ZFIN ID: ZDB-PUB-121010-28
Development of novel visual-plus quantitative analysis systems for studying DNA double-strand break repairs in zebrafish
Liu, J., Gong, L., Chang, C., Liu, C., Peng, J., and Chen, J.
Date: 2012
Source: Journal of genetics and genomics = Yi chuan xue bao   39(9): 489-502 (Journal)
Registered Authors: Peng, Jinrong
Keywords: DNA DSB repair, NHEJ, HR, SSA, I-Sce I, zebrafish
MeSH Terms:
  • Animals
  • CHO Cells
  • Cell Line
  • Cricetinae
  • DNA Breaks, Double-Stranded*
  • DNA End-Joining Repair
  • DNA Helicases/genetics
  • DNA Helicases/metabolism
  • DNA Ligases/genetics
  • DNA Ligases/metabolism
  • DNA, Single-Stranded/genetics
  • DNA-Binding Proteins/analysis*
  • Green Fluorescent Proteins
  • Homologous Recombination/genetics*
  • Zebrafish/embryology
  • Zebrafish/genetics
PubMed: 23021549 Full text @ J. Genet. Genomics
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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.

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