Two-staged nuclear transfer can enhance the developmental ability of goat–sheep interspecies nuclear transfer embryos in vitro

  • Li-Bing MaEmail author
  • Lu Cai
  • Jia-Jia Li
  • Xiu-Li Chen
  • Feng-Yu Ji


The technique of interspecies somatic cell nuclear transfer, in which interspecies cloned embryos can be reconstructed by using domestic animal oocytes as nuclear recipients and endangered animal or human somatic cells as nuclear donors, can afford more opportunities in endangered animal rescue and human tissue transplantation, but the application of this technique is limited by extremely low efficiency which may be attributed to donor nucleus not fully reprogrammed by xenogenic cytoplasm. In this study, goat fetal fibroblasts (GFFs) were used as nuclear donors, in vitro-matured sheep oocytes were used as nuclear recipients, and a two-stage nuclear transfer procedure was performed to improve the developmental ability of goat–sheep interspecies clone embryos. In the first stage nuclear transfer (FSNT), GFFs were injected into the ooplasm of enucleated sheep metaphase-II oocytes, then non-activated reconstructed embryos were cultured in vitro, so that the donor nucleus could be exposed to the ooplasm for a period of time. Subsequently, in the second stage nuclear transfer, FSNT-derived non-activated reconstructed embryo was centrifuged, and the donor nucleus was then transferred into another freshly enucleated sheep oocyte. Compared with the one-stage nuclear transfer, two-stage nuclear transfer could significantly enhance the blastocyst rate of goat–sheep interspecies clone embryos, and this result indicated that longtime exposure to xenogenic ooplasm benefits the donor nucleus to be reprogrammed. The two-stage nuclear transfer procedure has two advantages, one is that the donor nucleus can be exposed to the ooplasm for a long time, the other is that the problem of oocyte aging can be solved.


Nuclear transfer Interspecies Goat Sheep 



This work was supported by the Natural Science Foundation of Inner Mongolia Autonomous Region of China (no. 2009BS0503), the Research Program of Natural Science at Universities of Inner Mongolia Autonomous Region of China (no. NJ09093), and the Innovation Foundation of Inner Mongolia University of Science & Technology (no. 2009NC058).


