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Originally published as Genetics Published Articles Ahead of Print on December 18, 2006.
Genetics, Vol. 175, 2019-2028, April 2007, Copyright © 2007
doi:10.1534/genetics.106.065920
In Vivo Construction of Transgenes in Drosophila
Hajime Takeuchi1, Oleg Georgiev, Michael Fetchko, Michael Kappeler, Walter Schaffner and Dieter Egli2
Institute of Molecular Biology, University of Zurich, Zurich CH-8057, Switzerland
2 Corresponding author: Department of Molecular and Cellular Biology, Harvard University, 437 Fairchild, 7 Divinity Ave., Cambridge, MA 02138.
E-mail: degli{at}mcb.harvard.edu
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.
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