Functional Characterization of Tol2 Transposon Sequences Using a Somatic Transposition Assay in Zebrafish (A) Visualization of Tol2-mediated somatic transposition in zebrafish (see somatic transposition assay in Materials and Methods). Left, Injection of transposon DNA alone results in a highly mosaic expression pattern with fewer than 5% of the animals displaying eye expression [13,50]. Middle, Injection of SB transposase RNA does not significantly alter this somatic gene transfer distribution [12]. Right, Injection of Tol2 transposase RNA results in gene transfer into the larval eye with over 75% of the injected embryos displaying gene transfer in this tissue at 3 dpf (red arrows). (B) Molecular evidence of rapid Tol2-transposase activity in injected zebrafish embryos. Time-course analysis of excision from injected plasmid DNA [14] was conducted on animals co-injected with a mixture of GFP-marked Tol2 (pTol2/S2EF1a-GM2) and SB (pT2/S2EF1a-GM2 [13] transposons with either Tol2 or SB RNA. Note the kinetic delay in activity maturation by the presumptive obligate tetrameric SB transposase compared to Tol2 (see text). Red arrows indicate the resulting transposase-dependent excision product. (C) Deletion analysis identifies minimal Tol2 sequences required for gene transfer and transposon excision in zebrafish. Zebrafish embryos were injected with depicted deletion constructs and transposase RNA and scored for GFP fluorescence at 3 d postfertilization (eye GFP column). Twenty GFP-positive embryos were used to prepare DNA for excision PCR (Exc. Column). Striped boxes represent Tol2 transposon sequences, with red triangles indicating terminal inverted repeats. Structural elements present in the commonly used Tol2 vector are depicted, with exons shown as open arrows and internal inverted repeats as solid arrows. Restriction enzyme sites are indicated above the transposon drawing.