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Production of cloned pigs from in vitro systems

Nature Biotechnology volume 18, pages 1055–1059 (2000)Cite this article

Abstract

Here we describe a procedure for cloning pigs by the use of in vitro culture systems. Four healthy male piglets from two litters were born following nuclear transfer of cultured somatic cells and subsequent embryo transfer. The initiation of five additional pregnancies demonstrates the reproducibility of this procedure. Its important features include extended in vitro culture of fetal cells preceding nuclear transfer, as well as in vitro maturation and activation of oocytes and in vitro embryo culture. The cell culture and nuclear transfer techniques described here should allow the use of genetic modification procedures to produce tissues and organs from cloned pigs with reduced immunogenicity for use in xenotransplantation.

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Figure 1: Nuclear transfer piglets from two litters.
Figure 2: Porcine donor cells.
Figure 3: Multiplex electropherogram of three microsatellite markers.

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References

  1. Campbell, K.H., McWhir, J., Ritchie, W.A. & Wilmut, I. Sheep cloned by nuclear transfer from a cultured cell line. Nature 380, 64–66 (1996).

    Article  CAS  PubMed  Google Scholar 

  2. 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 

  3. Cibelli, J.B. et al. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280, 1256–1258 (1998).

    Article  CAS  PubMed  Google Scholar 

  4. Wells, D.N., Misica, P.M. & Tervit, H.R. Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. Biol. Reprod. 60, 996–1005 (1999).

    Article  CAS  PubMed  Google Scholar 

  5. Baguisi, A. et al. Production of goats by somatic cell nuclear transfer. Nat. Biotechnol. 17, 456–461 (1999).

    Article  CAS  PubMed  Google Scholar 

  6. 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 

  7. Prather, R.S., Sims, M.M. & First, N.L. Nuclear transplantation in early pig embryos. Biol. Reprod. 41, 414–418 (1989).

    Article  CAS  PubMed  Google Scholar 

  8. Prather, R.S., Tao, T. & Machaty, Z. Development of the techniques for nuclear transfer in pigs. Theriogenology 51, 487–498 (1999).

    Article  CAS  PubMed  Google Scholar 

  9. Hazeleger, W. & Kemp, B. State of the art in pig embryo transfer. Theriogenology 51, 81–90 (1999).

    Article  CAS  PubMed  Google Scholar 

  10. Niemann, H. & Kues, W.A. Transgenic livestock: premises and promises. Anim. Reprod. Sci. 60–61, 277–293 (2000).

    Article  PubMed  Google Scholar 

  11. Tao, T., Machaty, Z., Abeydeera, L.R., Day, B.N. & Prather, R.S. Optimisation of porcine oocyte activation following nuclear transfer. Zygote 8, 69–77 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Polejaeva, I.A. et al. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407, 505–509 (2000).

    Article  Google Scholar 

  13. Onishi, A. et al. Pig cloning by microinjection of fetal fibroblast nuclei. Science 289, 1188–1190 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Cozzi, E., Masroor, S., Soin, B., Vial, C. & White, D.J. Progress in xenotransplantation. Clin. Nephrol. 53, 13–18 (2000).

    Google Scholar 

  15. Cozzi, E. & White, D.J. The generation of transgenic pigs as potential organ donors for humans. Nat. Med. 1, 964–966 (1995).

    Article  CAS  PubMed  Google Scholar 

  16. Joziasse, D.H. & Oriol, R. Xenotransplantation: the importance of the Galalpha1,3Gal epitope in hyperacute vascular rejection. Biochim. Biophys. Acta 1455, 403–418 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. 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  PubMed  Google Scholar 

  18. Mueller, S. et al. Chimeric pigs following blastocyst injection of transgenic porcine primordial germ cells. Mol. Reprod. Dev. 54, 244–254 (1999).

    Article  CAS  PubMed  Google Scholar 

  19. Piedrahita, J.A. Targeted modification of the domestic animal genome. Theriogenology 53, 105–116 (2000).

    Article  CAS  PubMed  Google Scholar 

  20. McCreath, K.J. et al. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 405, 1066–1069 (2000).

    Article  CAS  PubMed  Google Scholar 

  21. Lanza, R.P. et al. Extension of cell life-span and telomere length in animals cloned from senescent somatic cells. Science 288, 665–669 (2000).

    Article  CAS  PubMed  Google Scholar 

  22. Gordon, I.R. Controlled reproduction in pigs. (CAB International, Wallingford, Oxon, UK, New York, NY; 1997).

    Google Scholar 

  23. Hunter, R.H. Chronological and cytological details of fertilization and early embryonic development in the domestic pig, Sus scrofa. Anat. Rec. 178, 169–185 (1974).

    Article  CAS  PubMed  Google Scholar 

  24. Papaioannou, V.E. & Ebert, K.M. The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro. Development 102, 793–803 (1988).

    CAS  PubMed  Google Scholar 

  25. Polge, C., Rowson, L.E. & Chang, M.C. The effect of reducing the number of embryos during early stages of gestation on the maintenance of pregnancy in the pig. J. Reprod. Fertil. 12, 395–397 (1966).

    Article  CAS  PubMed  Google Scholar 

  26. Saito, S. & Niemann, H. Effects of extracellular matrices and growth factors on the development of isolated porcine blastomeres. Biol. Reprod. 44, 927–936 (1991).

    Article  CAS  PubMed  Google Scholar 

  27. Reichelt, B. & Niemann, H. Generation of identical twin piglets following bisection of embryos at the morula and blastocyst stage. J. Reprod. Fertil. 100, 163–172 (1994).

