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Increased blastocyst formation of cloned porcine embryos produced with donor cells pre-treated with Xenopus egg extract and/or digitonin

Published online by Cambridge University Press:  08 February 2011

Ying Liu ,
Olga Østrup ,
Juan Li ,
Gábor Vajta ,
Lin Lin ,
Peter M. Kragh ,
Stig Purup ,
Poul Hyttel  and
Henrik Callesen
Ying Liu*
Affiliation:
Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus University, DK-8830 Tjele, Denmark.
Olga Østrup
Affiliation:
Department of Animal and Veterinary Basic Sciences, Faculty of Life Sciences, University of Copenhagen, Denmark.
Juan Li
Affiliation:
Department of Genetics and Biotechnology, Faculty of Health Science; Aarhus University, Denmark.
Gábor Vajta
Affiliation:
Department of Genetics and Biotechnology, Faculty of Health Science; Aarhus University, Denmark.
Lin Lin
Affiliation:
Department of Genetics and Biotechnology, Faculty of Health Science; Aarhus University, Denmark. Department of Human Genetics, Faculty of Health Science; Aarhus University, Denmark.
Peter M. Kragh
Affiliation:
Department of Genetics and Biotechnology, Faculty of Health Science; Aarhus University, Denmark.
Stig Purup
Affiliation:
Department of Animal Health and Bioscience, Faculty of Agricultural Sciences, Faculty of Health Science; Aarhus University, Denmark.
Poul Hyttel
Affiliation:
Department of Animal and Veterinary Basic Sciences, Faculty of Life Sciences, University of Copenhagen, Denmark.
Henrik Callesen
Affiliation:
Department of Genetics and Biotechnology, Faculty of Health Science; Aarhus University, Denmark.
*
All correspondence to: Ying Liu. Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus University, DK-8830 Tjele, Denmark. Tel: +45 8999 1149. Fax: +45 8999 1300. e-mail: [email protected]
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Summary

Pre-treating donor cells before somatic cell nuclear transfer (SCNT, ‘cloning’) may improve the efficiency of the technology. The aim of this study was to evaluate the early development of cloned embryos produced with porcine fibroblasts pre-treated with a permeabilizing agent and extract from Xenopus laevis eggs. In Experiment 1, fetal fibroblasts were permeabilized by digitonin, incubated in egg extract and, after re-sealing of cell membranes, cultured for 3 or 5 days before use as donor cells in handmade cloning (HMC). Controls were produced by HMC with non-treated donor cells. The blastocyst rate for reconstructed embryos increased significantly when digitonin-permeabilized, extract-treated cells were used after 5 days of culture after re-sealing. In Experiment 2, fetal and adult fibroblasts were treated with digitonin alone before re-sealing the cell membranes, then cultured for 3 or 5 days and used as donor cells in HMC. Treatment with digitonin alone increased the blastocyst rate, but only when fetal, and not adult fibroblasts, were used as donor cells, and only after 3 days of culture. In conclusion, we find a time window for increased efficiency of porcine SCNT using donor cells after pre-treatment with permeabilization/re-sealing and Xenopus egg extract. Interestingly, we observe a similar increase in cloning efficiency by permeabilization/re-sealing of donor cells without extract treatment that seems to depend on choice of donor cell type. Thus, pre-treatment of donor cells using permeabilizing treatment followed by re-sealing and in vitro culture for few days could be a simple way to improve the efficiency of porcine cloning.

Keywords

PermeabilizationPorcinePre-treatmentSomatic cell nuclear transferXenopus egg extract
Type
Research Article
Information
Zygote , Volume 20 , Issue 1 , February 2012 , pp. 61 - 66
Copyright
Copyright © Cambridge University Press 2011

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