摘要
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 l-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.
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 l-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.
基金
supported by the National Natural Science Foundation of China(No.30971677)
the National Basic Research Program of China(973 Program)(No. 2012CB944500)
the Fundamental Research Funds for the Central Universities(No.2011XZZX006)
