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

背景：人类基因组不断受到（1）外源性DNA损伤，如电离辐射造成的双链断裂，和（2）内源性DNA损伤，起源于停滞或崩溃的复制叉。目前尚不清楚DNA损伤如DNA双链断裂在哺乳动物细胞中实际上是如何修复的。其原因是缺乏适当的技术来同时在确定的染色体位点诱导DSB。方法：本研究利用两种技术构建了HO内切酶在酵母中的表达系统：（1）从大肠杆菌表达系统中纯化出带有膜转位序列达特的重组内切酶I-SceI;通过将这种蛋白质添加到培养基中，预计它将移位细胞膜并最终被转运到细胞核。(2)I-SceI融合突变蛋白的稳定表达 关于我们 雌激素受体（Mer）的艾德配体结合域。在正常情况下，这种蛋白质维持在细胞质中，但在加入雌激素类似物他莫昔芬后，该蛋白质预计将迅速进入细胞核。我们使用鸡DT 40细胞系，其中重组底物SCneo被敲入到OVA基因座。结果：（1）我们从大肠杆菌中纯化了TAT-I-SceI融合蛋白，但该融合蛋白有沉淀现象，可能是由于沉淀而导致其失去功能，目前我们还没有解决这个问题。(2)我们制备了三种构建体（Mer-I-SceI、Mer-I-EceI-Mer、I-SceI-Mer），并将它们中的每一种表达到含有SCneo的DT 40中。我们可以使用免疫细胞化学技术显示Mer-I-EceI-Mer蛋白从细胞质快速转移到细胞核，然而，新耐药集落的出现比预期的要少。在所有的融合构建体中，HR修复（转化为neo抗性）的速率与瞬时转染I-SceI相比是低的。一致，我们能够检测DSB连接介导的PCR，但不是通过Southern blotting.Conclusion：我们创建了一个系统，可以诱导染色体DSB添加他莫昔芬培养基，但是，它的效率是不够高，让真实的时间监测DSB修复。少

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