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；(2)内源性DNA损伤，源于停滞或崩溃的复制叉。哺乳动物细胞中DNA损伤（如DNA双链断裂）是如何修复的，目前尚不清楚。其原因是缺乏适当的技术，以诱导DSB同时在确定的染色体位点。在酵母中，利用HO内切酶在强诱导启动子的调控下表达单染色体DSB是可行的。方法：本研究采用两种技术：(1)从大肠杆菌表达系统中纯化膜易位序列TAT标记的重组核酸内切酶I-SceI。通过将这种蛋白加入培养基，它有望转移细胞膜并最终被运送到细胞核。(2) I-SceI与突变的雌激素受体配体结合域（Mer）融合稳定表达。在正常情况下，该蛋白维持在细胞质中，但在加入雌激素类似物他莫昔芬后，该蛋白有望迅速进入细胞核。我们使用鸡DT40细胞系，将重组底物SCneo敲入OVA位点。SCneo对I-SceI具有18bp的识别序列，并且I-SceI诱导的DSB可以通过同源重组修复，以上游无功能的neo片段为模板，将细胞转化为抗reo细胞。结果：(1)我们从大肠杆菌中纯化了TAT-I-SceI融合蛋白，但由于其沉淀，可能导致其无功能，在我们的手中，我们无法解决这个问题。(2)制备了3种构建体（Mer-I-SceI、Mer-I-EceI-Mer、I-SceI-Mer），分别表达到携带SCneo的DT40中。利用免疫细胞化学技术，我们可以发现Mer-I-EceI-Mer蛋白从细胞质到细胞核的快速易位，但新耐药菌落的出现比预期的要少。在所有的融合构建中，与瞬时转染I-SceI相比，HR修复率（转化为新抗性）较低。一致地，我们能够通过连接介导的PCR检测DSB，但不能通过Southern印迹法检测。结论：我们通过在培养基中添加他莫昔芬，建立了一种诱导染色体DSB的系统，但其效率不够高，无法实时监测DSB的修复情况。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