zTrap and NIGKOF: Resources for Gene Trap and Enhancer Trap Lines and Knockout Fish

K. Kawakami2,1, G. Abe2, K, Asakawa2,1, R. Fukuda2, P. Lal2,1, A. Muto2, M. Suster3, H. Takakubo2, A. Urasaki2

1)Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), 2)Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka 411- 8540, Japan, 3)Sars International Centre, Bergen, Norway

 We have developed genetic methods by using the Tol2 transposable element, i.e., gene trapping and enhancer trapping utilizing the GFP gene or the Gal4FF gene, a modified yeast Gal4 transcription activator. To date, we have generated and collected 173 and 572 transgenic fish that express GFP and Gal4FF in temporally and spatially restricted fashions. These transgenic fish are a useful resource to study genetics, development and behavior. To improve the availability of the transgenic fish resource, we developed a web-based database named zTrap. zTrap contains images of expression patterns of GFP and Gal4FF visualized with UAS:GFP, and is equipped with the functionality to search the expression patterns effectively. Therefore researchers can find fish with expression patterns of their interests quickly. Furthermore, these lines have been analyzed by Southern blot hybridization and outcrossed to generate fish with single insertions, which is necessary to reduce unwanted effects by irrelevant insertions and make further molecular characterization easy. To date, zTrap contains DNA sequences surrounding 439 integration sites of the gene trap and enhancer trap constructs thus analyzed. These are mapped on the genome by in-house Blat analysis and can be viewed both on our zTrap browser and on the Ensembl browser. zTrap is also equipped with the functionality to search the integration sites effectively and researchers can find transgenic fish which carry transposon insertions near genes or loci of their interests. zTrap also contains the information on the UAS reporter and effector fish.
 To facilitate knocking out all of zebrafish genes, we recently started collecting not only fish with single insertions but also fish with multiple insertions. We set up systems for sperm cryopreservation and cloning multiple insertions from such male fish. Currently we have found that 34% of the transposon insertions thus identified are located within annotated transcriptional units and 10% of the total insertions are located within exons, indicating that this approach is effective. We have launched the NIGKOF database to integrate these results. Thus, the zTrap and NIGKOF databases and resources should be useful to facilitate the zebrafish research (http://kawakami.lab.nig.ac.jp/).