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(如电离辐射引起的双链断裂)和(2)内源性DNA损伤(源于停滞或坍塌的复制叉子)的攻击。目前尚不清楚DNA双链断裂等DNA损伤在哺乳动物细胞中是如何修复的。造成这种情况的原因是缺乏适当的技术来同时在确定的染色体位置诱导DSB。在酵母中，在强诱导启动子的调控下，利用HO内切酶的表达来构建单染色体DSB是可行的。方法：(1)从大肠杆菌表达系统中纯化得到带有膜转位序列TAT的重组内切酶I-SCEI。通过将这种蛋白质添加到培养基中，它有望转移细胞膜，最终被运输到细胞核。(2)I-SCEI与变构体…融合表达雌激素受体(Mer)更多的配体结合结构域。在正常情况下，这种蛋白质保持在细胞质中，但在加入雌激素类似物他莫昔芬后，蛋白质预计会迅速进入细胞核。以鸡DT40细胞系为材料，将重组底物SCneo插入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，但由于TAT-I-SCEI融合蛋白的沉淀，可能正因为如此，我们无法解决这个问题。(2)我们制备了三种载体(Mer-I-SCEI、Mer-I-EceI-Mer、I-SCEI-Mer)，分别表达了含有SCneo的DT40。免疫细胞化学显示Mer-I-EceI-Mer蛋白从胞浆快速移位到胞核，但出现的neo抗性集落少于预期。在所有的融合构建体中，HR修复(转化为neo抗性)的比率与I-SCEI瞬时转染率相比是低的。结论：我们建立了一种通过在培养液中添加他莫昔芬来诱导染色体DSB的系统，但其效率还不够高，不足以实时监测DSB的修复。较少

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