  1. Beaujean N.; Taylor J.; Gardner J.; Wilmut I.; Meehan R.; Young L. Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer. Biol Reprod 71: 185–193; 2004.CrossRefPubMedGoogle Scholar
  2. Bui L. C.; Evsikov A. V.; Khan D. R.; Archilla C.; Peynot N.; Hénaut A.; Le Bourhis D.; Vignon X.; Renard J. P.; Duranthon V. Retrotransposon expression as a defining event of genome reprogramming in fertilized and cloned bovine embryos. Reproduction 138: 289–299; 2009.CrossRefPubMedGoogle Scholar
  3. Choi I.; Campbell K. H. Treatment of ovine oocytes with caffeine increases the accessibility of DNase I to the donor chromatin and reduces apoptosis in somatic cell nuclear transfer embryos. Reprod Fertil Dev 22: 1000–1014; 2010.CrossRefPubMedGoogle Scholar
  4. Cibelli J. B.; Stice S. L.; Golueke P. J.; Kane J. J.; Jerry J.; Blackwell C.; de Leon FA Ponce; Robl J. M. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280(Robl JM): 1256–1258; 1998.CrossRefPubMedGoogle Scholar
  5. Collas P.; Robl J. M. Relationship between nuclear remodeling and development in nuclear transplant rabbit embryos. Biol Reprod 45: 455–465; 1991.CrossRefPubMedGoogle Scholar
  6. De Sousa P. A.; Dobrinsky J. R.; Zhu J.; Archibald A. L.; Ainslie A.; Bosma W.; Bowering J.; Bracken J.; Ferrier P. M.; Fletcher J.; Gasparrini B.; Harkness L.; Johnston P.; Ritchie M.; Ritchie W. A.; Travers A.; Albertini D.; Dinnyes A.; King T. J.; Wilmut I. Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy. Biol Reprod 66: 642–650; 2002.CrossRefPubMedGoogle Scholar
  7. Du F.; Xu J.; Zhang J.; Gao S.; Carter M. G.; He C.; Sung L. Y.; Chaubal S.; Fissore R. A.; Tian X. C.; Yang X.; Chen Y. E. Beneficial effect of young oocytes for rabbit somatic cell nuclear transfer. Cloning Stem Cells 11: 131–140; 2009.CrossRefPubMedGoogle Scholar
  8. Fulka Jr. J. Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning. Adv Exp Med Biol 591: 93–102; 2007.CrossRefPubMedGoogle Scholar
  9. Hall V. J.; Compton D.; Stojkovic P.; Nesbitt M.; Herbert M.; Murdoch A.; Stojkovic M. Developmental competence of human in vitro aged oocytes as host cells for nuclear transfer. Hum Reprod 22: 52–62; 2007.CrossRefPubMedGoogle Scholar
  10. Heindryckx B.; Rybouchkin A.; Van Der Elst J.; Dhont M. Serial pronuclear transfer increases the developmental potential of in vitro-matured oocytes in mouse cloning. Biol Reprod 67: 1790–1795; 2002.CrossRefPubMedGoogle Scholar
  11. Jiang Y.; Chen T.; Nan C. L.; Ouyang Y. C.; Sun Q. Y.; Chen D. Y. In vitro culture and mtDNA fate of ibex–rabbit nuclear transfer embryos. Zygote 13: 233–240; 2005.CrossRefPubMedGoogle Scholar
  12. Kawahara M.; Wakai T.; Yamanaka K.; Kobayashi J.; Sugimura S.; Shimizu T.; Matsumoto H.; Kim J. H.; Sasada H.; Sato E. Caffeine promotes premature chromosome condensation formation and in vitro development in porcine reconstructed embryos via a high level of maturation promoting factor activity during nuclear transfer. Reproduction 130: 351–357; 2005.CrossRefPubMedGoogle Scholar
  13. Lanza R. P.; Cibelli J. B.; Diaz F. Cloning of endangered species (Bosgaurus) using interspecies nuclear transfer. Cloning 2: 79–90; 2000.CrossRefPubMedGoogle Scholar
  14. Lee B. C.; Kim M. K.; Jang G.; Oh H. J.; Yuda F.; Kim H. J.; Hossein M. S.; Kim J. J.; Kang S. K.; Schatten G.; Hwang W. S. Dogs cloned from adult somatic cells. Nature 436: 641; 2005.CrossRefPubMedGoogle Scholar
  15. Li J. S.; Cheng D. Y.; Hang Z. M.; Zhu Z. Y.; Weng D. C.; Sun Q. Y.; Liu Z. H.; Wang M. K.; Lian L.; Dou J.; Wang P. Y.; Zhang H. M. Serial nuclear transfer improves the development of interspecies reconstructed giant panda (Aluropoda melanoleuca) embryos. Chin Sci Bull 47: 467–469; 2002.CrossRefGoogle Scholar
