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.
Similar content being viewed by others
References
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. 1385–1393
Campbell, K. H., McWhir, J., Ritchie W. A., andWilmut, I. (1996): ‘Sheep cloned by nuclear transfer from a cultured cell line’,Nature,380, pp. 64–66
Colman, A., andKind, A. (2000): ‘Therapeutic cloning: concepts and practicalities’,Trends Biotechnol.,18, pp. 192–196
Jones, T. (1995): ‘Electromechanics of particles’ (Cambridge University Press, USA, 1995)
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. 889–894
Lagutina, I., Crotti, G., Colleoni, S., Ponderato, N., Duchi, R., Lazzari, G., andGalli, C. (2003): ‘Nuclear transfer in horses’,Theriogenology,59, p. 269
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. 689–696
Neil, G., andZimmermann, U. (1993): ‘Electrofusion’,Methods Enzymol.,220, pp. 174–196
Neumann E., Sowers, A., andJordan, C. (Eds) (1989): ‘Electroporation and electrofusion in cell biology’, (Plenum Press, New York, 1989)
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. 3–12
Oback, B., andWells, D. N. (2003): ‘Cloning cattle’,Cloning Stem Cells,5, pp. 243–256
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. 103–134
Solter, D. (2000): ‘Mammalian cloning: advances and limitations’,Nat. Rev. Genet.,1, pp. 199–207
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. 47–55
Wells, D., Misica, P., andTervit, H. (1999): ‘Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells’,Biol. Reprod.,60, pp. 996–1005
Wells, D. N. (2003): ‘Cloning in livestock agriculture’,Reproduction, Suppl. 61, pp. 131–150
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Gaynor, P., Wells, D.N. & Oback, B. Couplet alignment and improved electrofusion by dielectrophoresis for a zona-free high-throughput cloned embryo production system. Med. Biol. Eng. Comput. 43, 150–154 (2005). https://doi.org/10.1007/BF02345137
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02345137