Medical and Biological Engineering and Computing

, Volume 43, Issue 1, pp 150–154

Couplet alignment and improved electrofusion by dielectrophoresis for a zona-free high-throughput cloned embryo production system

Article

Abstract

Mammalian cloning by somatic nuclear transfer has great potential for developing medical applications such as biopharmaceuticals and generation of tissues for transplantation. For agricultural applications, it allows the rapid dissemination of genetic gain in livestock breeding. The maximisation of that potential requires improvements to overall cloning technology, especially with respect to increasing cloning efficiency and throughput rates in cloned embryo production. A zona-free embryo reconstruction system was developed to increase cloning throughput and ease of operation. Central to this system is a modified electrofusion procedure for nuclear transfer. Cytoplast-donor cell couplets were placed in a custom-designed ‘parallel plate’ electrode chamber. A 1 MHz sinusoidal AC dielectrophoresis alignment electric field of 6–10 kVm−1 was applied for 5–10 s. The couplets were then fused using 2×10 μs rectangular DC-field pulses (150–200 kVm−1), followed by application of the AC field (6–10 kVm−1) for another 5–10 s. Fusion was performed in hypoosmolar buffer (210 mOsm). Automated alignment of up to 20 couplets at a time has been achieved, resulting in greatly improved fusion throughput rates (2.5-fold increase) and improved fusion yields (1.3-fold increase), compared with commonly followed zona-intact protocols.

Keywords

Cloning Dielectrophoresis Electrofusion Zona-free Nuclear transfer 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Campbell, K. H., Loi, P., Cappai, P., andWilmut, I. (1994): ‘Improved development to blastocyst of ovine nuclear transfer embryos reconstructed during the presumptive S-phase of enucleated activated oocytes’,Biol. Reprod.,50, pp. 1385–1393CrossRefGoogle Scholar
  2. Campbell, K. H., McWhir, J., Ritchie W. A., andWilmut, I. (1996): ‘Sheep cloned by nuclear transfer from a cultured cell line’,Nature,380, pp. 64–66Google Scholar
  3. Colman, A., andKind, A. (2000): ‘Therapeutic cloning: concepts and practicalities’,Trends Biotechnol.,18, pp. 192–196CrossRefGoogle Scholar
  4. Jones, T. (1995): ‘Electromechanics of particles’ (Cambridge University Press, USA, 1995)Google Scholar
  5. Kuroiwa, Y., Kasinathan, P., Choi, Y. J., Naeem, R., Tomizuka, K., Sullivan, E. J., Knott, J. G., Duteau, A., Goldsby, R. A., Osborne, B. A., Ishida, I., andRobl, J. M. (2002): ‘Cloned transchromosomic calves producing human immunoglobulin’,Nat. Biotechnol.,20, pp. 889–894CrossRefGoogle Scholar
  6. Lagutina, I., Crotti, G., Colleoni, S., Ponderato, N., Duchi, R., Lazzari, G., andGalli, C. (2003): ‘Nuclear transfer in horses’,Theriogenology,59, p. 269Google Scholar
  7. Lanza, R. P., Chung, H. Y., Yoo, J. J., Wettstein, P. J., Blackwell, C., Borson, N., Hofmeister, E., Schuch, G., Soker, S., Moraes, C. T., West, M. D., andAtala, A. (2002): ‘Generation of histocompatible tissues using nuclear transplantation’,Nat. Biotechnol.,20, pp. 689–696Google Scholar
  8. Neil, G., andZimmermann, U. (1993): ‘Electrofusion’,Methods Enzymol.,220, pp. 174–196Google Scholar
  9. Neumann E., Sowers, A., andJordan, C. (Eds) (1989): ‘Electroporation and electrofusion in cell biology’, (Plenum Press, New York, 1989)Google Scholar
  10. Oback, B., Wiersma, A., Gaynor, P., Laible, G., Tucker, F., Oliver, J., Miller, A., Troskie, H., Wilson, K., Forsyth, J., Berg, M., Cockrem, K., McMillan, V., Tervit, H., andWells, D. (2003): ‘Cloned cattle derived from a novel zona-freein vitro production system’,Cloning Stem Cells,5, pp. 3–12Google Scholar
  11. Oback, B., andWells, D. N. (2003): ‘Cloning cattle’,Cloning Stem Cells,5, pp. 243–256Google Scholar
  12. Robertson, I., andNelson, R. (1998): ‘Certification and identification of the embryo’, inStringfellow, D. A., andSeidel, S. M. (Eds) ‘Manual of the International Embryo Transfer Society’ (International Embryo Transfer Society, Illinois, 1998), pp. 103–134Google Scholar
  13. Solter, D. (2000): ‘Mammalian cloning: advances and limitations’,Nat. Rev. Genet.,1, pp. 199–207CrossRefGoogle Scholar
  14. Thompson, J. G., McNaughton, C., Gasparrini, B., McGowan, L. T., andTervit, H. R. (2000): ‘Effect of inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos culturedin vitro’,J. Reprod. Fertil.,118, pp. 47–55CrossRefGoogle Scholar
  15. Wells, D., Misica, P., andTervit, H. (1999): ‘Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells’,Biol. Reprod.,60, pp. 996–1005CrossRefGoogle Scholar
  16. Wells, D. N. (2003): ‘Cloning in livestock agriculture’,Reproduction, Suppl. 61, pp. 131–150Google Scholar

Copyright information

© IFMBE 2005

Authors and Affiliations

  1. 1.Department of Physics & Electronic EngineeringUniversity of WaikatoHamiltonNew Zealand
  2. 2.Reproductive Technologies, AgResearch Ltd.Ruakura Research CentreHamiltonNew Zealand

Personalised recommendations