  16. Li Y.; Dai Y.; Du W.; Zhao C.; Wang H.; Wang L.; Li R.; Liu Y.; Wan R.; Li N. Cloned endangered species takin (Budorcas taxicolor) by interspecies nuclear transfer and comparison of the blastocyst development with yak (Bos grunniens) and bovine. Mol Reprod Dev 73: 189–195; 2006.CrossRefPubMedGoogle Scholar
  17. Liu G.; Kato Y.; Tsunoda Y. Aging of recipient oocytes reduces the development of cloned embryos receiving cumulus cells. J Reprod Dev 53: 785–790; 2007.CrossRefPubMedGoogle Scholar
  18. Liu L.; Keefe D. L. Aging-associated aberration in meiosis of oocytes from senescence-accelerated mice. Hum Reprod 17: 2678–2685; 2002.CrossRefPubMedGoogle Scholar
  19. Loi P.; Ptak G.; Barboni B.; Fulka J. J.; Cappai P.; Clinton M. Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells. Nat Biotechnol 19: 962–964; 2001.CrossRefPubMedGoogle Scholar
  20. Lorthongpanich C.; Laowtammathron C.; Chan A. W.; Ketudat-Cairns M.; Parnpai R. Development of interspecies cloned monkey embryos reconstructed with bovine enucleated oocytes. J Reprod Dev 54: 306–313; 2008.CrossRefPubMedGoogle Scholar
  21. Ma L. B.; Yang L.; Hua S.; Cao J. W.; Li J. X.; Zhang Y. Development in vitro and mitochondrial fate of interspecies cloned embryos. Reprod Domest Anim 43: 279–285; 2008a.CrossRefPubMedGoogle Scholar
  22. Ma L. B.; Yang L.; Zhang Y.; Cao J. W.; Hua S.; Li J. X. Quantitative analysis of mitochondrial RNA in goat–sheep cloned embryos. Mol Reprod Dev 75: 33–39; 2008b.CrossRefPubMedGoogle Scholar
  23. McLean C. A.; Wang Z.; Babu K.; Edwards A.; Kasinathan P.; Robl J.; Sheppard A. M. Normal development following chromatin transfer correlates with donor cell initial epigenetic state. Anim Reprod Sci 118: 388–393; 2010.CrossRefPubMedGoogle Scholar
  24. Miao Y. L.; Kikuchi K.; Sun Q. Y.; Schatten H. Oocyte aging: cellular and molecular changes, developmental potential and reversal possibility. Hum Reprod Update 15: 573–585; 2009.CrossRefPubMedGoogle Scholar
  25. Mitalipov S. M.; Zhou Q.; Byrne J. A.; Ji W. Z.; Norgren R. B.; Wolf D. P. Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling. Hum Reprod 22: 2232–2242; 2007.CrossRefPubMedGoogle Scholar
  26. Miyoshi K.; Rzucidlo S. J.; Pratt S. L.; Stice S. L. Improvements in cloning efficiencies may be possible by increasing uniformity in recipient oocytes and donor cells. Biol Reprod 68: 1079–1086; 2003.CrossRefPubMedGoogle Scholar
  27. Miyoshi K.; Taguchi Y.; Sendai Y.; Hoshi H.; Sato E. Establishment of a porcine cell line from in vitro-produced blastocysts and transfer of the cells into enucleated oocytes. Biol Reprod 62: 1640–1646; 2000.CrossRefPubMedGoogle Scholar
  28. Murakami M.; Otoi T.; Wongsrikeao P.; Agung B.; Sambuu R.; Suzuki T. Development of interspecies cloned embryos in yak and dog. Cloning Stem Cells 7: 77–81; 2005.CrossRefPubMedGoogle Scholar
  29. Niemann H.; Tian X. C.; King W. A.; Lee R. S. Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning. Reproduction 135: 151–163; 2008.CrossRefPubMedGoogle Scholar
  30. 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.CrossRefPubMedGoogle Scholar
  31. Palmieri C.; Loi P.; Ptak G.; Della Salda L. A review of the pathology of abnormal placentae of somatic cell nuclear transfer clone pregnancies in cattle, sheep, and mice. Vet Pathol 45: 865–880; 2008.CrossRefPubMedGoogle Scholar
