Physical monitoring of repair process of chromosomal double stand break induced by simultaneous nuclear introduction of I-SceI endonuclease

在这项研究中，我们试图开发一种方法，同时将单双链DNA断裂(DSB)引入到定义的染色体座位中。如果这成为可能，那么我们将能够通过基因组Southern杂交、修复和/或检查点因子的招募、修复过程本身的进展等来持续监测末端的物理状态。在酵母细胞中，这种技术是可用的，对我们理解DSB修复的基本过程有很大的帮助。为此，我们计划使用稀有限制性内切酶I-SCEI和在其染色体上含有18个碱基对特定识别序列的细胞。通过I-SCEI在集成重组底物SCneo中引入DSB，修复效率可以通过G418敏感细胞到抗性细胞的转换来确定。为了高效、同步地导入I-SCEI，将I-SCEI编码序列与雌激素受体…的两个配体结合域融合从HIV TAT序列和6XHis序列中衍生出更多或更多的蛋白转导结构域，用于纯化标签。在前一种情况下，融合蛋白I-SCEI-ER在细胞系中稳定表达。(1)在鸡B细胞系DT40中成功表达了I-SCEI-ER，并检测了其在雌激素类似物他莫昔芬处理前后的定位。I-SCEI-ER蛋白可在15min内从胞浆快速动员到胞核。然而，DSB修复事件的发生相对较少，这与ER可能干扰内切酶活性的可能性是一致的。尽管如此，这种细胞罚款对一些研究是有用的。我们现在正试图通过制造新的融合蛋白来提高效率。(2)我们纯化了重组TAT-I-SCEI蛋白，但蛋白似乎可以自发聚集。我们正在努力寻找纯化和储存蛋白质的最佳条件。较少

In this study we tried to develop a method to simultaneously introduce a single double strand DNA break (DSB) into defined chromosomal locus. If this is made possible, then we would be able to continuously monitor physical status of the ends by genomic Southern blotting, recruitment of repair and/or checkpoint factors, progression of the repair process itself and so on. In yeast cells, such technology is available and contributes enormously to our understanding of the basic process of the DSB repair.To this end, we plan to use rare restriction endonuclease I-SceI and cells harbouring the18 base-pair specific recognition sequence in its chromosome. The DSB is introduced in the integrated recombination substrate SCneo by I-SceI, and the repair efficiency can be determined by conversion of the G418-sensitive cells to resistant cells. To introduce I-SceI in high efficiency and in a synchronous manner, the I-SceI coding sequence was fused with two ligand binding domains of estrogen receptor … More or the protein transduction domain derived from HIV TAT sequence and 6XHis sequence for purification tag. In the former case, the fusion protein I-SceI-ER was stably expressed in a cell line. The latter case, the plasmid was introduced into E.coli and the fusion protein TAT-I-SceI was biochemically purifed.(1)We successfully expressed the I-SceI-ER in the chicken B cell line DT40, and examined localization of the protein before and after treatment with estrogen analog tamoxifen. I-SceI-ER protein was rapidly (within 15min) mobilized from cytoplasm to nucleus with high efficiency. However, relatively few DSB repair events occurred, which is consistent with the possibility that ER may interfere with the endonuclease activity. Nonetheless, this cell fine would be useful for some studies. We are now trying to improve the efficiency by making new fusion proteins.(2)We purified the recombinant TAT-I-SceI protein but the protein appeared to aggregate spontaneously. We are trying to find optimal condition to purigy and store the protein. Less