Systems for gene targeting and producing stable genomic transgene insertions

Abstract

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.

1 : A method for producing a heritable integration of a transgene within a genome of a somatic or germ line cell of an invertebrate organism, the method comprising:
 providing a first DNA cassette within said genome, wherein said first cassette comprises a first flanking transposon half side, a second flanking transposon half side, and an internal transposon half side, wherein said internal transposon half side and said first flanking transposon half side form a pair of excisable transposon half-sides, and wherein said first cassette further comprises said transgene in-between the internal transposon half side and said second flanking transposon half side; and   mobilizing said excisable transposon half-sides.   
     
     
         2 : The method of  claim 1 , wherein said internal transposon half side and said second flanking transposon half side are TransposonL half sides, and wherein said first flanking transposon half side is a TransposonR half side. 
     
     
         3 : The method of  claim 1 , wherein said internal transposon half side and said second flanking transposon half side are TransposonR half sides, and wherein said first flanking transposon half side is a TransposonL half side. 
     
     
         4 : The method of  claim 1 , wherein said excisable transposon half-sides and corresponding transposase enzyme are from a transposable element, wherein said transposable element has terminal inverted sequences, and wherein said transposable element transposes via a DNA-mediated process. 
     
     
         5 : The method of  claim 1 , wherein said first DNA cassette further comprises a first selectable marker gene located between said internal transposon half side and said first flanking transposon half side, and a second selectable marker gene located between said internal transposon half side and said second flanking transposon half side, and wherein said first and second selectable marker genes are phenotypically distinguishable. 
     
     
         6 : The method of  claim 5 , wherein said first and second marker genes are, in either order, any combination of marker genes producing distinguishable fluorescent or other visible dominant phenotypes. 
     
     
         7 : The method of  claim 5  wherein said first and second marker genes are, in either order, a combination of the transformation marker genes PUbDsRed1 and 3×P3-ECFP. 
     
     
         8 : The method of  claim 1 , wherein said internal transposon half side is provided in reverse orientation, wherein said excisable transposon is formed by inversion of said internal transposon half side relative to said first flanking transposon half side, wherein said internal transposon half side further comprises flanking recombinase sites, and wherein said inversion is catalyzed by a site-specific recombinase. 
     
     
         9 : The method of  claim 8 , wherein said recombinase sites are FRT sites in opposite or reverse orientation. 
     
     
         10 : The method of  claim 1 , wherein said excisable transposon is mobilized by a source of transposase corresponding to said excisable transposon to render the remaining genomic DNA immobilizable. 
     
     
         11 - 22 . (canceled) 
     
     
         23 : An invertebrate organism comprising the heritable transgene produced according to  claim 1 . 
     
     
         24 . (canceled) 
     
     
         25 : A method for producing a heritable integration of a transgene within a genome of a somatic or germ line cell of an organism, the method comprising:
 providing a first DNA cassette within said genome, wherein said first cassette comprises a first flanking transposon half side, a second flanking transposon half side, and an internal transposon half side, wherein said internal transposon half side and said first flanking transposon half side form a pair of excisable transposon half-sides, and wherein said first cassette further comprises said transgene in-between the internal transposon half side and said second flanking transposon half side; and   mobilizing said excisable transposon half-sides.   
     
     
         26 : The method of  claim 25 , wherein said internal transposon half side and said second flanking transposon half side are TransposonL half sides, and wherein said first flanking transposon half side is a TransposonR half side. 
     
     
         27 : The method of  claim 25 , wherein said internal transposon half side and said second flanking transposon half side are TransposonR half sides, and wherein said first flanking transposon half side is a TransposonL half side. 
     
     
         28 : The method of  claim 25 , wherein said excisable transposon half-sides and corresponding transposase enzyme are from a transposable element, wherein said transposable element has terminal inverted sequences, and wherein said transposable element transposes via a DNA-mediated process. 
     
     
         29 : The method of  claim 25 , wherein said first DNA cassette further comprises a first selectable marker gene located between said internal transposon half side and said first flanking transposon half side, and a second selectable marker gene located between said internal transposon half side and said second flanking transposon half side, and wherein said first and second selectable marker genes are phenotypically distinguishable. 
     
     
         30 : The method of  claim 29 , wherein said first and second marker genes are, in either order, any combination of marker genes producing distinguishable fluorescent or other visible dominant phenotypes. 
     
     
         31 : The method of  claim 29 , wherein said first and second marker genes are, in either order, a combination of the transformation marker genes PUbDsRed1 and 3×P3-ECFP. 
     
     
         32 : The method of  claim 25 , wherein said internal transposon half side is provided in reverse orientation, wherein said excisable transposon is formed by inversion of said internal transposon half side relative to said first flanking transposon half side, wherein said internal transposon half side further comprises flanking recombinase sites, and wherein said inversion is catalyzed by a site-specific recombinase. 
     
     
         33 : The method of  claim 32 , wherein said recombinase sites are FRT sites in opposite or reverse orientation. 
     
     
         34 : The method of  claim 25 , wherein said excisable transposon is mobilized by a source of transposase corresponding to said excisable transposon to render the remaining genomic DNA immobilizable. 
     
     
         35 - 46 . (canceled) 
     
     
         47 : An organism comprising the heritable transgene produced according to  claim 25 . 
     
     
         48 . (canceled)