I-SceI meganuclease mediates highly efficient transgenesis in fish. - Archive ouverte HAL Access content directly
Journal Articles Mech Dev Year : 2002

I-SceI meganuclease mediates highly efficient transgenesis in fish.

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Abstract

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.

Dates and versions

hal-00122203 , version 1 (28-12-2006)

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Violette Thermes, Clemens Grabher, Filomena Ristoratore, Franck Bourrat, André Choulika, et al.. I-SceI meganuclease mediates highly efficient transgenesis in fish.. Mech Dev, 2002, 118 (1-2), pp.91-8. ⟨10.1016/S0925-4773(02)00218-6⟩. ⟨hal-00122203⟩

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