| Techniques available for the generation of transgenic pigs have problems associated with random chromosomal insertion of DNA. While these problems can be overcome by the precise genetic manipulation of the DNA by homologous recombination, due to the extremely low efficiency of homologous recombination, large numbers of cells are required. Unfortunately, germ line competent embryonic stem (ES) cells, a critical element of homologous recombination, are only available in mice at this time. An alternative source of pluripotent stem cells are embryonic germ (EG) cells derived from primordial germ cells (PGCs). Isolation of such cell lines from domestic livestock would allow production of transgenic animals via homologous recombination. However, one of the problems associated with culture of PGCs is the large loss of cells during the initial period of culture.The objective of this study was to establish EG-like cells from porcine, cattle, goats, rabbits and rat PGCs, characterize the initial loss of porcine PGCs, and determine the effectiveness of two classes of apoptosis inhibitors, protease inhibitors and antioxidants, on the ability of porcine PGCs to survive in culture. Using the culture system from porcine EG cell isolation, EG cells from cattle, goats, rabbits and rats were successfully isolated. Results from electron microscopic analysis and in situ DNA fragmentation assay indicated that porcine PGCs rapidly undergo apoptosis when placed in culture. Additionally, alpha2-macroglobulin, a protease inhibitor and cytokine carrier, and N-acetylcysteine, an antioxidant, increased the survival of PGCs in vitro. While other protease inhibitors tested did not affect survival of PGCs, all antioxidants tested improved survival of PGCs. Further, results indicated that the beneficial effect of the antioxidants was critical only during the initial period of culture. Finally, it was determined that in short-term culture, in the absence of feeder layers, antioxidants could partially replace the effect(s) of growth factors and reduce apoptosis. Collectively, these results indicate that the addition of alpha2-macroglobulin and antioxidants can increase the number of PGCs in vitro by suppressing apoptosis. In conclusion, this method could improve the quality and quantity of the porcine PGCs which can be used to generate transgenic pigs via gene targeting and nuclear transplantation. |
