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. It 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 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.

Key Words: I-SceI, gap repair, homologous recombination, transgenesis

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