  32. Peura T. T. Improved in vitro development rates of sheep somatic nuclear transfer embryos by using a reverse-order zona-free cloning method. Cloning Stem Cells 5: 13–24; 2003.CrossRefPubMedGoogle Scholar
  33. Rodriguez-Osorio N.; Wang Z.; Kasinathan P.; Page G. P.; Robl J. M.; Memili E. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos. BMC Genomics 10: 190; 2009.CrossRefPubMedGoogle Scholar
  34. Sha H. Y.; Chen J. Q.; Chen J.; Zhang P. Y.; Wang P.; Chen L. P.; Cheng G. X.; Zhu J. H. Fates of donor and recipient mitochondrial DNA during generation of interspecies SCNT-derived human ES-like cells. Cloning Stem Cells 11: 497–507; 2009.CrossRefPubMedGoogle Scholar
  35. Shen P. C.; Lee S. N.; Liu B. T.; Chu F. H.; Wang C. H.; Wu J. S.; Lin H. H.; Cheng W. T. The effect of activation treatments on the development of reconstructed bovine oocytes. Anim Reprod Sci 106: 1–12; 2008.CrossRefPubMedGoogle Scholar
  36. Stice S. L.; Keefer C. L. Multiple generational bovine embryo cloning. Biol Reprod 48: 715–719; 1993.CrossRefPubMedGoogle Scholar
  37. Sugawara A.; Sugimura S.; Hoshino Y.; Sato E. Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes. Zygote 17: 195–202; 2009.CrossRefPubMedGoogle Scholar
  38. Sullivan E. J.; Kasinathan S.; Kasinathan P.; Robl J. M.; Collas P. Cloned calves from chromatin remodeled in vitro. Biol Reprod 70: 146–153; 2004.CrossRefPubMedGoogle Scholar
  39. Sumer H.; Liu J.; Tat P.; Heffernan C.; Jones K. L.; Verma P. J. Somatic cell nuclear transfer: pros and cons. J Stem Cells 4: 85–93; 2009.PubMedGoogle Scholar
  40. Thuan N. V.; Kishigami S.; Wakayama T. How to improve the success rate of mouse cloning technology. J Reprod Dev 56: 20–30; 2010.CrossRefPubMedGoogle Scholar
  41. Vajta G.; Gjerris M. Science and technology of farm animal cloning: state of the art. Anim Reprod Sci 92: 211–230; 2006.CrossRefPubMedGoogle Scholar
  42. Wakayama T.; Perry A. C. F.; 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.CrossRefPubMedGoogle Scholar
  43. Wells D. N.; Misica P. M.; Forsyth J. T.; Berg M. C.; Lange J. M.; Tervit H. R.; Vivanco W. H. The use of adult somatic cell nuclear transfer to preserve the last surviving cow of the Enderby Island cattle breed. Theriogenology 51: 217; 1999.CrossRefGoogle Scholar
  44. Wells D. N.; Misica P. M.; McMillan W. H.; Tervit H. R. Production of cloned bovine fetuses following nuclear transfer with cells from a fetal fibroblast cell line. Theriogenology 49: 330; 1998.CrossRefGoogle Scholar
  45. Wilcox A. J.; Weinberg C. R.; Baird D. D. Post-ovulatory aging of the human oocyte and embryo failure. Hum Reprod 13: 394–397; 1998.CrossRefPubMedGoogle Scholar
  46. Yang X.; Smith S. L.; Tian X. C.; Lewin H. A.; Renard J. P.; Wakayama T. Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning. Nat Genet 39: 295–302; 2007.CrossRefPubMedGoogle Scholar
  47. Yin X. J.; Cho S. K.; Park M. R.; Im Y. J.; Park J. J.; Bhak J. S.; Kwon D. N.; Jun S. H.; Kim N. H.; Kim J. H. Nuclear remodelling and the developmental potential of nuclear transferred porcine oocytes under delayed-activated conditions. Zygote 11: 167–174; 2003.CrossRefPubMedGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2010

Authors and Affiliations

  • Li-Bing Ma
    • 1
    • 2
    Email author
  • Lu Cai
    • 1
    • 2
  • Jia-Jia Li
    • 1
  • Xiu-Li Chen
    • 1
  • Feng-Yu Ji
    • 1
  1. 1.School of Mathematics, Physics and Biological EngineeringInner Mongolia University of Science & TechnologyBaotouChina
  2. 2.Institute of Bioengineering & TechnologyInner Mongolia University of Science & TechnologyBaotouChina

Personalised recommendations