title: Captured segment exchange: a strategy for custom engineering large genomic regions in Drosophila melanogaster.
abstract: 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.
authors.affiliation: Biology Department, Bowdoin College, Brunswick, ME 04011, USA. jbateman@bowdoin.edu
authors.forename: Jack R
authors.initials: JR
authors.lastname: Bateman
authors.forename: Michael F
authors.initials: MF
authors.lastname: Palopoli
authors.forename: Sarah T
authors.initials: ST
authors.lastname: Dale
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Stauffer
authors.forename: Anita L
authors.initials: AL
authors.lastname: Shah
authors.forename: Justine E
authors.initials: JE
authors.lastname: Johnson
authors.forename: Conor W
authors.initials: CW
authors.lastname: Walsh
authors.forename: Hanna
authors.initials: H
authors.lastname: Flaten
authors.forename: Christine M
authors.initials: CM
authors.lastname: Parsons
mesh_terms: D000818:Animals; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics*; D018390:Gene Targeting; D049750:Genome, Insect; D019426:Integrases / Q000235:genetics; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D012097:Repressor Proteins / Q000235:genetics
chemical_list: D029721:Drosophila Proteins; D012097:Repressor Proteins; C066830:tsh protein, Drosophila; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases; C077570:Site-specific recombinase