Non-Homologous End Joining Plays a Key Role in Transgene Concatemer Formation in Transgenic Zebrafish Embryos
文献类型：期刊论文
| 作者 | Dai, Jun1,2; Cui, Xiaojuan1,2; Zhu, Zuoyan1; Hu, Wei1 |
| 刊名 | INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES
 |
| 出版日期 | 2010 |
| 卷号 | 6期号:7页码:756-768 |
| 关键词 | Transgene Concatemer DSB (double strand breaks) NHEJ (non-homologous end joining) Zebrafish |
| ISSN号 | 1449-2288 |
| 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 |
| 中文摘要 | 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. |
| 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. |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| 学科主题 | Biochemistry & Molecular Biology; Biology |
| 类目[WOS] | Biochemistry & Molecular Biology ; Biology |
| 研究领域[WOS] | Biochemistry & Molecular Biology ; Life Sciences & Biomedicine - Other Topics |
| 关键词[WOS] | STRAND BREAK REPAIR ; CARP CYPRINUS-CARPIO ; MAMMALIAN-CELLS ; V(D)J RECOMBINATION ; CHROMOSOMAL INTEGRATION ; DNA ; MECHANISM ; PATTERNS ; DMC1 |
| 收录类别 | SCI |
| 资助信息 | state Key Fundamental Research of China [2007CB109206]; National Natural Science Foundation [30930069, 30430540]; '863' High Technology Project of China [2006AA10Z141] |
| 语种 | 英语 |
| WOS记录号 | WOS:000285770600014 |
| 公开日期 | 2011-04-14 |
| 源URL | [http://ir.ihb.ac.cn/handle/342005/15494]  |
| 专题 | 水生生物研究所_鱼类生物学及渔业生物技术研究中心_期刊论文 |
| 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 |
| 推荐引用方式 GB/T 7714 | Dai, Jun,Cui, Xiaojuan,Zhu, Zuoyan,et al. Non-Homologous End Joining Plays a Key Role in Transgene Concatemer Formation in Transgenic Zebrafish Embryos[J]. INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES,2010,6(7):756-768. |
| APA | Dai, Jun,Cui, Xiaojuan,Zhu, Zuoyan,&Hu, Wei.(2010).Non-Homologous End Joining Plays a Key Role in Transgene Concatemer Formation in Transgenic Zebrafish Embryos.INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES,6(7),756-768. |
| MLA | Dai, Jun,et al."Non-Homologous End Joining Plays a Key Role in Transgene Concatemer Formation in Transgenic Zebrafish Embryos".INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES 6.7(2010):756-768. |
入库方式： OAI收割
来源：水生生物研究所
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