Kumar Sandeep, AlAbed Diaa, Worden Andrew, Novak Stephen, Wu Huixia, Ausmus Carla, Beck Margaret, Robinson Heather, Minnicks Tatyana, Hemingway Daren, Lee Ryan, Skaggs Nicole, Wang Lizhen, Marri Pradeep, Gupta Manju
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
J Biotechnol. 2015 Aug 10;207:12-20. doi: 10.1016/j.jbiotec.2015.04.006. Epub 2015 Apr 23.
A modular, selection-based method was developed for site-specific integration of transgenes into a genomic locus to create multigene stacks. High-frequency gene targeting was obtained using zinc finger nuclease (ZFN)-mediated double-strand break (DSB) formation at a pre-defined target genomic location using a unique intron directly downstream of a promoter driving a selectable marker gene to facilitate homology between target and donor sequences. In this system, only insertion into the target locus leads to a functional selectable marker, and regeneration from random insertions of the promoterless donor construct are reduced on selection media. A new stack of transgenes can potentially be loaded with each successive cycle of gene targeting by exchanging the selectable marker gene using the intron homology. This system was tested in maize using the pat selectable marker gene, whereby up to 30% of the plants regenerated on Bialaphos-containing medium were observed to have the donor construct integrated into the target locus. Unlike previous gene targeting methods that utilize defective or partial genes for selecting targeted events, the present method exchanges fully functional genes with every cycle of targeting, thereby allowing the recycling of selectable marker genes, hypothetically for multiple generations of gene targeting.
开发了一种基于模块化选择的方法，用于将转基因位点特异性整合到基因组位点以创建多基因堆叠。通过锌指核酸酶（ZFN）介导在预定义的目标基因组位置形成双链断裂（DSB），利用驱动选择标记基因的启动子直接下游的独特内含子来促进目标序列与供体序列之间的同源性，从而获得高频基因靶向。在该系统中，只有插入目标位点才能产生功能性选择标记，并且在选择培养基上，无启动子供体构建体随机插入后的再生会减少。通过使用内含子同源性交换选择标记基因，每一轮连续的基因靶向都有可能加载新的转基因堆叠。该系统在玉米中使用pat选择标记基因进行了测试，结果观察到在含有双丙氨膦的培养基上再生的植株中，高达30%的植株的供体构建体已整合到目标位点。与以往利用缺陷或部分基因来选择靶向事件的基因靶向方法不同，本方法在每个靶向循环中交换的都是功能完全正常的基因，从而允许选择标记基因循环利用，理论上可用于多代基因靶向。
