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Nucleic Acids Res. 2013 Sep;41(17):e163. doi: 10.1093/nar/gkt638. Epub 2013 Jul 22.

An efficient strategy for TALEN-mediated genome engineering in Drosophila.

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1
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.

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.

PMID:
23877243
PMCID:
PMC3783190
DOI:
10.1093/nar/gkt638
[Indexed for MEDLINE]
Free PMC Article
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