使用锌指核酸酶将DNA靶向先前整合的转基因位点。

Targeting DNA to a previously integrated transgenic locus using zinc finger nucleases.

作者信息

Strange Tonya L, Petolino Joseph F

机构信息

Discovery R&D, Dow AgroSciences, Indianapolis, IN, USA.

出版信息

Methods Mol Biol. 2012;847:391-7. doi: 10.1007/978-1-61779-558-9_31.

DOI:10.1007/978-1-61779-558-9_31

PMID:22351024

Abstract

Targeting exogenously supplied DNA to a predetermined location within a plant genome provides a powerful tool for basic studies of plant gene function and opens up some intriguing possibilities for crop improvement. The induction of double-strand DNA breaks at specific genomic loci via the use of designed zinc finger nucleases (ZFNs) allows for targeted transgene integration. Preintegrating a reporter construct containing a nonfunctional herbicide resistance gene flanked by ZFN binding sites results in a locus capable of being targeted. Retransformation with a corresponding ZFN-expressing cassette and a donor DNA with sequences homologous to the integrated construct and capable of functionalizing the herbicide resistance gene following site-specific integration results in targeted DNA addition. Targeted DNA integration can be confirmed in herbicide-resistant plant cells using PCR analysis.

摘要

将外源提供的DNA靶向植物基因组内的预定位置，为植物基因功能的基础研究提供了一个强大的工具，并为作物改良开辟了一些引人入胜的可能性。通过使用设计的锌指核酸酶（ZFN）在特定基因组位点诱导双链DNA断裂，可实现靶向转基因整合。预整合一个报告构建体，该构建体包含一个无功能的除草剂抗性基因，两侧为ZFN结合位点，这样就形成了一个能够被靶向的位点。用相应的ZFN表达盒和供体DNA进行再转化，供体DNA具有与整合构建体同源的序列，并且在位点特异性整合后能够使除草剂抗性基因功能化，从而实现靶向DNA添加。使用PCR分析可以在抗除草剂植物细胞中确认靶向DNA整合。