Nuclear Donor Choice,Sperm-Mediated Activation AndEmbryo Aggregation: AMultipronged Approach ToSequentially Improve Cattle CloningEfficiency



Cloning by somatic cell nuclear transfer (SCNT) remains very inefficient with only Abstract of the 8th Royan International Twin Congress, Tehran, Iran, 5-7 September 2007 Yakhteh Medical Journal, Vol 9, Sup 1, Summer 2007 31 about 1-5% of cloned blast cysts developing into viable offspring. In order to improve cloning efficiency, I first developed a zonafree NT procedure which doubles the throughput in cloned embryo and offspring production in cattle and mouse, increasing both ease of operation and reproducibility. I then used this method to determine at which step the NT procedure could be improved to increase cloning efficiency. I focused on the choice of nuclear donor cell type and cell cycle stage, the artificial activation method and cloned embryo culture conditions. Firstly, I hypothesized that cloning efficiency is inversely correlated with donor cell differentiation status and could be increased by using undifferentiated somatic stem cells as donors. By cloning the world,s first red deer from multipotent antler stem cells and their differentiated progeny, we showed that this was not the case. This finding was confirmed in cattle where myogenic cells of divergent differentiation status resulted in very similar cattle cloning efficiency, suggesting that cell plasticity and epigenetic reprogramming are biologically unrelated and somatic donor cell type is not critical for cloning success.we further demonstrated that, independent of donor cell type and cll line, cloning efficiency can be more than doubled by simple serum-starvation of donor cells. The next step towards improving cloning efficiency was using sperm rather than artificial means to activate cloned embryos. Implantation and birth of live offspring were significantly improved after sperm- mediated activation. Finally, we aggregated individual cloned embryos during in vitro culture to further increase cloning efficiency. In embryonic clones, aggregation led to a2-3 fold significant increase in survival to term. Somatic clones, however, showed compromised in vivo survival, revealing striking biological differences between embryonic and somatic clones in response to aggregation.