Choice of Donor Cells. It seems clear that cloning from adult somatic cells is possible for a variety of mammalian species. For most (if not all) biomedical applications of cloning it is probably not important if the cells are of adult origin or even somatic cells for that matter. What is most important is that cells readily cultured and genetically manipulated in vitro can be used to generate viable and reproductively sound animals. Fetal derived cells are a likely choice with these conditions in mind, though embryo derived cells (so called ES cells) might also be likely candidates. The first paper from the Roslin group, which demonstrated cloning from cultured cells, did so with embryo derived cells. The potential value of such cells might be the tendency of such cells to survive in long-term culture. It may not matter if such cells go through some sort of morphological differentiation during this culture if they can be genetically manipulated and latter result in viable offspring.

Cell cycle. A lot has been said about the cell cycle stage of donor cells necessary for viable young to be produced by nuclear transplantation. Unfortunately, much of what has been said has been more for the purpose of establishing intellectual property positions within the patent office than interpretation the data available. I find it difficult to conclude that because a few (one to two in several reports) cells from a population of millions treated in a particular manner result in viable offspring, that those few cells have any particular characteristic. From the technical point of view it probably does not matter. If a particular method yields live offspring then the objective has been reached regardless of the cell cycle stage. On the other hand it could be very difficult if one must prove the cell cycle stage of each cell resulting in live birth in order to avoid patent infringement. I personally would find it unsatisfactory if intellectual property were established on such an undefinable point.

Despite this bit of grumbling, this is an important area of future investigation. If G0 arrest increases the efficiency of nuclear transplantation then various methods of establishing this arrest are worthy of investigation. If such arrest is not beneficial then it is probably best avoided. The more treatments cells are exposed to, the greater the probability of abnormalities. The problem of abnormalities in nuclear transfer experiments can not be overstated. Nuclear transfer experiments have been characterized by low embryo viability, poor pregnancy rates, high abortion rates and a high incidence of unexplained deaths.

Oocyte activation. I do not feel that oocyte activation is or will be a limiting factor in developing cloning technology for the pig. In my experience oocytes have been readily activated by electrical stimulation and by chemical means. I have found the activation method described by Machaty et al (BOR 57:1123, 1997) using a combined thimerosal/DTT treatment to be highly effective.

Embryo transfer. The transfer of nuclear transfer embryos of the pig is likely to be more challenging than for other species. It is my assumption that some period of culture after fusion will be necessary and transfer will occur at the blastocyst stage into the uterus. If only nuclear transfer embryos are transferred it is unlikely that less than 20 embryos would result in a pregnancy. Obtaining 20 or more nuclear transfer embryos at any single point in time will be challenging. Some of the options include, transfer into pregnant females, transfer of "normal" embryos as carriers or transfer of in vitro produced embryos as carriers. Each of these options raise unique questions related to the optimum number of embryos to transfer, the stage of development of the carrier embryos relative to the nuclear transfer embryos and the optimum synchrony of the recipient. Such questions will be crucial to the ultimate goal of producing live offspring from cultured cells and will be expensive and time consuming to address.