| Simple and efficient transgenesis with meganuclease constructs in zebrafish. | |
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MedLine Citation:
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PMID: 19378101 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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In the past, microinjection of plasmid DNA into early embryos represented the state of the art to generate transgenic zebrafish. However, this approach suffers significant drawbacks (mosaic distribution of the injected transgene, late transgene integration at high copy numbers, low transgenesis frequency), making the generation of transgenic lines a laborious task. Coinjection of I-SceI meganuclease with a reporter construct flanked by I-SceI sites overcomes these problems by earlier transgene integration into the host genome. Here, we provide an optimized protocol for I-SceI meganuclease-mediated transgenesis in zebrafish. This simple protocol provides a reliable method to transiently test tissue-specific reporter expression of meganuclease constructs in injected embryos (F0). Furthermore, it substantially facilitates the generation of multiple stable transgenic lines increasing transgenesis frequencies up to 45%, compared with 5% without I-SceI. The reliable reporter activity in F0 and the improved transgenesis frequency make this protocol a powerful tool for use in gain- and loss-of-function, cell tracing, and cell labeling experiments. |
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Authors:
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Daniele Soroldoni; Benjamin M Hogan; Andrew C Oates |
Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Methods in molecular biology (Clifton, N.J.) Volume: 546 ISSN: 1064-3745 ISO Abbreviation: Methods Mol. Biol. Publication Date: 2009 |
Date Detail:
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Created Date: 2009-04-20 Completed Date: 2009-06-30 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 9214969 Medline TA: Methods Mol Biol Country: United States |
Other Details:
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Languages: eng Pagination: 117-30 Citation Subset: IM |
Affiliation:
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Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Pfotenhauerstrasse 108, 01307, Dresden, Germany. soroldon@mpi-cbg.de |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Animals DNA / genetics, metabolism Deoxyribonucleases, Type II Site-Specific / administration & dosage, genetics*, metabolism Female Gene Transfer Techniques* Genes, Reporter Genetic Engineering / methods Germ-Line Mutation Green Fluorescent Proteins Male Microinjections Saccharomyces cerevisiae Proteins / administration & dosage, genetics*, metabolism Zebrafish / embryology, genetics*, metabolism |
| Chemical | |
Reg. No./Substance:
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0/Saccharomyces cerevisiae Proteins; 147336-22-9/Green Fluorescent Proteins; 9007-49-2/DNA; EC 3.1.21.-/SCEI protein, S cerevisiae; EC 3.1.21.4/Deoxyribonucleases, Type II Site-Specific |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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