Conclusions and Future Directions
It is very likely that transposon technology will have an impact in one or more of the three applications described in this review over the next several years. This progress may involve use of one or more of the transposon systems described above or may involve new transposon systems. The precedent set by work on SB shows that evolutionarily defunct transposons can be "resurrected" using reverse evolutionary principals. This means that many other potentially useful transposons could be derived from a purely informatics based approach using sequenced genomes. A rich source of new transposon systems could be generated in this way, some of which may have attributes more suited to one or another application. Alternately, it is likely that many species contain active elements, such as Tol2 and L1, since we see the evidence of their past activity in all genomes examined. Again, the search for these active elements will be a useful outcome of ongoing genome projects. Mammalian or avian germline transgenesis by transposition could have an impact on several agriculturally relevant species. Transgenesis for most of these species is currently very difficult or impossible. Given the technical challenges of harvesting, manipulating, and injecting early embryos from some of these species, it is worth considering sperm transgenesis by transposition. This process has been achieved in mice via micro-injection [57
], and might be made more efficient using transposon vectors. While the risk of insertional mutagenesis, specifically activation of endogenous proto-oncogenes and cancer, are present with transposons used in gene therapy [58
], it remains to be determined if they are low of enough compared to the benefits that transposon-based gene therapy may bring. However, it is clear from work using SB that the concept is sound from a technical standpoint. Finally, transposon-mediated insertional mutagenesis of the mouse germline is clearly possible with SB, Minos and probably Tol2. Transposon-based germline mutagenesis might also be considered for other species, particularly those for which ES cells cannot be easily obtained. In the mouse, however, the best use of each system probably depends upon the type of screen desired. The maximal number of insertions per gamete that can be reliably obtained must be determined. The ideal sequences for gene-trapping must be identified. Applications such as local saturation mutagenesis or chromosome engineering by placement of LoxP sites within transposon vectors are certainly possible. It is hoped that such approaches can led to significant contributions to functional annotation of the mammalian genome in the future. Acknowledgments
The author thanks Drs. Perry Hackett, Steve Ekker, Scott McIvor, and other members of the University of Minnesota Arnold and Mabel Beckman Center for Transposon Research for helpful suggestions and technical review. This work was partially supported by The Arnold and Mabel Beckman Foundation, the University of Minnesota Academic Health Center and Graduate School, and NIH grant R01 DA14764.