Summary
The Tol2 transposon system can create insertions in the zebrafish genome efficiently. By using this system, the gene trap and enhancer trap methods have been developed. The gene trap and enhancer trap constructs contain the green fluorescent protein (GFP) reporter gene or the yeast Gal4 transcription activator gene. By creating random integrations of these constructs in the genome, transgenic fish expressing the GFP gene or the Gal4 gene in specific cells, tissues or organs are generated. These fish are valuable resources for developmental biology. Especially, the Gal4-expressing transgenic fish can be used to ectopically express any gene of interest placed downstream of the Gal4 recognition sequence, UAS, and thereby allow visualization, modification or ablation of the Gal4-expressing cells. In this chapter, we will describe how the gene trap and enhancer trap screens can be performed and how the transposon insertions created by these methods can be analyzed.
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References
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, 133–44.
Kawakami, K. (2005). Transposon tools and methods in zebrafish. Dev Dyn 234, 244–54.
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, 449–59.
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, 159–69.
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, 3799–811.
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, 811–24.
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, 323–6.
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, 1255–60.
Acknowledgments
We thank the National BioResource Project and grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Urasaki, A., Kawakami, K. (2009). Analysis of Genes and Genome by the Tol2-Mediated Gene and Enhancer Trap Methods. 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_6
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DOI: https://doi.org/10.1007/978-1-60327-977-2_6
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