I-SceI meganuclease mediates highly efficient transgenesis in fish
- Thermes, V., Grabher, C., Ristoratore, F., Bourrat, F., Choulika, A., Wittbrodt, J., and Joly, J.S.
- Mechanisms of Development 118(1-2): 91-98 (Journal)
- Registered Authors
- Bourrat, Franck, Grabher, Clemens, Joly, Jean-Stephane, Wittbrodt, Jochen
- medaka; zebrafish; fish; transgenesis; embryo; endonuclease; I-SceI; meganuclease; DNA injection
- MeSH Terms
- Animals, Genetically Modified
- Blotting, Southern
- Deoxyribonucleases, Type II Site-Specific/genetics*
- Deoxyribonucleases, Type II Site-Specific/physiology*
- Enhancer Elements, Genetic
- Green Fluorescent Proteins
- Luminescent Proteins/metabolism
- Microscopy, Fluorescence
- Promoter Regions, Genetic
- Saccharomyces cerevisiae Proteins
- Time Factors
- 12351173 Full text @ Mech. Dev.
Thermes, V., Grabher, C., Ristoratore, F., Bourrat, F., Choulika, A., Wittbrodt, J., and Joly, J.S. (2002) I-SceI meganuclease mediates highly efficient transgenesis in fish. Mechanisms of Development. 118(1-2):91-98.
The widespread use of fish as model systems is still limited by the mosaic distribution of cells transiently expressing transgenes leading to a low frequency of transgenic fish. Here we present a strategy that overcomes this problem. Transgenes of interest were flanked by two I-SceI meganuclease recognition sites, and co-injected together with the I-SceI meganuclease enzyme into medaka embryos (Oryzias latipes) at the one-cell stage. First, the promoter dependent expression was strongly enhanced. Already in F0, 76% of the embryos exhibited uniform promoter dependent expression compared to 26% when injections were performed without meganuclease. Second, the transgenesis frequency was raised to 30.5%. Even more striking was the increase in the germline transmission rate. Whereas in standard protocols it does not exceed a few percent, the number of transgenic F1 offspring of an identified founder fish reached the optimum of 50% in most lines resulting from meganuclease co-injection. Southern blot analysis showed that the individual integration loci contain only one or few copies of the transgene in tandem. At a lower rate this method also leads to enhancer trapping effects, novel patterns that are likely due to the integration of the transgene in the vicinity of enhancer elements. Meganuclease co-injection thus provides a simple and highly efficient tool to improve transgenesis by microinjection.
Genes / Markers
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Engineered Foreign Genes
Errata and Notes