With regards to pluripotent/totipotent cell lines, in spite of considerable efforts it has not been possible to establish ES cell lines in the bovine and porcine that can be used to introduce genetic modifications into the germ line. Fortunately, in the case of the bovine species, advances in cloning by nuclear transfer indicate that fetal fibroblasts can be manipulated in vitro and still be used to generate a normal offspring. In order to test the use of fibroblasts as targets for homologous recombination we isolated the bovine PrP gene, and obtained sequence information necessary for generation of targeting constructs. Utilizing isogenic DNA, targeting constructs were made and introduced into fetal fibroblasts by electroporation. Transgenic colonies are being analyzed for the presence of the targeted allele. By far the greatest problem affecting the outcome of this experiment is the inability to maintain the fetal fibroblasts in a proliferative state for longer than 25-30 doublings. This, combined with the lower targeting efficiency of somatic cells versus embryonic stem cells, makes identification of targeted clones extremely difficult. It is clear, therefore, that for wide applicability of the technology cell lines that can be used for the cloning procedure but can also be cultured for greater than 30 doublings are required. Alternately, gene targeting system of much greater efficiency than the existing ones need to be developed to ensure than even with a limited cellular life span, targeted events are identified.

Although it is likely that the cloning procedure will soon be applicable to swine, at this time the only available option for precise genetic modification in this species is the use of EG cell lines. Recently, we have been able to isolate EG cells from swine, and utilize them for the generation of transgenic chimeras. While these cell lines may be of use as carrier systems, they are difficult to maintain, have low cloning efficiency, and require the generation of chimeras before a founder can be established; an additional generation that can have significant economic impact in species with relatively long gestation and reproductive maturity periods. However, in contrast to fetal fibroblast, they do not readily undergo senescence and can be maintained for prolonged periods of time, although with some loss of undifferentiated characteristics. Additionally, they may be of use for favoring entrance into the germ line by use of tetraploid embryos for chimera generation, or by using the cells as nuclear donors in a cloning procedure.

While at this time the greatest barriers to the application of this technology are mostly technical, it is likely that with a concerted effort and federal support most if not all of these barriers will soon be overcome. Thus, it is critical that in parallel with efforts to complete development of the technology, efforts to identify single genes that can have significant economic impact in phenotypes of agricultural importance, such as muscle composition and disease resistance, must be supported. Otherwise, we run the risk of ending up with a technique for manipulating genes but no genes to manipulate.