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Injection of an SV40 transcriptional terminator causes embryonic lethality: a possible zebrafish model for screening nonhomologous end-joining inhibitors

Authors Yang Z, Chen S, Xue S, Li X, Hu J, Sun Z, Cui H

Received 9 October 2017

Accepted for publication 30 May 2018

Published 17 August 2018 Volume 2018:11 Pages 4945—4953

DOI https://doi.org/10.2147/OTT.S153576

Checked for plagiarism Yes

Review by Single-blind

Peer reviewers approved by Dr Lucy Goodman

Peer reviewer comments 3

Editor who approved publication: Professor Jianmin Xu


Zhe Yang,1,2 Shihao Chen,1,2 Songlei Xue,1,2 Xinxiu Li,1,2 Jiang Hu,1,2 Zhen Sun,1,2 Hengmi Cui1–5

1Institute of Epigenetics and Epigenomics, Yangzhou University, Yangzhou, Jiangsu 225009, People’s Republic of China; 2College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, People’s Republic of China; 3Institute of Comparative Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, People’s Republic of China; 4Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, Jiangsu 225009, People’s Republic of China; 5Joint International Research Laboratory of Agricultural & Agri-Product Safety of Educational Ministry of China, Yangzhou University, Yangzhou, Jiangsu 225009, People’s Republic of China

Introduction:
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.
Materials and methods: 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.
Results: 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.
Conclusion: 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.

Keywords: nonhomologous end joining, NHEJ, zebrafish embryo, SV40 terminator, SCR7
 

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