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Originally published as Genetics Published Articles Ahead of Print on March 17, 2006.

Genetics, Vol. 173, 769-777, June 2006, Copyright © 2006
doi:10.1534/genetics.106.056945

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Site-Specific Transformation of Drosophila via {phi}C31 Integrase-Mediated Cassette Exchange

Jack R. Bateman*,1, Anne M. Lee*,1 and C.-ting Wu*,{dagger},2

* Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115 and {dagger} Division of Genetics and Division of Molecular Medicine, Harvard Medical School, Boston, Massachusetts 02115

2 Corresponding author: Harvard Medical School, Department of Genetics, 77 Avenue Louis Pasteur, New Research Building, Room 264, Boston, MA 02115.
E-mail: twu{at}genetics.med.harvard.edu

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 {phi}C31 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, {phi}C31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs.




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