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.
Nucleic Acids Res. 2013 Sep;41(17):e163. doi: 10.1093/nar/gkt638. Epub 2013 Jul 22.
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.
在反向遗传学中，可以通过靶向修饰编码区或相关的 DNA 顺式调控元件来阐明基因的功能。为此，最近开发的可定制转录激活样效应物核酸酶（TALENs）已被证明是一种非常有价值的工具，可在基因组的预定位置引入双链断裂。在这里，我们描述了一种在果蝇中进行靶向基因组工程的实用且高效的方法。我们利用可追踪的标记表型证明了 TALEN 介导的靶向基因整合和高效的突变体果蝇鉴定。此外，我们开发了一种简单的 TALEN 组装（easyT）方法，该方法依赖于 DNA Bae I 消化和连接的同时反应，能够在一天内从单体单元文库中构建完整的 TALEN。总之，我们的 easyT 和 TALEN 质粒显微注射策略简化了突变体的产生，并能够在 F1 代中分离到所需的突变果蝇品系。
