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Transgenesis in Zebrafish with the Tol2 Transposon System

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Transgenesis Techniques

Part of the book series: Methods in Molecular Biology ((MIMB,volume 561))

Summary

The zebrafish (Danio rerio) is a useful model for genetic studies of vertebrate development. Its embryos are transparent and develop rapidly outside the mother, making it feasible to visualize and manipulate specific cell types in the living animal. Zebrafish is well suited for transgenic manipulation since it is relatively easy to collect large numbers of embryos from adult fish. Several approaches have been developed for introducing transgenes into the zebrafish germline, from the injection of naked DNA to transposon-mediated integration. In particular, the Tol2 transposable element has been shown to create insertions in the zebrafish genome very efficiently. By using Tol2, gene trap and enhancer trap vectors containing the GFP reporter gene or yeast transcription activator Gal4 gene have been developed. Here we outline methodology for creating transgenic zebrafish using Tol2 vectors, and their applications to visualization and manipulation of specific tissues or cells in vivo and for functional studies of vertebrate neural circuits.

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References

  1. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., and Schilling, T. F. (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203, 253–310

    Article  PubMed  CAS  Google Scholar 

  2. Haffter, P., Granato, M., Brand, M., Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J., van Eeden, F. J., Jiang, Y. J., Heisenberg, C. P., Kelsh, R. N., Furutani-Seiki, M., Vogelsang, E., Beuchle, D., Schach, U., Fabian, C., and Nüsslein-Volhard, C. (1996) The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123, 1–36

    PubMed  CAS  Google Scholar 

  3. Driever, W., Solnica-Krezel, L., Schier, A. F., Neuhauss, S. C., Malicki, J., Stemple, D. L., Stainier, D. Y., Zwartkruis, F., Abdelilah, S., Rangini, Z., Belak, J., and Boggs, C. (1996) A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123, 37–46

    PubMed  CAS  Google Scholar 

  4. Amsterdam, A., Nissen, R. M., Sun, Z., Swindell, E. C., Farrington, S., and Hopkins, N. (2004) Identification of 315 genes essential for early zebrafish development. Proc Natl Acad Sci U S A 101, 12792–12797

    Article  PubMed  CAS  Google Scholar 

  5. Nasevicius, A. and Ekker, S. C. (2000) Effective targeted gene ‘knockdown’ in zebrafish. Nat Genet 26, 216–220

    Article  PubMed  CAS  Google Scholar 

  6. Xu, Q., Alldus, G., Holder, N., and Wilkinson, D. G. (1995) Expression of truncated Sek-1 receptor tyrosine kinase disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain. Development 121, 4005–4016

    PubMed  CAS  Google Scholar 

  7. Hammerschmidt, M., Bitgood, M. J., and McMahon, A. P. (1996) Protein kinase A is a common negative regulator of Hedgehog signaling in the vertebrate embryo. Genes Dev 10, 647–658

    Article  PubMed  CAS  Google Scholar 

  8. Xu, Q., Alldus, G., Macdonald, R., Wilkinson, D. G., and Holder, N. (1996) Function of the Eph-related kinase rtk1 in patterning of the zebrafish forebrain. Nature 381, 319–322

    Article  PubMed  CAS  Google Scholar 

  9. Wada, H., Iwasaki, M., Sato, T., Masai, I., Nishiwaki, Y., Tanaka, H., Sato, A., Nojima, Y., and Okamoto, H. (2005) Dual roles of zygotic and maternal Scribble1 in neural migration and convergent extension movements in zebrafish embryos. Development 132, 2273–2285

    Article  PubMed  CAS  Google Scholar 

  10. Xiao, T., Roeser, T., Staub, W., and Baier, H. (2005) A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection. Development 132, 2955–2967

    Article  PubMed  CAS  Google Scholar 

  11. 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

    PubMed  CAS  Google Scholar 

  12. Amsterdam, A., Lin, S., and Hopkins, N. (1995) The Aequorea victoria green fluorescent protein can be used as a reporter in live zebrafish embryos. Dev Biol 171, 123–129

    Article  PubMed  CAS  Google Scholar 

  13. Long, Q., Meng, A., Wang, H., Jessen, J. R., Farrell, M. J., and Lin, S. (1997) GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene. Development 124, 4105–4111

