Captured segment exchange: A strategy for custom engineering large genomic regions in Drosophila melanogaster
Site-specific recombinases (SSRs) are valuable tools for manipulating genomes. In Drosophila, thousands of transgenic insertions carrying SSR recognition sites have been distributed throughout the genome by several large-scale projects. Here we describe a method with the potential to use these insertions to make custom alterations to the Drosophila genome in vivo. Specifically, by employing recombineering techniques and a dual recombinase-mediated cassette exchange 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. © 2013 by the Genetics Society of America.
Bateman, Jack R.; Palopoli, Michael F.; Dale, Sarah T.; Stauffer, Jennifer E.; Shah, Anita L.; Johnson, Justine E.; Walsh, Conor W.; Flaten, Hanna; and Parsons, Christine M., "Captured segment exchange: A strategy for custom engineering large genomic regions in Drosophila melanogaster" (2013). Biology Faculty Publications. 17.