Targeted transgene integration in plant cells using designed zinc finger nucleases
Targeted transgene integration in plant cells using designed zinc finger nucleases
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DOI：
10.1007/s11103-008-9449-7
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发表时间：
2009-04-01
影响因子：
5.1
通讯作者：
Petolino, Joseph F.
中科院分区：
文献类型：
--
作者：
Cai, Charles Q.;Doyon, Yannick;Petolino, Joseph F.
在植物中实现靶向转基因整合对于基础研究和应用研究来说仍然是一项重大的技术挑战。本文报道，设计的锌指核酸酶（ZFNs）能够驱动定点DNA整合到转基因和天然基因位点。由设计的4指ZFNs组成的二聚体能够在烟草细胞培养物与供体构建体共转化后，实现失活的绿色荧光蛋白（gfp）报告基因的染色体内重组以及位点特异性转基因整合到染色体报告基因位点，该供体构建体包含补充无功能的抗草铵膦除草剂基因所需的序列。此外，利用一种基于酵母的检测方法鉴定出能够切割天然内切几丁质酶基因的ZFNs。农杆菌递送同时含有ZFNs和供体DNA构建体的Ti质粒，该供体DNA构建体包含一个抗草铵膦除草剂基因盒，其两侧是与内切几丁质酶基因座同源的短片段，可使高达10%的靶向、同源导向的转基因精确整合到ZFN切割位点。鉴于ZFNs可被设计用于识别多种靶序列，这些数据指向一种在植物中进行靶向基因添加、替换和性状叠加的新方法。
Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.