I-SceI meganuclease mediates highly efficient transgenesis in fish

被引:392
作者
Thermes, V
Grabher, C
Ristoratore, F
Bourrat, F
Choulika, A
Wittbrodt, J
Joly, JS
机构
[1] Cellectis SA, F-75724 Paris, France
[2] European Mol Biol Lab, Dev Biol Programme, D-69012 Heidelberg, Germany
[3] INRA, Inst Neurobiol A Fessard, Jr Grp, CNRS,UPR2197, F-91198 Gif Sur Yvette, France
基金
澳大利亚研究理事会;
关键词
medaka; zebrafish; fish; transgenesis; embryo; endonuclease; I-SceI; meganuclease; DNA injection;
D O I
10.1016/S0925-4773(02)00218-6
中图分类号
Q [生物科学];
学科分类号
07 ; 0710 ; 09 ;
摘要
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. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.
引用
收藏
页码:91 / 98
页数:8
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