An efficient strategy for TALEN-mediated genome engineering in Drosophila

作者：Katsuyama Tomonori; Akmammedov Arslan; Seimiya Makiko; Hess Samuel C; Sievers Cem; Paro Renato^*

来源：Nucleic Acids Research, 2013, 41(17): e163.

DOI：10.1093/nar/gkt638

In reverse genetics, a gene%26apos;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 F-1 generation.

• 出版日期2013-9