Functional Characterization of <i>Tol2</i> Transposon Sequences Using a Somatic Transposition Assay in Zebrafish

<div><p>(A) Visualization of <i>Tol2</i>-mediated somatic transposition in zebrafish (see somatic transposition assay in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020169#s4" target="_blank">Materials and Methods</a>). Left, Injection of transposon DNA alone results in a highly mosaic expression pattern with fewer than 5% of the animals displaying eye expression [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020169#pgen-0020169-b013" target="_blank">13</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020169#pgen-0020169-b050" target="_blank">50</a>]. Middle, Injection of <i>SB</i> transposase RNA does not significantly alter this somatic gene transfer distribution [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020169#pgen-0020169-b012" target="_blank">12</a>]. Right, Injection of <i>Tol2</i> 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).</p><p>(B) Molecular evidence of rapid <i>Tol2</i>-transposase activity in injected zebrafish embryos. Time-course analysis of excision from injected plasmid DNA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020169#pgen-0020169-b014" target="_blank">14</a>] was conducted on animals co-injected with a mixture of GFP-marked <i>Tol2</i> (pTol2/S2EF1a-GM2) and <i>SB</i> (pT2/S2EF1a-GM2 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020169#pgen-0020169-b013" target="_blank">13</a>] transposons with either <i>Tol2</i> or <i>SB</i> RNA. Note the kinetic delay in activity maturation by the presumptive obligate tetrameric <i>SB</i> transposase compared to <i>Tol2</i> (see text). Red arrows indicate the resulting transposase-dependent excision product.</p><p>(C) Deletion analysis identifies minimal <i>Tol2</i> 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 <i>Tol2</i> transposon sequences, with red triangles indicating terminal inverted repeats. Structural elements present in the commonly used <i>Tol2</i> 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.</p>