September 1, 2006;
Tol2 transposon-mediated transgenesis in Xenopus tropicalis.
The diploid frog Xenopus tropicalis is becoming a powerful developmental genetic model system. Sequencing of the X. tropicalis genome is nearing completion and several labs are embarking on mutagenesis screens. We are interested in developing insertional mutagenesis strategies in X. tropicalis. Transposon-mediated insertional mutagenesis, once used exclusively in plants and invertebrate systems, is now more widely applicable to vertebrates. The first step in developing transposons as tools for mutagenesis is to demonstrate that these mobile elements function efficiently in the target organism. Here, we show that the Medaka fish transposon, Tol2, is able to stably integrate into the X. tropicalis genome and will serve as a powerful tool for insertional mutagenesis strategies in the frog.
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FIG. 1. Tol2 transgenesis in Xenopus tropicalis. (a) Schematic of the Tol2 transposon (Tol2) and the modified non-autonomous substrate (Tol2XIG) that was used in this study (not to scale) (Kawakami and Shima, 1999). The Xenopus EF-1a promoter drives expression of GFP. The mini-gene is cloned in the antisense orientation relative to the Tol2 sequence. An intron is included upstream of the reporter to promote correct processing of the transcript. SA, splice acceptor; SD, splice donor. (b) Schematic of the microinjection procedure and subsequent screening. (c) Representative examples of GFP-positive tadpoles after injection of Tol2 plasmid DNA and Tol2 mRNA at the one cell stage. Reproduced with permission of the Publisher, John Wiley & Sons.
FIG. 2. Germline transmission of Tol2 transposon transgenes. (a) Stage 50 sibling GFP-positive F1 tadpoles from founder 12M. Note the difference in intensity of GFP expression likely reflects either transposon integration position effects or the number of independently segre- gating alleles present in each individual (see text and Southern analysis in Fig. 3b). (b) Stage 40 GFP-positive F1 tadpoles from founder 10M. (c) Stage 40 GFP-positive and GFP-negative F2 tadpoles generated by outcross of 10M F1 adults. Insert shows bright-field photo- micrograph of the same group of tadpoles and reveals the GFP-negative siblings. Reproduced with permission of the Publisher, John Wiley & Sons.