Skip to main content Accessibility help

In vitro development of mouse somatic nuclear transfer embryos: effects of donor cell passages and electrofusion

  • Gang Zhang (a1) (a2), Qing-Yuan Sun (a1) and Da-Yuan Chen (a3) (a1)


In this study, C57BL/6 adult male mouse ear fibroblast cells and Kunming mouse M2 oocytes were used as donors and recipients, respectively, to investigate the effect of passage number on donor cells and electrofusion times on the in vitro development of nuclear transfer (NT) embryos. The results demonstrated firstly that when the ear fibroblast cells from either 2–4, 5–7 or 8–10 passages were used as donors, respectively, to produce NT embryos, the number of passages undergone by the donor cells had no significant effect on the in vitro development of NT embryos. The developmental rates for morula/blastocyst were 15.2, 13.3 and 14.0%, respectively, which were not significantly difference (p > 0.05). Secondly, when the NT embryos were electrofused, there was no significant difference between the fusion ratio for the first electrofusion and the second electrofusion (p > 0.05). The developmental rates of the 2-cell and 4-cell stages that had undergone only one electrofusion, however, were significantly higher than those that had had two electrofusions (65.7% compared with 18.4% and 36.4% compared with 6.1%; p < 0.01), furthermore the NT embryos with two electrofusions could not develop beyond the 4-cell stage. This study suggests that this protocol might be an alternative method for mouse somatic cloning, even though electrofusion can exert negative effects on the development of NT embryos.


Corresponding author

All correspondence to: Da-Yuan Chen. State Key Laboratory of Reproductive Biology, Institute of Zoology, The Chinese Academy of Sciences, Beijing, 100101, P. R. China. Tel: +86 010 64807052. Fax: +86 010 64807090. e-mail:


Hide All
Baguisi, A., Behboodi, E., Melican, D.T., Pollock, J.S., Destrempes, M.M., Cammuso, C., Williams, J.L., Nims, S.D., Porter, C.A., Midura, P., Palacios, M.J., Ayres, S.L., Denniston, R.S., Hayes, M.L., Ziomek, C.A., Meade, H.M., Godke, R.A., Gavin, W.G., Overstrom, E.W. & Echelard, Y. (1999). Production of goats by somatic cell nuclear transfer. Nat. Biotechnol. 17, 456–61.
Chesne, P., Adenot, P.G., Viglietta, C., Baratte, M., Boulanger, L. & Renard, J.P. (2002). Cloned rabbits produced by nuclear transfer from adult somatic cells. Nat. Biotechnol. 20, 366–9.
Eggan, K., Baldwin, K., Tackett, M., Osborne, J., Gogos, J., Chess, A., Axel, R. & Jaenisch, R. (2004). Mice cloned from olfactory sensory neurons. Nature 428, 44–9.
Galli, C., Lagutina, I., Crotti, G., Colleoni, S., Turini, P., Ponderato, N., Duchi, R. & Lazzari, G. (2003). Pregnancy: a cloned horse born to its dam twin. Nature 424, 635.
Hochedlinger, K. & Jaenisch, R. (2002). Monoclonal mice generated by nuclear transfer from mature B and T donor cells. Nature 415, 1035–8.
Kato, Y., Tani, T., Sotomaru, Y., Kurokawa, K., Kato, J., Doguchi, H., Yasue, H. & Tsunoda, Y. (1998). Eight calves cloned from somatic cells of a single adult. Science 282, 2095–8.
Kim, M.K., Jang, G., Oh, H.J., Yuda, F., Kim, H.J., Hwang, W.S., Hossein, M.S., Kim, J.J., Shin, N.S., Kang, S.K. & Lee, B.C. (2007). Endangered wolves cloned from adult somatic cells. Cloning Stem Cells 9, 130–7.
Kong, F.Y., Zhang, G., Zhong, Z.S., Li, Y.L., Sun, Q.Y. & Chen, D.Y. (2005). Transplantation of male pronucleus derived from in vitro fertilization of enucleated oocyte into parthenogenetically activated oocyte results in live offspring in mouse. Zygote 13, 35–8.
Kubota, C., Yamakuchi, H., Todoroki, J., Mizoshita, K., Tabara, N., Barber, M. & Yang, X. (2000). Six cloned calves produced from adult fibroblast cells after long-term culture. Proc. Natl. Acad. Sci. USA 97, 990–5.
Lee, B.C., Kim, M.K., Jang, G., Oh, H.J., Yuda, F., Kim, H.J., Hossein, M.S., Shamim, M.H., Kim, J.J., Kang, S.K., Schatten, G. & Hwang, W.S. (2005). Dogs cloned from adult somatic cells. Nature 436, 641.
Li, J., Ishii, T., Feinstein, P. & Mombaerts, P. (2004). Odorant receptor gene choice is reset by nuclear transfer from mouse olfactory sensory neurons. Nature 428, 393–9.
Ogura, A., Inoue, K., Ogonuki, N., Noguchi, A., Takano, K., Nagano, R., Suzuki, O., Lee, J., Ishino, F. & Matsuda, J. (2000a). Production of male cloned mice from fresh, cultured and cryopreserved immature Sertoli cells. Biol. Reprod. 62, 1579–84.
Ogura, A., Inoue, K., Takano, K., Wakayama, T. & Yanagimachi, R. (2000b). Birth of mice after nuclear transfer by electrofusion using tail tip cells. Mol. Reprod. Dev. 57, 55–9.
Ono, Y., Shimozawa, N., Ito, M. & Kono, T. (2001). Cloned mice from fetal fibroblast cells arrested at metaphase by serial nuclear transfer. Biol. Reprod. 64, 4450.
Parker, H.G., Kruglyak, L. & Ostrander, E.A. (2006). Molecular genetics: DNA analysis of a putative dog clone. Nature 440, E12.
Polejaeva, I.A., Chen, S.H., Vaught, T.D., Page, R.L., Mullins, J., Ball, S., Dai, Y., Boone, J., Walker, S., Ayares, D.L., Colman, A. & Campbell, K.H. (2000). Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407, 8690.
Shin, T., Kraemer, D., Pryor, J., Liu, L., Rugila, J., Howe, L., Buck, S., Murphy, K., Lyons, L. & Westhenin, M. (2002). A cat cloned by nuclear transplantation. Nature 415, 859.
Tsunoda, Y., Yasui, T., Nakamura, K., Uchida, T. & Sugie, T. (1986). Effect of cutting the zona pellucida on the pronuclear transplantation in the mouse. J. Exp. Zool. 240, 119–25.
Wakayama, T., Perry, A.C.F., Zuccotti, M., Johnson, K.R. & Yanagimachi, R. (1998). Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–74.
Wakayama, T. & Yanagimachi, R. (1999b). Cloning of male mice from adult tail-tip cells. Nat. Genet. 22, 127–8.
Wakayama, T., Rodriguez, I., Perry, A.C., Yanagimachi, R. & Mombaerts, P. (1999a). Mice cloned from embryonic stem cells. Proc. Natl. Acad. Sci. USA 96, 14984–9.
Wang, M.K., Chen, D.Y., Liu, J.L., Li, G.P. & Sun, Q.Y. (2001a). In vitro fertilization of mouse oocytes reconstructed by transfer of metaphase 2 chromosomes results in live births. Zygote 9, 914.
Wang, M.K., Liu, J.L., Li, G.P., Lian, L. & Chen, D.Y. (2001b). Sucrose pretreatment for enucleation: an efficient and non-damage method for removing the spindle of the mouse M2 oocytes. Mol. Reprod. Dev. 58, 432–6.
Wilmut, I., Schnieke, A.E., Mcwhir, J., Kind, A.J. & Campbell, K.H. (1997). Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–3.
Woods, G.L., White, K.L., Vanderwall, D.K., Li, G.P., Aston, K.I., Bunch, T.D., Meerdo, L.N. & Pate, B.J. (2003). A mule cloned from fetal cells by nuclear transfer. Science 301, 1063.
Zhang, G., Sun, Q.Y. & Chen, D.Y. (2007). Effects of sucrose treatment on the development of mouse nuclear transfer embryos with morula blastomeres as donors. Zygote (in press).
Zhou, Q., Renard, J.P., Le, F.G., Brochard, V., Beaujean, N., Cherifi, Y., Fraichard, A. & Cozzi, J. (2003). Generation of fertile cloned rats by regulating oocytes activation. Science 302, 1179.


In vitro development of mouse somatic nuclear transfer embryos: effects of donor cell passages and electrofusion

  • Gang Zhang (a1) (a2), Qing-Yuan Sun (a1) and Da-Yuan Chen (a3) (a1)


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed