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学科主题: Biochemistry & Molecular Biology; Biology
题名: Non-Homologous End Joining Plays a Key Role in Transgene Concatemer Formation in Transgenic Zebrafish Embryos
作者: Dai, Jun1, 2; Cui, Xiaojuan1, 2; Zhu, Zuoyan1; Hu, Wei1
通讯作者: Hu, W, Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China ; huwei@ihb.ac.cn
关键词: Transgene ; Concatemer ; DSB (double strand breaks) ; NHEJ (non-homologous end joining) ; Zebrafish
刊名: INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES
发表日期: 2010
卷: 6, 期:7, 页:756-768
收录类别: SCI
文章类型: Article
部门归属: [Dai, Jun; Cui, Xiaojuan; Zhu, Zuoyan; Hu, Wei] Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China; [Dai, Jun; Cui, Xiaojuan] Chinese Acad Sci, Grad Sch, Beijing 100049, Peoples R China
WOS标题词: Science & Technology ; Life Sciences & Biomedicine
资助者: state Key Fundamental Research of China [2007CB109206]; National Natural Science Foundation [30930069, 30430540]; '863' High Technology Project of China [2006AA10Z141]
类目[WOS]: Biochemistry & Molecular Biology ; Biology
研究领域[WOS]: Biochemistry & Molecular Biology ; Life Sciences & Biomedicine - Other Topics
摘要: This study focused on concatemer formation and integration pattern of transgenes in zebrafish embryos. A reporter plasmid based on enhanced green fluorescent protein (eGFP) driven by Cytomegalovirus (CMV) promoter, pCMV-pax6in-eGFP, was constructed to reflect transgene behavior in the host environment. After removal of the insertion fragment by double digestion with various combinations of restriction enzymes, linearized pCMV-pax6in-eGFP vectors were generated with different combinations of 5'-protruding, 3'-protruding, and blunt ends that were microinjected into zebrafish embryos. Repair of double-strand breaks (DSBs) was monitored by GFP expression following religation of the reporter gene. One-hundred-and-ninety-seven DNA fragments were amplified from GFP-positive embryos and sequenced to analyze the repair characteristics of different DSB end combinations. DSBs involving blunt and asymmetric protruding ends were repaired efficiently by direct ligation of blunt ends, ligation after blunting and fill-in, or re moved by cutting. Repair of DSBs with symmetric 3'-3' protrusions was less efficient and utilized template-directed repair. The results suggest that non-homologous end joining (NHEJ) was the principal mechanism of exogenous gene concatemer formation and integration of transgenes into the genome of transgenic zebrafish.
英文摘要: This study focused on concatemer formation and integration pattern of transgenes in zebrafish embryos. A reporter plasmid based on enhanced green fluorescent protein (eGFP) driven by Cytomegalovirus (CMV) promoter, pCMV-pax6in-eGFP, was constructed to reflect transgene behavior in the host environment. After removal of the insertion fragment by double digestion with various combinations of restriction enzymes, linearized pCMV-pax6in-eGFP vectors were generated with different combinations of 5'-protruding, 3'-protruding, and blunt ends that were microinjected into zebrafish embryos. Repair of double-strand breaks (DSBs) was monitored by GFP expression following religation of the reporter gene. One-hundred-and-ninety-seven DNA fragments were amplified from GFP-positive embryos and sequenced to analyze the repair characteristics of different DSB end combinations. DSBs involving blunt and asymmetric protruding ends were repaired efficiently by direct ligation of blunt ends, ligation after blunting and fill-in, or re moved by cutting. Repair of DSBs with symmetric 3'-3' protrusions was less efficient and utilized template-directed repair. The results suggest that non-homologous end joining (NHEJ) was the principal mechanism of exogenous gene concatemer formation and integration of transgenes into the genome of transgenic zebrafish.
关键词[WOS]: STRAND BREAK REPAIR ; CARP CYPRINUS-CARPIO ; MAMMALIAN-CELLS ; V(D)J RECOMBINATION ; CHROMOSOMAL INTEGRATION ; DNA ; MECHANISM ; PATTERNS ; DMC1
语种: 英语
WOS记录号: WOS:000285770600014
ISSN号: 1449-2288
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.ihb.ac.cn/handle/342005/15494
Appears in Collections:鱼类生物学及渔业生物技术研究中心_期刊论文

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作者单位: 1.Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China
2.Chinese Acad Sci, Grad Sch, Beijing 100049, Peoples R China

Recommended Citation:
Dai, Jun; Cui, Xiaojuan; Zhu, Zuoyan; Hu, Wei.Non-Homologous End Joining Plays a Key Role in Transgene Concatemer Formation in Transgenic Zebrafish Embryos,INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES,2010,6(7):756-768
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