In vivo modification of a maize engineered minichromosome
In vivo modification of a maize engineered minichromosome
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DOI：
10.1007/s00412-013-0403-3
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发表时间：
2013-06-01
期刊：
影响因子：
1.6
通讯作者：
Birchler, James A.
中科院分区：
文献类型：
--
作者：
Gaeta, Robert T.;Masonbrink, Rick E.;Birchler, James A.
工程微染色体提供了在作物中堆叠转基因的有效平台。用于在体内修饰这些染色体的方法对于开发用于去除选择基因或其他序列以及用于添加新基因的可定制系统是必不可少的。先前的研究已经证明，Cre，一种位点特异性重组酶，可以用于修饰玉米微型染色体上的转基因上的lox位点;然而，这些研究证明了体细胞重组，并且修饰的微型染色体不能被回收。我们描述了一个工程染色体组成的恢复比一个着丝粒加转基因是由端粒介导的截断。我们使用的纤维荧光原位杂交技术，并检测到一个转基因的微小染色体插入之间延伸的CentC着丝粒重复序列，这个插入是足够大的，建议串联插入。通过将微型染色体与表达Cre重组酶的植物杂交，Bar选择基因被去除，留下单个loxP位点。这项研究表明，工程染色体可以在体内使用位点特异性重组酶进行修饰，这是在植物中开发可重复染色体平台所必需的证明。
Engineered minichromosomes provide efficient platforms for stacking transgenes in crop plants. Methods for modifying these chromosomes in vivo are essential for the development of customizable systems for the removal of selection genes or other sequences and for the addition of new genes. Previous studies have demonstrated that Cre, a site-specific recombinase, could be used to modify lox sites on transgenes on maize minichromosomes; however, these studies demonstrated somatic recombination only, and modified minichromosomes could not be recovered. We describe the recovery of an engineered chromosome composed of little more than a centromere plus transgene that was derived by telomere-mediated truncation. We used the fiber fluorescence in situ hybridization technique and detected a transgene on the minichromosome inserted among stretches of CentC centromere repeats, and this insertion was large enough to suggest a tandem insertion. By crossing the minichromosome to a plant expressing Cre-recombinase, the Bar selection gene was removed, leaving behind a single loxP site. This study demonstrates that engineered chromosomes can be modified in vivo using site-specific recombinases, a demonstration essential to the development of amendable chromosome platforms in plants.