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Although technically established in mammalian species more than 15 year ago, the efficiency of embryo cloning by nuclear transfer in terms of offspring production has been invariably low. This article reviews a series of interrelated experiments carried out from 1992 to 1997 regarding the co-ordination of nuclear and cytoplasmic events after embryo reconstruction. The experiments were undertaken at the Istituto Zootecnico e Caseario per la Sardegna (IZCS) and the sheep used belonged to the Sardinian dairy breed. The use of enucleated, pre-activated recipient cytoplasts resulted in an increased frequency of development to blastocyst of embryos reconstructed with unsynchronized blastomere nuclei. This increase in development was due to the absence of chromatin damage and unbalanced ploidy in nuclei transferred after the decay of MPF (Maturation Promoting Factor). The combination of synchronous S-phase cytoplast-karyoplast nuclear transfer together with the reduction of embryo losses from the oviduct of temporary recipients allowed for the first time the production of large numbers of clones of genetically identical lambs, thus bringing nuclear transfer closer to practical application. Finally, the last series of experiments described deals with the establishment of an alternative protocol for embryonic nuclear transfer where the combined use of a chemical activating agent, ionomycin, with a protein kinase inhibitor, 6-Dimethylaminopurine, resulted in the highest development rates to blastocyst stage of metaphase II enucleated oocytes reconstructed with embryonic nuclei described so far. |
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