Screening of factors affecting gene targeting efficiency.

建立了具有I-SceI识别位点和诱导型I-SceI表达系统的拟南芥测试系，用于监测DNA双链断裂（DSB）位点处的蛋白质附着和监测基因打靶事件。通过使用该实验材料，揭示了在基因打靶载体的转化过程中，通过DSB诱导，基因打靶效率提高了5-10倍。VirD 2是根癌农杆菌（Agrobacterium tumefaciens）中参与T-DNA加工和转移的关键蛋白之一，对T-DNA整合到植物基因组中具有重要作用。然而，没有直接证据表明VirD 2在T-DNA整合步骤中的作用。为了检测体内VirD 2在DSB位点的附着，我建立了染色质免疫沉淀法，使用该拟南芥测试系的原生质体转染VirD 2表达盒和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.