Captured segment exchange: A strategy for custom engineering large genomic regions in Drosophila melanogaster. Jack R. Bateman, Michael F. Palopoli, Sarah T. Dale, Jennifer E. Stauffer, Anita L. Shah, Justine E. Johnson, Conor W. Walsh, Hanna Flaten, Christine M. Parsons. Biology Department, Bowdoin College, Brunswick, ME.

   Thousands of transgenic insertions carrying site-specific recombinase (SSR) recognition sites have been distributed throughout the Drosophila genome by several large-scale projects. Here we describe a method aimed at using these insertions to make custom alterations to Drosophila genomic sequences in vivo. Specifically, by employing recombineering techniques and a dual RMCE strategy based on the phiC31 integrase and FLP recombinase, we show that a large genomic segment that lies between two SSR recognition site insertions can be captured as a target cassette and exchanged for a sequence that was engineered in bacterial cells. We demonstrate this approach by targeting a 50 kb segment spanning the tsh gene, replacing the existing segment with corresponding recombineered sequences through simple and efficient manipulations. Given the high density of SSR recognition site insertions in Drosophila, our method affords a straightforward and highly efficient approach to explore gene function in situ for a substantial portion of the Drosophila genome.