    PubMed  CAS  Google Scholar 

  14. Higashijima, S., Okamoto, H., Ueno, N., Hotta, Y., and Eguchi, G. (1997) High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin. Dev Biol 192, 289–299

    Article  PubMed  CAS  Google Scholar 

  15. Lin, S., Gaiano, N., Culp, P., Burns, J. C., Friedmann, T., Yee, J. K., and Hopkins, N. (1994) Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish. Science 265, 666–669

    Article  PubMed  CAS  Google Scholar 

  16. Gaiano, N., Allende, M., Amsterdam, A., Kawakami, K., and Hopkins, N. (1996) Highly efficient germ-line transmission of proviral insertions in zebrafish. Proc Natl Acad Sci U S A 93, 7777–7782

    Article  PubMed  CAS  Google Scholar 

  17. Ellingsen, S., Laplante, M. A., Konig, M., Kikuta, H., Furmanek, T., Hoivik, E. A., and Becker, T. S. (2005) Large-scale enhancer detection in the zebrafish genome. Development 132, 3799–3811

    Article  PubMed  CAS  Google Scholar 

  18. Raz, E., van Luenen, H. G., Schaerringer, B., Plasterk, R. H., and Driever, W. (1998) Transposition of the nematode Caenorhabditis elegans Tc3 element in the zebrafish Danio rerio. Curr Biol 8, 82–88

    Article  PubMed  CAS  Google Scholar 

  19. Fadool, J. M., Hartl, D. L., and Dowling, J. E. (1998) Transposition of the mariner element from Drosophila mauritiana in zebrafish. Proc Natl Acad Sci U S A 95, 5182–5186

    Article  PubMed  CAS  Google Scholar 

  20. Kawakami, K., Koga, A., Hori, H., and Shima, A. (1998) Excision of the Tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio. Gene 225, 17–22

    Article  PubMed  CAS  Google Scholar 

  21. Davidson, A. E., Balciunas, D., Mohn, D., Shaffer, J., Hermanson, S., Sivasubbu, S., Cliff, M. P., Hackett, P. B., and Ekker, S. C. (2003) Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon. Dev Biol 263, 191–202

    Article  PubMed  CAS  Google Scholar 

  22. Kawakami, K. (2005) Transposon tools and methods in zebrafish. Dev Dyn 234, 244–254

    Article  PubMed  CAS  Google Scholar 

  23. Kawakami, K. (2007) Tol2: a versatile gene transfer vector in vertebrates. Genome Biol 8 Suppl 1, S7

    Article  PubMed  Google Scholar 

  24. Urasaki, A., Morvan, G., and Kawakami, K. (2006) Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics 174, 639–649

    Article  PubMed  CAS  Google Scholar 

  25. Balciunas, D., Wangensteen, K. J., Wilber, A., Bell, J., Geurts, A., Sivasubbu, S., Wang, X., Hackett, P. B., Largaespada, D. A., McIvor, R. S., and Ekker, S. C. (2006) Harnessing a high cargo-capacity transposon for genetic applications in vertebrates. PLoS Genet 2, e169

    Article  PubMed  Google Scholar 

  26. Fisher, S., Grice, E. A., Vinton, R. M., Bessling, S. L., Urasaki, A., Kawakami, K., and McCallion, A. S. (2006) Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish. Nat Protoc 1, 1297–1305

    Article  PubMed  CAS  Google Scholar 

  27. Kwan, K. M., Fujimoto, E., Grabher, C., Mangum, B. D., Hardy, M. E., Campbell, D. S., Parant, J. M., Yost, H. J., Kanki, J. P., and Chien, C. B. (2007) The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev Dyn 236, 3088–3099

    Article  PubMed  CAS  Google Scholar 

  28. Villefranc, J. A., Amigo, J., and Lawson, N. D. (2007) Gateway compatible vectors for analysis of gene function in the zebrafish. Dev Dyn 236, 3077–3087

    Article  PubMed  CAS  Google Scholar 

  29. Kawakami, K., Takeda, H., Kawakami, N., Kobayashi, M., Matsuda, N., and Mishina, M. (2004) A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev Cell 7, 133–144

