The Tol2-mediated genetic methodologies in zebrafish: gene trapping, enhancer trapping and remobilization

Urasaki, A., Asakawa, K., Kotani, T.,Nagayoshi, S.,Kishimoto, Y.,Kawakami, K.

　We have been developing the gene trap method using the Tol2 transposon in zebrafish. To date, we have identified and characterized 102 unique GFP expression patterns. 75% of the genes trapped by the transposon insertions turned out to be previously uncharacterized genes, indicating that the gene trap method is useful to identify novel developmental genes. Also we have constructed various types of trap vectors and compared their efficiencies; i.e., a vector with a zebrafish endogenous splice acceptor, a vector to trap a coding frame and an enhancer trap vector.
　Currently, chromosomal insertions of Tol2 are created by plasmid injection. We injected the transposase mRNA into embryos carrying a single transposon insertion and found that the integrated genomic copy could be mobilized. To determine whether new trap events can be detected by remobilization, we injected the transposase mRNA into homozygous embryos carrying an insertion of the gene trap construct in the hoxc cluster. Among progeny from 40 injected fish, we identified 10 new GFP expression patterns, different from that of the hoxc insertion, indicating that the insertion was remobilized very efficiently, and the new insertions could entrap endogenous transcripts. Thus, the remobilization system should be an alternative method to create transposon insertions in new loci. Further, we are currently constructing transgenic fish expressing the transposase, and testing whether remobilization can be induced by mating.