In vivo construction of transgenes in Drosophila.
Transgenic flies are generated by transposon-mediated transformation. A drawback of this approach is the size limit of transposable elements. Here, we propose a novel method that allows the extension of transgenes in vivo. This method is based on an incomplete transgene that has been constructed in vitro and integrated into the Drosophila genome by conventional transgenesis. The incomplete transgene contains two short stretches of DNA homologous to the 5'- and 3'-ends of a larger DNA segment of interest. Between the short stretches of homology an I-SceI recognition site is located. Once activated, I-SceI endonuclease introduces a DNA double-strand break, which triggers ectopic recombination between the stretches of homology and the endogenous locus. Through gap repair, the transgene obtains the complete region of interest in vivo. Our results show that this method is effective for copying up to 28 kb of genomic DNA into the transgene, thereby eliminating the technical difficulties associated with the in vitro construction of large transgenes and extending the size limits of current transgenesis protocols. In general, this method may be a useful technique for genetic engineering of eukaryotic model organisms.
Takeuchi
Hajime
H
Institute of Molecular Biology, University of Zurich, Zurich CH-8057, Switzerland.
Georgiev
Oleg
O
Fetchko
Michael
M
Kappeler
Michael
M
Schaffner
Walter
W
Egli
Dieter
D
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D004331:Drosophila melanogaster / Q000235:genetics*; D005260:Female; D017344:Genes, Insect; D017930:Genes, Reporter; D005821:Genetic Techniques; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D008957:Models, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D011995:Recombination, Genetic
D017931:DNA Primers; D008164:Luminescent Proteins; D011994:Recombinant Proteins