Yang Zhe, Chen Shihao, Xue Songlei, Li Xinxiu, Hu Jiang, Sun Zhen, Cui Hengmi
Institute of Epigenetics and Epigenomics, Yangzhou University, Yangzhou, Jiangsu 225009, People's Republic of China,
College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, People's Republic of China,
Onco Targets Ther. 2018 Aug 17;11:4945-4953. doi: 10.2147/OTT.S153576. eCollection 2018.
DNA repair by the nonhomologous end joining (NHEJ) pathway promotes tumor recurrence after chemotherapy and radiotherapy. Discovery of rapid and high-throughput techniques to screen for an effective NHEJ inhibitor drug is imperative for the suppression of NHEJ during tumor treatment. However, traditional screening methods are too cumbersome to meet the current need. Zebrafish is an ideal model for drug screening due to the specificity of its early embryonic development and similarity of tumor cell generation. By exploiting the high frequency of NHEJ in early embryonic development, we established a model that uses a transcriptional terminator signal fragment from the Simian virus 40 (SV40) to cause embryonic lethality. SV40 fragment-induced embryonic lethality was alleviated by 5,6-bis ((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol or C18H14N4OS (SCR7), an NHEJ inhibitor.
A 122 bp SV40 terminator fragment (10 ng/µL) was microinjected into zebrafish zygotes. SV40 fragment integration into the zebrafish embryonic genome was detected by Southern blot using a DNA probe for the SV40 terminator. Embryonic lethality rates were observed 24 and 48 h after microinjection. A nonhomologous recombinant inhibitor, SCR7 (5 µM), was used to alleviate embryonic lethality.
Microinjection of zebrafish embryos with the SV40 terminator fragment (10 ng/µL) caused a progressive increase in mortality over time. Using Southern blots, we confirmed that SV40 terminator sequences were integrated into the zebrafish embryonic genome. This phenomenon was effectively alleviated by addition of SCR7.
Injection of an SV40 terminator into zebrafish embryos may cause embryonic lethality due to NHEJ during early zebrafish development. The high mortality of zebrafish embryos could be alleviated by using the NHEJ inhibitor, SCR7. The zebrafish model presented here is simpler and more convenient than traditional methods of screening for NHEJ inhibitors and can be utilized in large-scale drug screens for NHEJ inhibitors and for the development of novel anticancer drugs.
通过非同源末端连接（NHEJ）途径进行的DNA修复会促进化疗和放疗后肿瘤复发。发现快速且高通量的技术来筛选有效的NHEJ抑制剂药物对于在肿瘤治疗期间抑制NHEJ至关重要。然而，传统的筛选方法过于繁琐，无法满足当前需求。斑马鱼因其早期胚胎发育的特异性和肿瘤细胞生成的相似性，是药物筛选的理想模型。通过利用早期胚胎发育中NHEJ的高频率，我们建立了一个模型，该模型使用来自猿猴病毒40（SV40）的转录终止子信号片段来导致胚胎致死。5,6-双（（E）-亚苄基氨基）-2-巯基嘧啶-4-醇或C18H14N4OS（SCR7），一种NHEJ抑制剂，可减轻SV40片段诱导的胚胎致死率。
将122 bp的SV40终止子片段（10 ng/µL）显微注射到斑马鱼受精卵中。使用针对SV40终止子的DNA探针，通过Southern印迹法检测SV40片段整合到斑马鱼胚胎基因组中的情况。在显微注射后24小时和48小时观察胚胎致死率。使用非同源重组抑制剂SCR7（5 µM）来减轻胚胎致死率。
用SV40终止子片段（10 ng/µL）显微注射斑马鱼胚胎会导致死亡率随时间逐渐增加。通过Southern印迹法，我们证实SV40终止子序列已整合到斑马鱼胚胎基因组中。添加SCR7可有效缓解这一现象。
在斑马鱼胚胎发育早期，向斑马鱼胚胎注射SV40终止子可能由于NHEJ导致胚胎致死。使用NHEJ抑制剂SCR7可减轻斑马鱼胚胎的高死亡率。此处呈现的斑马鱼模型比传统的NHEJ抑制剂筛选方法更简单、方便，可用于大规模筛选NHEJ抑制剂药物以及开发新型抗癌药物。
