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High-throughput transgenesis in Xenopus using I-SceI meganuclease

Nature Protocols volume 1, pages 1703–1710 (2006)Cite this article

Abstract

In this report we describe an easy, highly efficient transgenesis method for Xenopus. The method is very simple; a commercially available meganuclease, I-SceI, is incubated with a transgene construct carrying its recognition sites, and is subsequently microinjected into fertilized eggs. Approximately 30% (in Xenopus tropicalis) or 20% (in Xenopus laevis) of injected embryos exhibit non-mosaic, promoter-dependent transgene expression, and transgenes from the founder animals are transmitted to offspring. The method is compatible with mRNA or antisense morpholino oligonucleotide injection, and these secondary reagents can be introduced simultaneously or sequentially with a transgene to test their interaction. This high-throughput transgenic technique will be a powerful tool for studying the complex wiring of regulatory networks at the genome-wide level, as well as for facilitating genetic studies in the rapidly breeding diploid frog, X. tropicalis.

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Figure 1: Examples of transgenic founder embryos (Xenopus tropicalis and Xenopus laevis) generated by the I-SceI method.

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Acknowledgements

We wish to thank V. Thermes, J. S. Joly, J. Wittbrodt and E. Amaya for kindly providing plasmids. We also thank J. Wittbrodt, T. Hollemann and A. Brandli for helpful discussions about the I-SceI procedure. We also are grateful to members of the Grainger laboratory for continuous positive discussions regarding this study. This research was supported by US National Institutes of Health grants RR13221, EY06675, EY10283 and EY17400.

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  1. Department of Biology, University of Virginia, Charlottesville, 22904, Virginia, USA

    Hajime Ogino, William B McConnell & Robert M Grainger

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  1. Hajime Ogino
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  2. William B McConnell
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  3. Robert M Grainger
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Correspondence to Robert M Grainger.

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Ogino, H., McConnell, W. & Grainger, R. High-throughput transgenesis in Xenopus using I-SceI meganuclease. Nat Protoc 1, 1703–1710 (2006). https://doi.org/10.1038/nprot.2006.208

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