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Production of Cloned Calves Using Roscovitine-Treated Adult Somatic Cells as Donors

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Nuclear Transfer Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 348))

Abstract

The stage of the donor cell cycle is a major factor in the success of cloning. Quiescent cells arrested in the G0/G1 phases of the cell cycle by either serum starvation or growth arrest when cultured cells reach confluence have been used as donors to produce cloned animals. Recently, we have developed a novel and effective method using roscovitine to synchronize adult bovine granulosa cells in the G0/G1 cell cycle stage. The resulting fetal and calf survival after transfer of cloned embryos was enhanced in the roscovitine-treated group compared with serum-starved controls. The methods described in this chapter outline (1) the preparation of donor cells, (2) the preparation of recipient oocytes, and (3) the production of cloned embryos. The first section involves methods for the preparation of donor cell stocks from isolated granulosa cells and the roscovitine treatment of the cells before nuclear transfer. The second section explains procedures of in vitro maturation of recipient oocytes. The last section involves methods for the production of cell-oocyte complexes, the fusion of the complexes, and the activation, in vitro culture, and transfer into recipient females of cloned embryos.

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Author information

Authors and Affiliations

  1. Faculty of Agriculture, Kagoshima University, Kagoshima, Japan

    Kazuchika Miyoshi

  2. Animal Biotechnology Research Group, TUBIAK-RIGEB, Turkey

    Sezen Arat

  3. Department of Animal and Dairy Science, University of Georgia, Athens, GA

    Steven L. Stice

Authors
  1. Kazuchika Miyoshi
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  2. Sezen Arat
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  3. Steven L. Stice
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Editor information

Editors and Affiliations

  1. Centre for Reproduction and Development Monash Institute of Medical Research, Monash University, Melbourne, Victoria, Australia

    Paul J. Verma

  2. Monash Immunology and Stem Cell Laboratories, Monash University, Melbourne, Victoria, Australia

    Alan O. Trounson

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© 2006 Humana Press Inc.

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Miyoshi, K., Arat, S., Stice, S.L. (2006). Production of Cloned Calves Using Roscovitine-Treated Adult Somatic Cells as Donors. In: Verma, P.J., Trounson, A.O. (eds) Nuclear Transfer Protocols. Methods in Molecular Biology™, vol 348. Humana Press. https://doi.org/10.1007/978-1-59745-154-3_8

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  • DOI: https://doi.org/10.1007/978-1-59745-154-3_8

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-280-3

  • Online ISBN: 978-1-59745-154-3

  • eBook Packages: Springer Protocols

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