The novel germ-line
transformation systems disclosed in this
patent application allow the physical deletion of transposon
DNA following the transformation process, and the targeting of
transgene integrations into predefined target sites. In this way,
transposase-mediated mobilization of genes-of-interest is excluded mechanistically and random genomic integrations eliminated. In contrast to conventional germ-line transformation technology, our systems provide enhanced stability to the
transgene insertion. Furthermore,
DNA sequences required for the
transgene modification (e.g. transformation marker genes,
transposase or
recombinase target sites), are largely removed from the
genome after the final transgene
insertion, thereby eliminating the possibility for
instability generated by these processes. The RMCE technology, which is disclosed in this
patent application for
invertebrate organisms (exemplified in
Drosophila melanogaster) represents an extremely versatile tool with application potential far beyond the goal of transgene immobilization. RMCE makes possible the targeted integration of
DNA cassettes into a specific genomic loci that are pre-defined by the integration of the RMCE
acceptor plasmid. The loci can be characterized prior to a targeting experiment allowing optimal integration sites to be pre-selected for specific applications, and allowing selection of host strains with optimal fitness. In addition, multiple cassette exchange reactions can be performed in a repetitive way where an
acceptor cassette can be repetitively exchanged by multiple donor cassettes. In this way several different transgenes can be placed precisely at the same genomic locus, allowing, for the first time, the ability to eliminate genomic positional effects and to comparatively study the biological effects of different transgenes.