Development of Novel Visual-Plus Quantitative Analysis Systems for Studying DNA Double-Strand Break Repairs in Zebrafish

Jingang Liu; Lu Gong; Changqing Chang; Cong Liu; Jinrong Peng; Jun Chen

doi:10.1016/j.jgg.2012.07.009

Volume 39 Issue 9

Sep. 2012

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Article Navigation > Journal of Genetics and Genomics > 2012 > 39(9): 489-502

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Development of Novel Visual-Plus Quantitative Analysis Systems for Studying DNA Double-Strand Break Repairs in Zebrafish

doi: 10.1016/j.jgg.2012.07.009

Jingang Liu^{a, #},
Lu Gong^{a, #},
Changqing Chang^{b, #},
Cong Liu^c,
Jinrong Peng^b,
Jun Chen^a

a
College of Life Sciences, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou 310058, China
b
College of Animal Sciences, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou 310058, China
c
Developmental and Stem Cell Institute, West China Second University Hospital, Sichuan University, Chengdu 610041, China

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Corresponding author: E-mail address: pengjr@zju.edu.cn (Jinrong Peng); E-mail address: chenjun2009@zju.edu.cn (Jun Chen)
Received Date: 2012-05-02
Accepted Date: 2012-07-18
Rev Recd Date: 2012-06-26

Available Online: 2012-08-23

Publish Date: 2012-09-20

Abstract

Abstract

The use of reporter systems to analyze DNA double-strand break (DSB) repairs, based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce I, is usually carried out with cell lines. In this study, we developed three visual-plus quantitative assay systems for homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA) DSB repair pathways at the organismal level in zebrafish embryos. To initiate DNA DSB repair, we used two I-Sce I recognition sites in opposite orientation rather than the usual single site. The NHEJ, HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions, and the repair of DNA lesion caused by I-Sce I could be tracked by EGFP expression in the embryos. Apart from monitoring the intensity of green fluorescence, the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction (qPCR). Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos. Furthermore, while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52, respectively, NHEJ could only be impaired by the knockdown of ligaseIV (lig4) when the NHEJ construct was cut by I-Sce I in vivo. More interestingly, blocking NHEJ with lig4-MO increased the frequency of HR, but decreased the frequency of SSA. Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal, and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.
- DNA DSB repair,
- NHEJ,
- HR,
- SSA,
- Zebrafish

Present address: College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510650, China.

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References(26)

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