reprod. Dev. 51, 765–772 (2005).

    Article  PubMed  Google Scholar 

  30. Rideout, W.M., Hochedlinger, K., Kyba, M., Daley, G.Q. & Jaenisch, R. Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell 109, 17–27 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Hochedlinger, K. & Jaenisch, R. Monoclonal mice generated by nuclear transfer from mature B and T donor cells. Nature 415, 1035–1038 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Eggan, K. et al. Mice cloned from olfactory sensory neurons. Nature 428, 44–49 (2004).

    Article  CAS  PubMed  Google Scholar 

  33. Li, J., Ishii, T., Feinstein, P. & Mombaerts, P. Odorant receptor gene choice is reset by nuclear transfer from mouse olfactory sensory neurons. Nature 428, 393–399 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Blelloch, R.H. et al. Nuclear cloning of embryonal carcinoma cells. Proc. Natl. Acad. Sci. USA 101, 13985–13990 (2004).

    CAS  PubMed  Google Scholar 

  35. Hochedlinger, K. et al. Reprogramming of a melanoma genome by nuclear transplantation. Genes Dev. 18, 1875–1885 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Wakayama, S. et al. Establishment of male and female nuclear transfer embryonic stem cell lines from different mouse strains and tissues. Biol. Reprod. 72, 932–936 (2005).

    Article  CAS  PubMed  Google Scholar 

  37. Wakayama, S. et al. Propagation of an infertile hermaphrodite mouse lacking germ cells by using nuclear transfer and embryonic stem cell technology. Proc. Natl. Acad. Sci. USA 102, 29–33 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Kishigami, S. et al. Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer. Biochem. Biophys. Res. Commun. 340, 183–189 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Rybouchkin, A., Kato, Y. & Tsunoda, Y. Role of histone acetylation in reprogramming of somatic nuclei following nuclear transfer. Biol. Reprod., 74, 1089–1098 (2006).

    Article  Google Scholar 

  40. Boiani, M., Eckardt, S., Leu, N.A., Scholer, H.R. & McLaughlin, K.J. Pluripotency deficit in clones overcome by clone-clone aggregation: epigenetic complementation? EMBO J. 22, 5304–5312 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Nagy, A., Gertsenstein, M., Vintersten, K. & Behringer, R. Manipulating the Mouse Embryo. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2003).

    Google Scholar 

  42. Wakayama, S. et al. Production of offspring from one-day-old oocytes stored at room temperature. J. Reprod. Dev. 50, 627–637 (2004).

    Article  PubMed  Google Scholar 

  43. Schatten, G., Smith, J., Navara, C., Park, J.H. & Pedersen, R. Culture of human embryonic stem cells. Nat. Methods 2, 455–463 (2005).

    Article  CAS  PubMed  Google Scholar 

  44. Wakayama, T. & Yanagimachi, R. Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei. Reproduction 122, 49–60 (2001).

    Article  CAS  PubMed  Google Scholar 

  45. Cavaleri, F., Gentile, L., Schö ler, H.R. & Boiani, M. Recombinant human albumin supports development of somatic cell nuclear transfer embryos in mice: toward the establishment of a chemically defined cloning protocol. Cloning Stem Cells 8, 24–40 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. Elsheikh, A.S., Takahashi, Y., Nagano, M. & Kanagawa, H. Manipulated mouse embryos as bioassay system for water quality control. Reprod. Domest. Anim. 38, 204–208 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. 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  PubMed  Google Scholar 

  48. Wakayama, T. & Yanagimachi, R. Cloning the laboratory mouse. Semin. Cell Dev. Biol. 10, 253–258 (1999).

    Article  CAS  PubMed  Google Scholar 

  49. Ogawa, K., Matsui, H., Ohtsuka, S. & Niwa, H. A novel mechanism for regulating clonal propagation of mouse ES cells. Genes Cells 9, 471–477 (2004).

    Article  CAS  Google Scholar 

  50. Wakayama, T. Establishment of ES cell lines from adult somatic cells by nuclear transfer. in Cell Biology: A Laboratory Handbook 3rd edn vol. 1 (ed. Celis, J.) 87–95 (Academic Press, San Diego, 2006).

    Chapter  Google Scholar 

  51. Kishigami, S. et al. Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids. Dev. Biol. 289, 195–205 (2006).

    Article  CAS  PubMed  Google Scholar 

  52. Wakayama, T. & Yanagimachi, R. Development of normal mice from oocytes injected with freeze-dried spermatozoa. Nat. Biotechnol. 16, 639–641 (1998).

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Center for Developmental Biology RIKEN Kobe, 650-0047, Japan

    Satoshi Kishigami, Sayaka Wakayama, Nguyen Van Thuan, Hiroshi Ohta, Eiji Mizutani, Takafusa Hikichi, Hong-Thuy Bui & Teruhiko Wakayama

  2. Max Planck Institute for Molecular Biomedicine, Mendelstrasse 7, Muenster, 48149, Germany

    Sebastian Balbach & Michele Boiani

  3. RIKEN Bioresource Center, Tsukuba, 305-0074, Ibaraki, Japan

    Atsuo Ogura

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  1. Satoshi Kishigami
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Correspondence to Michele Boiani or Teruhiko Wakayama.

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Kishigami, S., Wakayama, S., Van Thuan, N. et al. Production of cloned mice by somatic cellnuclear transfer. Nat Protoc 1, 125–138 (2006). https://doi.org/10.1038/nprot.2006.21

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