Excision of the tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio.

The Tol2 element is a transposable element in Oryzias latipes (the medaka fish) found in the tyrosinase gene locus of the tyrosinase-deficient mutant medaka fish and has been shown to be excised from the genome during medaka embryogenesis (Koga, A., Suzuki, M., Inagaki, H., Bessho, Y., Hori, H., 1996. Transposon element in fish. Nature 383, 30). It is, however, not known whether the Tol2 element is an autonomous element. To determine whether the cloned Tol2 element is an autonomous element and whether excision can occur also in the other fish species, the plasmid DNA harboring the Tol2 element was injected to fertilized eggs of zebrafish, Danio rerio, and the total DNA extracted from the embryos 9-10h after the injection was analyzed by PCR. When a plasmid with the full-length Tol2 element was used for the microinjection, in 39 out of 43 injected embryos, we found generation of short PCR products indicative of the loss of the Tol2 element from the injected plasmid. Ten of these cases were analyzed at the DNA sequence level, and nine of them showed either precise excision of the Tol2 element (three cases) or nearly precise excision of the element with the addition of a few nucleotides of the target duplication (six cases). When a deletion version of the Tol2 element that retained the terminal inverted repeats but lacked about one-fourth of the open reading frame-coding region was used for the microinjection, such short PCR products could not be amplified from any of the injected embryos (0 out of 30). Thus, the Tol2 element is capable of excision in zebrafish embryos, presumably dependent on a putative transposase encoded by the Tol2 element itself. This transient embryonic excision assay using zebrafish should be useful to analyze the structure and the function of the transposase and cis-elements necessary for excision. Also, this study implies the potential use of the Tol2 element in transgenesis and insertional mutagenesis in both zebrafish and the medaka fish.