    Article  PubMed  CAS  Google Scholar 

  30. Parinov, S., Kondrichin, I., Korzh, V., and Emelyanov, A. (2004) Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo. Dev Dyn 231, 449–459

    Article  PubMed  CAS  Google Scholar 

  31. Nagayoshi, S., Hayashi, E., Abe, G., Osato, N., Asakawa, K., Urasaki, A., Horikawa, K., Ikeo, K., Takeda, H., and Kawakami, K. (2008) Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes: tcf7 and synembryn-like. Development 135, 159–169

    Article  PubMed  CAS  Google Scholar 

  32. Davison, J. M., Akitake, C. M., Goll, M. G., Rhee, J. M., Gosse, N., Baier, H., Halpern, M. E., Leach, S. D., and Parsons, M. J. (2007) Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish. Dev Biol 304, 811–824

    Article  PubMed  CAS  Google Scholar 

  33. Scott, E. K., Mason, L., Arrenberg, A. B., Ziv, L., Gosse, N. J., Xiao, T., Chi, N. C., Asakawa, K., Kawakami, K., and Baier, H. (2007) Targeting neural circuitry in zebrafish using GAL4 enhancer trapping. Nat Methods 4, 323–326

    PubMed  CAS  Google Scholar 

  34. Asakawa, K., Suster, M. L., Mizusawa, K., Nagayoshi, S., Kotani, T., Urasaki, A., Kishimoto, Y., Hibi, M., and Kawakami, K. (2008) Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish. Proc Natl Acad Sci U S A 105, 1255–1260

    Article  PubMed  CAS  Google Scholar 

  35. Kawakami, K. and Shima, A. (1999) Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene 240, 239–244

    Article  PubMed  CAS  Google Scholar 

  36. Kotani, T. and Kawakami, K. (2008) Misty somites, a maternal effect gene identified by transposon-mediated insertional mutagenesis in zebrafish that is essential for the somite boundary maintenance. Dev Biol 316, 383–396

    Article  PubMed  CAS  Google Scholar 

  37. Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polverino de Laureto, P., DasGupta, B. R., and Montecucco, C. (1992) Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359, 832–835

    Article  PubMed  CAS  Google Scholar 

  38. Kawakami, K. (2004) Transgenesis and gene trap methods in zebrafish by using the Tol2 transposable element. Methods Cell Biol 77, 201–222

    Article  PubMed  CAS  Google Scholar 

  39. Kotani, T., Nagayoshi, S., Urasaki, A., and Kawakami, K. (2006) Transposon-mediated gene trapping in zebrafish. Methods 39, 199–206

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank postdoctoral fellowships from the Japan Society for the Promotion of Science (MLS and KA) and the Takeda Research Foundation (MLS) and from National Institute of Genetics (HK), and the National BioResource Project and grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Authors and Affiliations

  1. Division of Molecular and Developmental Biology, National Institute of Genetics, 1111 Yata, Mishima, 411-8540, Shizuoka, Japan

    Maximiliano L. Suster, Hiroshi Kikuta, Akihiro Urasaki, Kazuhide Asakawa & Koichi Kawakami

  2. Department of Genetics, The Graduate University of Advanced Studies (SOKENDAI), 1111 Yata, Mishima, 411-8540, Shizuoka, Japan

    Koichi Kawakami

Authors
  1. Maximiliano L. Suster
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  2. Hiroshi Kikuta
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  3. Akihiro Urasaki
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  4. Kazuhide Asakawa
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  5. Koichi Kawakami
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Correspondence to Koichi Kawakami .

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    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

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    Suster, M.L., Kikuta, H., Urasaki, A., Asakawa, K., Kawakami, K. (2009). Transgenesis in Zebrafish with the Tol2 Transposon System. In: Cartwright, E. (eds) Transgenesis Techniques. Methods in Molecular Biology, vol 561. Humana Press. https://doi.org/10.1007/978-1-60327-019-9_3

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    • DOI: https://doi.org/10.1007/978-1-60327-019-9_3

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    • Publisher Name: Humana Press

    • Print ISBN: 978-1-60327-018-2

    • Online ISBN: 978-1-60327-019-9

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