title: An efficient strategy for TALEN-mediated genome engineering in Drosophila.
abstract: In reverse genetics, a gene's function is elucidated through targeted modifications in the coding region or associated DNA cis-regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila. We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F1 generation.
authors.affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zürich, Mattenstrasse 26, 4058 Basel, Switzerland and Faculty of Science, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
authors.forename: Tomonori
authors.initials: T
authors.lastname: Katsuyama
authors.forename: Arslan
authors.initials: A
authors.lastname: Akmammedov
authors.forename: Makiko
authors.initials: M
authors.lastname: Seimiya
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Hess
authors.forename: Cem
authors.initials: C
authors.lastname: Sievers
authors.forename: Renato
authors.initials: R
authors.lastname: Paro
mesh_terms: D000818:Animals; D004268:DNA-Binding Proteins / Q000737:chemistry; D004331:Drosophila melanogaster / Q000235:genetics*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D049750:Genome, Insect; D059765:Homologous Recombination; D016296:Mutagenesis; D010957:Plasmids / Q000008:administration & dosage; D017434:Protein Structure, Tertiary
chemical_list: D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases