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ABSTRACT
Position effects can complicate transgene analyses. This is especially true when comparing transgenes that have inserted randomly into different genomic positions and are therefore subject to varying position effects. Here, we introduce a method for the precise targeting of transgenic constructs to predetermined genomic sites in Drosophila using the φC31 integrase system in conjunction with recombinase-mediated cassette exchange (RMCE). We demonstrate the feasibility of this system using two donor cassettes, one carrying the yellow gene and the other carrying GFP. At all four genomic sites tested, we observed exchange of donor cassettes with an integrated target cassette carrying the mini-white gene. Furthermore, because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify integrants simply by the loss of mini-white eye color. Importantly, this feature of the technology will permit integration of unmarked constructs into Drosophila, even those lacking functional genes. Thus, φC31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs.
BIOLOGICAL research is greatly facilitated by our ability to manipulate DNA sequences in vivo. In the model organism Drosophila melanogaster, exogenous sequences are routinely incorporated into the genome using the P-element transposon system developed by Rubin and Spradling (RUBIN and SPRADLING 1982; SPRADLING and RUBIN 1982). The harnessing of this technology afforded a new ability to alter the genome, allowing the construction of transgenic animals by the relatively simple method of embryonic injection. Furthermore, P elements have been indispensable as a platform for developing new technologies for Drosophila research including the GAL4/UAS system (BRAND and PERRIMON 1993), targeted deletions (RYDER et al. 2004), and high-resolution mapping of mutations (ZHAI et al. 2003).
Recently, strategies have been developed to repeatedly incorporate transgenes into a single position in the genome. This provides a significant advantage over conventional P-element transformation, which occurs in an untargeted fashion and therefore subjects transgenes to varying position effects. In general these new strategies make use of site-specific recombinases, which catalyze crossovers between defined target sequences (BRANDA and DYMECKI 2004). The most popular of these enzymes are FLP and Cre, which are widely used for many applications in Drosophila, including clonal analysis, targeted deletions, and tissue-specific excision (GOLIC and LINDQUIST 1989; GOLIC 1991; SIEGAL...