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.