Induction of single DNA double strand break in animal cell genome and its monitoring methodology

动物细胞基因组中单DNA双链断裂的诱导及其监测方法

基本信息

批准号：
17590279
负责人：
TAKATA Minoru
金额：
$ 2.24万
依托单位：
HIROSHIMA UNIVERSITY (2006)Kawasaki Medical School (2005)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590279/
关键词：
DNA double strand break DNA repair I-SceI homologous recombination non-homologous end joining estrogen receptor tamoxifen

项目摘要

Background :The human genome is under constant attack by (1) exogenous DNA damage such as double stranded breaks inflicted by ionizing irradiation, and (2) endogenous DNA damage originating in stalled or collapsed replication forks. It is still unknown how DNA lesions such as DNA double strand break are actually repaired in mammalian cells. The reason for this is lack of appropriate technology to induce DSB simultaneously at the defined chromosomal site. In yeast it is feasible to create a single chromosomal DSB by using expression of HO endonuclease under the regulation of strong inducible promoter.Methods :In this study, we wished to develop such system using two technologies:(1) Recombinant endonuclease I-SceI tagged with membrane translocation sequence TAT, which is purifed from E.coli expression system. By adding this protein to culture medium, it is expected to translocate cell membrane and eventually be transported to the nucleus. (2) Stable expression of I-SceI fused with mutat … More ed ligand-biding domain of estrogen receptor (Mer). In normal condition, this protein is maintained in cytoplasm, but upon addition of estrogen analog tamoxifen, the protein is expected to move rapidly into the nucleus. We used chicken DT40 cell line in which recombination substrate SCneo was knocked-in to OVA locus. The SCneo has 18-bp recognition sequence for I-SceI, and I-SceI induced DSB can be repaired through homologous recombination using upstream nonfunctional neo segment as a template, conversing the cell to reo-resistant.Results :(1) We purified TAT-I-SceI fusion protein from E.coli, however, it precipitated and probably because of this, it was nonfunctional, In our hands, we were not able to solve this problem.(2) We prepared three kinds of constructs (Mer-I-SceI, Mer-I-EceI-Mer, I-SceI-Mer), and expressed each of them into DT40 harboring SCneo. We could show rapid translocation of the Mer-I-EceI-Mer protein from cytoplasm to nucleus using immunocytochemistry, however, appearance of neo-resistant colony was less than expected. In all of the fusion constructs the rate of HR repair (conversion to neo resistance) was low compared to transient transfection of I-SceI. Consistently, we were able to detect DSB by ligation-mediated PCR but not by Southern blotting.Conclusion :We created a system that can induce chromosomal DSB by adding tamoxifen to culture media, however, its efficiency is not high enough to allow real time monitoring of DSB repair. Less

背景：（1）外源性DNA损伤（例如因电离照射而造成的双链断裂）以及（2）内源性DNA损伤造成的外源性DNA损伤的持续攻击，而内源性DNA损伤源自停滞或折叠的复制叉。在哺乳动物细胞中实际上如何修复了诸如DNA双链断裂之类的DNA病变仍然未知。原因是缺乏适当的技术来仅在定义的染色体部位诱导DSB。在酵母中，通过使用强诱导启动子的调节下使用HO核酸内切酶的表达来创建单个染色体DSB。通过将此蛋白质添加到培养基中，预计将转移（2）I-SCEI与Mutat融合的稳定表达……更多的ED雌激素受体抗球形结构域（MER）。在正常情况下，该蛋白质保持在细胞质中，但是加入雌激素模拟他莫昔芬后，该蛋白有望快速移动到细胞核中。我们使用了鸡DT40细胞系，其中重组底物scneo被撞向OVA基因座。 SCNEO对I-SCEI具有18 bp的识别顺序，并且I-SCEI诱导的DSB可以通过同源重组来维修，使用上游非功能性的NEO片段作为模板，将细胞与reo-neo-necersants进行交谈：（1）我们纯化的Tat-i-i-scei融合蛋白从E.coli中进行了启动，它是不合时宜的，我们的求解是不合时宜的。问题。（2）我们准备了三种构造（Mer-i-Scei，Mer-i-Ecei-Mer，I-Scei-Mer），并将它们表达为藏有Scneo的DT40。我们可以使用免疫细胞化学的Mer-i-Ecei-Mer蛋白从细胞质到核的快速易位，但是，新耐药菌落的出现少于预期。与I-SCEI的瞬时转染相比，在所有融合构建中，HR修复速率（转化为NEO耐药性）较低。一致地，我们能够通过连接介导的PCR来检测DSB，但不能通过Southern blotting。结论：我们创建了一个可以通过向培养基中添加他莫昔芬来诱导染色体DSB的系统，但是，其效率不足以实时监测DSB维修。较少的