title: Increased blastocyst formation of cloned porcine embryos produced with donor cells pre-treated with Xenopus egg extract and/or digitonin.
abstract: 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.
authors.affiliation: Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus University, DK-8830 Tjele, Denmark. Ying.Liu@agrsci.dk
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Olga
authors.initials: O
authors.lastname: Ostrup
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Gábor
authors.initials: G
authors.lastname: Vajta
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.forename: Peter M
authors.initials: PM
authors.lastname: Kragh
authors.forename: Stig
authors.initials: S
authors.lastname: Purup
authors.forename: Poul
authors.initials: P
authors.lastname: Hyttel
authors.forename: Henrik
authors.initials: H
authors.lastname: Callesen
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D002452:Cell Count; D018929:Cell Culture Techniques; D002463:Cell Membrane Permeability; D019976:Cloning, Organism / Q000379:methods; D004072:Digitonin / Q000494:pharmacology*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D047108:Embryonic Development; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D059471:In Vitro Oocyte Maturation Techniques; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D010063:Ovum / Q000166:cytology / Q000378:metabolism; D013552:Swine / Q000196:embryology* / Q000378:metabolism; D013997:Time Factors; D014020:Tissue Extracts / Q000494:pharmacology*; D029867:Xenopus Proteins / Q000494:pharmacology; D014982:Xenopus laevis / Q000378:metabolism*
chemical_list: D014020:Tissue Extracts; D029867:Xenopus Proteins; D004072:Digitonin