    Article  CAS  PubMed  Google Scholar 

  28. Tao, T., Reichelt, B. & Niemann, H. Ratio of inner cell mass and trophoblastic cells in demi- and intact pig embryos. J. Reprod. Fertil. 104, 251–258 (1995).

    Article  CAS  PubMed  Google Scholar 

  29. Susko-Parrish, J.L., Leibfried-Rutledge, M.L., Northey, D.L., Schutzkus, V. & First, N.L. Inhibition of protein kinases after an induced calcium transient causes transition of bovine oocytes to embryonic cycles without meiotic completion. Dev. Biol. 166, 729–739 (1994).

    Article  CAS  PubMed  Google Scholar 

  30. Strelchenko, N.S., Betthauser, J.M., Jurgella, G.L., Pace, M.P. & Bishop, M.D. Method of cloning bovines using reprogrammed non-embryonic bovine cells. US 6,011,197 (2000).

  31. Dalmasso, A.P., Vercellotti, G.M., Platt, J.L. & Bach, F.H. Inhibition of complement-mediated endothelial cell cytotoxicity by decay-accelerating factor. Potential for prevention of xenograft hyperacute rejection. Transplantation 52, 530–533 (1991).

    Article  CAS  PubMed  Google Scholar 

  32. Bhatti, F.N. et al. Three-month survival of HDAFF transgenic pig hearts transplanted into primates. Transplant. Proc. 31, 958 (1999).

    Article  CAS  PubMed  Google Scholar 

  33. Petters, R.M. & Wells, K.D. Culture of pig embryos. J. Reprod. Fertil. Suppl. 48, 61–73 (1993).

    CAS  PubMed  Google Scholar 

  34. Yoshida, M., Ishigaki, K., Nagai, T., Chikyu, M. & Pursel, V.G. Glutathione concentration during maturation and after fertilization in pig oocytes: relevance to the ability of oocytes to form male pronucleus. Biol. Reprod. 49, 89–94 (1993).

    Article  CAS  PubMed  Google Scholar 

  35. Grupen, C.G., Nagashima, H. & Nottle, M.B. Role of epidermal growth factor and insulin-like growth factor-I on porcine oocyte maturation and embryonic development in vitro. Reprod. Fertil. Dev. 9, 571–575 (1997).

    Article  CAS  PubMed  Google Scholar 

  36. Naito, K., Fukuda, Y. & Toyoda, Y. Effects of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in vitro. Gamete Res. 21, 289–295 (1988).

    Article  CAS  PubMed  Google Scholar 

  37. Funahashi, H., Cantley, T.C. & Day, B.N. Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization. Biol. Reprod. 57, 49–53 (1997).

    Article  CAS  PubMed  Google Scholar 

  38. Funahashi, H. & Day, B.N. Effects of the duration of exposure to hormone supplements on cytoplasmic maturation of pig oocytes in vitro. J. Reprod. Fertil. 98, 179–185 (1993).

    Article  CAS  PubMed  Google Scholar 

  39. Rosenkrans, C.F.J. & First, N.L. Culture of bovine zygotes to the blastocyst stage: effects of amino acids and vitamins. Theriogenology 35, 266 (1991).

  40. Long, C.R., Dobrinsky, J.R. & Johnson, L.A. In vitro production of pig embryos: comparisons of culture media and boars. Theriogenology 51, 1375–1390 (1999).

    Article  CAS  PubMed  Google Scholar 

  41. Grupen, C.G. & Nottle, M.B. A simple modification of the in vitro fertilization procedure improves the efficiency of in vitro pig embryo production. Theriogenology 53, 422 (2000).

    Google Scholar 

  42. Rath, D., Johnson, L., Dobrinsky, J., Welch, G. & Niemann, H. Production of piglets preselected for sex following in vitro fertilization with X and Y chromosome-bearing spermatozoa sorted by flow cytometry. Theriogenology 47, 795–800 (1997).

    Article  CAS  PubMed  Google Scholar 

  43. USDA supported U.S. Pig Genome Project, http://sol.marc.usda.gov/genome/swine/

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Acknowledgements

We would like to acknowledge Drs. Neal First and Tom Crenshaw of the University of Wisconsin, Madison, for their assistance in coordinating porcine embryo transfer at the University of Wisconsin Swine Research Facility and Dr. Matthew Wheeler of the University of Illinois, Champagne-Urbana, for discussions on porcine reproduction. We would also like to thank Dr. Heiner Niemann of the Institut für Tierzucht und Tierverhalten, Neustadt, Germany, for discussions on embryo transfer, and Dr. Max F. Rothschild of the US Pig Genome Project, Iowa State University, for the kind gift of porcine microsatellite markers.

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Authors and Affiliations

  1. Infigen, Inc., 1825 Infinity Drive, DeForest, 53532, WI

    Jeff Betthauser, Erik Forsberg, Monica Augenstein, Lynette Childs, Kenneth Eilertsen, Joellyn Enos, Todd Forsythe, Paul Golueke, Gail Jurgella, Richard Koppang, Tiffany Lesmeister, Kelly Mallon, Greg Mell, Pavla Misica, Marvin Pace, Martha Pfister-Genskow, Nikolai Strelchenko, Gary Voelker, Steven Watt & Michael Bishop

  2. Imutran, Ltd., Cambridge, United Kingdom

    Simon Thompson

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Correspondence to Michael Bishop.

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Betthauser, J., Forsberg, E., Augenstein, M. et al. Production of cloned pigs from in vitro systems. Nat Biotechnol 18, 1055–1059 (2000). https://doi.org/10.1038/80242

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