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

I-SceI核酸内切酶同时入核诱导染色体双链断裂修复过程的物理监测

基本信息

批准号：
15590259
负责人：
TAKATA Minoru
金额：
$ 2.3万
依托单位：
Kawasaki Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

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

在这项研究中，我们试图开发一种方法，将单个双链DNA断裂（DSB）引入定义的染色体基因座。如果使它成为可能，那么我们将能够通过基因组南部印迹，修复和/或检查点因素，维修过程本身的进展等等，不断地监测目的的身体状态。在酵母细胞中，此类技术可用，并为我们对DSB修复的基本过程的理解做出了巨大贡献。为此，我们计划使用罕见的限制性核酸内切酶I-SCEI和具有18个基本对碱基识别序列的细胞。 DSB由I-SCEI在集成的重组底物SCNEO中引入，并且可以通过将G418敏感细胞转换为抗性细胞来确定修复效率。为了以高效率和同步的方式引入I-SCEI，将I-SCEI编码序列与雌激素受体的两个配体结合结构域融合在一起……更多或源自HIV TAT序列和6xHis序列的蛋白质转移结构域的纯化标签。在前一种情况下，融合蛋白I-SCEIER在细胞系中稳定地表达。后一种情况是将质粒引入大肠杆菌，并在生化上纯化了融合蛋白Tat-i-SCEI。（1）我们在鸡B细胞系DT40中成功表达了I-SCEI-ER，并检查了用雌激素模拟tamoxifen治疗前后蛋白质的定位。 I-SCEI-ER蛋白迅速（在15分钟内）以高效率从细胞质动员到核动员。但是，发生的DSB修复事件相对较少，这与ER可能干扰内切清除酶活性的可能性一致。但是，该细胞罚款对于某些研究将很有用。现在，我们正在尝试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，但该蛋白似乎是由赞助的。我们正在尝试找到最佳的纯净和存储蛋白质的条件。较少的