Screening of factors affecting gene targeting efficiency.

建立了具有I-SCEI识别位点和可诱导的I-SCEI表达系统的拟南芥测试系，用于监测DNA双链断裂(DSB)处的蛋白附着和监测基因靶向事件。结果表明，在基因打靶载体转化过程中，通过DSB的诱导，基因打靶效率提高了5~10倍，证明T-DNA优先整合在DNA的DSB位点。先前的研究表明，VirD2是参与T-DNA加工和转移的关键农杆菌蛋白之一，对于T-DNA有效整合到植物基因组中是重要的。然而，没有直接证据表明VirD2在T-DNA整合步骤中的作用。为了检测体内VirD2在DSB位点的附着，我建立了利用该拟南芥测试系的原生质体进行染色质免疫沉淀的方法，并将其导入表达盒并进行DSB诱导。

Arabidopsis tester lines possessing an I-SceI recognition site and an inducible I-SceI expression system were established for monitoring protein attachment at DNA double strand break (DSB) site and for monitoring gene targeting events. By using this experimental material, it was revealed that gene targeting efficiency was increased by a factor of 5-10 by DSB induction during transformation of gene targeting vector.It is known that T-DNAs preferentially integrate at DNA DSB sites. Previous studies show that VirD2, which is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer is important for efficient T-DNA integration in to the plant genome. However there is no direct evidence to show the role of VirD2 in T-DNA integration step. For detecting in vivo VirD2 attachment at DSB sites, I established chromatin immunoprecipitation assay using protoplast of this Arabidopsis tester line with transfection of VirD2 expression cassette and DSB induction.