Document Detail


I-SceI meganuclease mediates highly efficient transgenesis in fish.
MedLine Citation:
PMID:  12351173     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Violette Thermes; Clemens Grabher; Filomena Ristoratore; Franck Bourrat; André Choulika; Jochen Wittbrodt; Jean-Stéphane Joly
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Mechanisms of development     Volume:  118     ISSN:  0925-4773     ISO Abbreviation:  Mech. Dev.     Publication Date:  2002 Oct 
Date Detail:
Created Date:  2002-09-27     Completed Date:  2003-05-29     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  9101218     Medline TA:  Mech Dev     Country:  Ireland    
Other Details:
Languages:  eng     Pagination:  91-8     Citation Subset:  IM    
Affiliation:
INRA Junior Group, UPR2197, Institut de Neurobiologie A. Fessard, CNRS, Avenue de la Terrasse, 91 198 Gif-Sur-Yvette, France.
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MeSH Terms
Descriptor/Qualifier:
Animals
Animals, Genetically Modified
Blotting, Southern
DNA / metabolism
Deoxyribonucleases, Type II Site-Specific / genetics*,  physiology*
Enhancer Elements, Genetic
Fishes
Green Fluorescent Proteins
Luminescent Proteins / metabolism
Microscopy, Fluorescence
Plasmids / metabolism
Promoter Regions, Genetic
Saccharomyces cerevisiae Proteins
Time Factors
Transgenes
Zebrafish
Chemical
Reg. No./Substance:
0/Luminescent Proteins; 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
Comments/Corrections
Erratum In:
Mech Dev. 2003 Feb;120(2):267

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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