Zebrafish, Danio rerio, is a small teleost that has been developed as a model animal to study vertebrate genetics and development. Although it has been widely recognized that zebrafish is a model vertebrate animal suitable for forward genetics approaches, methodologies available for forward genetics in zebrafish have been limited. One can mutagenize the zebrafish gene only by a chemical mutagen or by a pseudotyped retrovirus. It is not easy to clone the mutated gene induced by the chemical mutagen since it requires laborious and time consuming positional cloning approaches. Also it is not easy to manipulate and handle the pseudotyped retrovirus.
We aim to develop gene trap and enhancer trap methods in zebrafish using the To12 transposable element identified from the genome of the Japanese medaka fish. The development of these methods should facilitate genetic studies in zebrafish, i.e., isolation of mutants and identification of the mutated genes, and thereby facilitate our understanding of the function of not only fish genes but also vertebrate genes, which should lead to better insights into the function of human genes related to diseases. In the gene and enhancer trap methods in zebrafish, one can easily identify insertions in important genes as specific GFP expression by examining living embryos under a fluorescent microscope while, in a gene trap method in mouse, such insertions are first screened in ES cells and then the embryo derived from the ES cell has to be examined. Thus, it is possible to screen a large number of insertions and animals fairly easily by using the methods in zebrafish. We have already established a method to generate transposon-insertions in the zebrafish genome through transposition very efficiently. Taking advantage of this transposition system, we will construct various gene trap and enhancer trap vectors based on the To12 element and test them for the frequencies of gene trapping and of zygotic embryonic lethal mutations.
We aim to develop the Gal4/UAS system as an application of these methods, and also to develop the jump-starter system, in which transposition is induced by mating, and a method to construct deletion mutations upon excision of the transposon. Finally, as a consequence of these studies, we will establish a large number of fish expressing GFP in a spatially and temporally regulated fashion and various mutant fish, which will be useful resources to study vertebrate development.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-CDF-5 (50))
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Greenberg, Judith H
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National Institute of Genetics
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Asakawa, Kazuhide; Kawakami, Koichi (2009) The Tol2-mediated Gal4-UAS method for gene and enhancer trapping in zebrafish. Methods 49:275-81
Urasaki, Akihiro; Asakawa, Kazuhide; Kawakami, Koichi (2008) Efficient transposition of the Tol2 transposable element from a single-copy donor in zebrafish. Proc Natl Acad Sci U S A 105:19827-32
Nagayoshi, Saori; Hayashi, Eriko; Abe, Gembu et al. (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
Asakawa, Kazuhide; Suster, Maximiliano L; Mizusawa, Kanta et al. (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
Kotani, Tomoya; Kawakami, Koichi (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 316:383-96
Fan, Xiang; Hagos, Engda G; Xu, Bo et al. (2007) Nodal signals mediate interactions between the extra-embryonic and embryonic tissues in zebrafish. Dev Biol 310:363-78
Kawakami, Koichi (2007) Tol2: a versatile gene transfer vector in vertebrates. Genome Biol 8 Suppl 1:S7
Nakada, Tsutomu; Hoshijima, Kazuyuki; Esaki, Masahiro et al. (2007) Localization of ammonia transporter Rhcg1 in mitochondrion-rich cells of yolk sac, gill, and kidney of zebrafish and its ionic strength-dependent expression. Am J Physiol Regul Integr Comp Physiol 293:R1743-53
Urasaki, Akihiro; Morvan, Ghislaine; Kawakami, Koichi (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 174:639-49
Kotani, Tomoya; Nagayoshi, Saori; Urasaki, Akihiro et al. (2006) Transposon-mediated gene trapping in zebrafish. Methods 39:199-206

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