We have now produced cloned transgenic cattle derived from two fetal fibroblast cell lines, one male and one female and have obtained embryonic developmental rate data on several other cell lines. The one obvious conclusion that can be made is that the efficiency of the process varies greatly among cell lines. The highest efficiencies so far were achieved with a male cell line. Approximately one percent of these embryos developed to term (seven calves). On the other extreme are fetal fibroblast cell lines that have a low rate of development to blastocyst stage (<10%) and these embryos do not develop to sixty days of gestation or have a very low capacity of developing to 60 days of gestation. The reasons for this variation among genotypes is unknown. However, these results are similar to previous cattle studies that utilized donor nuclei from embryonic cell lines (Stice et al., Biol. Reprod., 54, 100, 1996) and embryo recloning studies (Stice & Keefer, Biol. Reprod., 48, 715, 1994).

When bovine embryonic cell lines were used as donor nuclei, developmental rates varied greatly among cell lines, and all pregnancies were consistently lost before 60 days of gestation. Similarly, recloning studies indicated that some donor embryos had consistently higher developmental rates in each recloning round than others. Also, no bovine embryonic genome that has gone beyond three generations of recloning develops past 60 days of gestation in cattle. Taken together, the data from several studies using several lines of various degrees of differentiation suggests that donor nuclei characteristics affect developmental rates to the blastocyst stage and later in vivo development. Although cloning technology can increase the efficiency of making transgenic cattle 10 to 100 fold, there are certainly opportunities to increase the efficiency further by determining why some donor nuclei result in clones having high rates of development. Also, efficiency gains can also be obtained by a better understanding of pregnancy losses at 60 days of gestation and term.

We have randomly inserted gene constructs into fibroblast cells and produced animals from these cells. However, gene targeting through homologous recombination will require further procedural refinements. The refinements will lead to products that can not be produced using current transgenic technology. However, gene targeting in fibroblast cells will not be a trivial task. First, fibroblast cells have limited lifespan. In our laboratory this is about 30 to 40 cell doublings for bovine fibroblasts. Also fibroblast cells are less capable of undergoing gene targeting events than embryonic stem cells. These combined limitations will require new and innovative methods of gene targeting in fibroblast cells. One possibility that we have considered is rejuvenating the fibroblast cell lines, thereby effectively increasing the number of cell doublings. Bovine fibroblast cells close to senescence (= 30 cell doublings) were used as nuclear donors. Resulting bovine fetuses were recovered and cloned fetal fibroblast cells were cultured. These cells also had an approximately 30 cell doubling life span. Therefore, the potential number of cell doubling to conduct genetic modifications increased from 30 to 60 through this process. However, as mentioned earlier, embryo blastomeres derived embryos undergoing multiple cloning rounds did lose viability, limiting their potential to develop beyond sixty days of gestation. The affects of multiple rounds of nuclear transfer on pregnancy rates will require careful, well thought out studies.

In conclusion, the cloning process has great promise for potential gains in biomedicine (pharmaceutical protein production and xenotransplantation) and agriculture (multiplication of genetically superior animals and increased production traits). Further development of the cloning process will advance the commercialization of this technology.