Summary
In the past, microinjection of plasmid DNA into early embryos represented the state of the art to generate transgenic zebrafish. However, this approach suffers significant drawbacks (mosaic distribution of the injected transgene, late transgene integration at high copy numbers, low transgenesis frequency), making the generation of transgenic lines a laborious task. Coinjection of I-SceI meganuclease with a reporter construct flanked by I-SceI sites overcomes these problems by earlier transgene integration into the host genome. Here, we provide an optimized protocol for I-SceI meganuclease-mediated transgenesis in zebrafish. This simple protocol provides a reliable method to transiently test tissue-specific reporter expression of meganuclease constructs in injected embryos (F0). Furthermore, it substantially facilitates the generation of multiple stable transgenic lines increasing transgenesis frequencies up to 45%, compared with 5% without I-SceI. The reliable reporter activity in F0 and the improved transgenesis frequency make this protocol a powerful tool for use in gain- and loss-of-function, cell tracing, and cell labeling experiments.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Houdebine, L. M., and Chourrout, D. (1991) Transgenesis in fish, Experientia 47, 891–897.
Iyengar, A., Muller, F., and Maclean, N. (1996) Regulation and expression of transgenes in fish – a review, Transgenic Res 5, 147–166.
Maclean, N. (1998) Regulation and exploitation of transgenes in fish, Mutat Res 399, 255–266.
Stuart, G. W., McMurray, J. V., and Westerfield, M. (1988) Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos, Development 103, 403–412.
Winkler, C., Vielkind, J. R., and Schartl, M. (1991) Transient expression of foreign DNA during embryonic and larval development of the medaka fish (Oryzias latipes), Mol Gen Genet 226, 129–140.
Culp, P., Nusslein-Volhard, C., and Hopkins, N. (1991) High-frequency germ-line transmission of plasmid DNA sequences injected into fertilized zebrafish eggs, Proc Natl Acad Sci U S A 88, 7953–7957.
Westerfield, M., Wegner, J., Jegalian, B. G., DeRobertis, E. M., and Puschel, A. W. (1992) Specific activation of mammalian Hox promoters in mosaic transgenic zebrafish, Genes Dev 6, 591–598.
Muller, F., Williams, D. W., Kobolak, J., Gauvry, L., Goldspink, G., Orban, L., and Maclean, N. (1997) Activator effect of coinjected enhancers on the muscle-specific expression of promoters in zebrafish embryos, Mol Reprod Dev 47, 404–412.
Muller, F., Chang, B., Albert, S., Fischer, N., Tora, L., and Strahle, U. (1999) Intronic enhancers control expression of zebrafish sonic hedgehog in floor plate and notochord, Development 126, 2103–2116.
Grabher, C., Joly, J. S., and Wittbrodt, J. (2004) Highly efficient zebrafish transgenesis mediated by the meganuclease I-SceI, Methods Cell Biol 77, 381–401.
Gupta, S., Zhu, H., Zon, L. I., and Evans, T. (2006) BMP signaling restricts hemato-vascular development from lateral mesoderm during somitogenesis, Development 133, 2177–2187.
Rembold, M., Lahiri, K., Foulkes, N. S., and Wittbrodt, J. (2006) Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct, Nat Protoc 1, 1133–1139.
Nyholm, M. K., Wu, S. F., Dorsky, R. I., and Grinblat, Y. (2007) The zebrafish zic2a-zic5 gene pair acts downstream of canonical Wnt signaling to control cell proliferation in the developing tectum, Development 134, 735–746.
Kawakami, K. (2007) Tol2: a versatile gene transfer vector in vertebrates, Genome Biol 8 Suppl 1, S7.
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, Mech Dev 118, 91–98.
Kawakami, K. (2004) Transgenesis and gene trap methods in zebrafish by using the Tol2 transposable element, Methods Cell Biol 77, 201–222.
Ogino, H., McConnell, W. B., and Grainger, R. M. (2006) Highly efficient transgenesis in Xenopus tropicalis using I-SceI meganuclease, Mech Dev 123, 103–113.
Pan, F. C., Chen, Y., Loeber, J., Henningfeld, K., and Pieler, T. (2006) I-SceI meganuclease-mediated transgenesis in Xenopus, Dev Dyn 235, 247–252.
Jacquier, A., and Dujon, B. (1985) An intron-encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene, Cell 41, 383–394.
Grabher, C., and Wittbrodt, J. (2007) Meganuclease and transposon mediated transgenesis in medaka, Genome Biol 8 Suppl 1, S10.
Koster, R. W., and Fraser, S. E. (2001) Tracing transgene expression in living zebrafish embryos, Dev Biol 233, 329–346.
Acknowledgments
We would like to thank F. Loosli and J. Wittbrodt for sharing reagents and knowledge concerning the I-SceI meganuclease. This work was supported by the Max Planck Society.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Soroldoni, D., Hogan, B.M., Oates, A.C. (2009). Simple and Efficient Transgenesis with Meganuclease Constructs in Zebrafish. In: Lieschke, G., Oates, A., Kawakami, K. (eds) Zebrafish. Methods in Molecular Biology, vol 546. Humana Press. https://doi.org/10.1007/978-1-60327-977-2_8
Download citation
DOI: https://doi.org/10.1007/978-1-60327-977-2_8
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-976-5
Online ISBN: 978-1-60327-977-2
eBook Packages: Springer Protocols