Simple and efficient transgenesis with meganuclease constructs in zebrafish
| Autor: | Soroldoni, D., Hogan, B. M., Oates, A. C. |
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| Předmět: |
green fluorescent protein
Saccharomyces cerevisiae Proteins Microinjections Green Fluorescent Proteins prenatal development Article male Saccharomyces cerevisiae protein zebra fish Genetics microinjection Animals animal gene transfer Reporter Germ-Line Mutation genetic engineering Deoxyribonucleases type II site specific deoxyribonuclease S cerevisiae Gene Transfer Techniques Methodology DNA Type II Site-Specific zebrafish reporter gene SCEI protein female Genes Mutation metabolism |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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