This observation suggested two different approaches to nuclear transfer. If an oocyte at metaphase II is to be used as the recipient, then normal ploidy, and hence presumably normal development, will only be maintained if the donor cell has a diploid nucleus awaiting DNA replication. By contrast, a recipient cell providing a suitable environment for a nucleus at any stage of the cell cycle could be prepared by activating and culturing the oocyte before nuclear transfer in order to allow MPF activity to decay. Both approaches were used in successful nuclear transfer with blastomeres. Other experiments focused upon donor cell cycle stage. Experiments with rabbit and mouse blastomeres showed an advantage in using donor cells in G1.

There appeared to be a difference between species in the response to nuclear transfer from blastomeres. Normal offspring were obtained from embryos at later stages of development in species such as the sheep and cow in which the embryonic genome is switched on relatively late. In the mouse, pups were obtained only from cleavage stage embryos, whereas in sheep offspring were obtained from blastocysts and cultured cells derived from late blastocysts.

Quiescent donor cells. The situation changed when quiescent (G0) donor cells were used. Live offspring have now been obtained following nuclear transfer from cells taken from adult sheep, cattle and mice. This contrast suggests profound differences in the response to nuclei in G1 and G0. Quiescent nuclei were originally selected because they are a more convenient form of diploid nuclei awaiting DNA replication. As the conventional checkpoints that may be used to block somatic cells in G1 are ineffective in cells from embryos, cells at this stage may only be obtained by arresting cells at mitosis and releasing groups as required to allow progression to G1 phase when required for nuclear transfer. By contrast, G0 is a comparatively stable state and donor cells may be stored for use over prolonged periods. However, it was quickly recognised that there are other differences between cells in G1 and G0. Quiescent cells are typically less active, may have destroyed specific mRNA species and might be expected to have different chromatin structure. It was hypothesised that these differences might facilitate reprogramming of gene expression in the transferred nucleus.

In the initial studies at Roslin, quiescence was induced by starving the cells in culture, but it was not envisaged that starvation as such would be the only means of obtaining suitable donor cells. It was never expected that all quiescent cells would prove to be suitable donors with the present nuclear transfer technique and protocols suitable for normal development from cumulus cells were ineffective with Sertoli cells and neurons. Future research will identify those cell types that are particularly suitable for use in nuclear transfer.

One report has claimed that calves were obtained following nuclear transfer from non-quiescent cells, however, the data presented do not substantiate the claim. The observations made by the authors to characterise the phases of the cell cycle were FACs sorting, (which does not distinguish between G1 and G0), and immunohistochemical analysis of PCNA (using a protocol which fixed the soluble form of PCNA present throughout the cell cycle). The fetal fibroblasts were grown to 70-80% confluence before use and that itself would cause some cells to exit the growth cycle and become quiescent. In these circumstances, the cell cycle stage of the cells is not adequately defined and the authors cannot justify their conclusion.

The recipients oocyte. An advantage has also been shown in adjusting the time of nuclear transfer in relation to oocyte activation. In many studies fusion of the donor and recipient cells was synchronised with oocyte activation. However, it is now recognised that for some cell types it may be beneficial to transfer the nucleus several hours before activation. The benefit is seen most clearly in the studies in which mouse cumulus cells were the donor cells. The mechanisms which account for this effect are not known. It has been suggested that it may allow more reprogramming of gene expression or that it make take time for cell machinery to re-assemble and that the time required may vary with cells at different depths of quiescence.