Bateman, J.R., Palopoli, M.F., Dale, S.T., Stauffer, J.E., Shah, A.L., Johnson, J.E., Walsh, C.W., Flaten, H., Parsons, C.M. (2013). Captured Segment Exchange: A Strategy for Custom Engineering Large Genomic Regions in Drosophila melanogaster.  Genetics 193(2): 421--430.

FBrf0220775

Research paper

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.

PMC3567733 (PMC) (EuropePMC)