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Xenopus is a powerful model for studying a diverse array of biological processes. However, despite multiple methods for transgenesis, relatively few transgenic reporter lines are available and commonly used. Previous work has demonstrated that transposon based strategies are effective for generating transgenic lines in both invertebrate and vertebrate systems. Here we show that the Tol2 transposon can be remobilized in the genome of X. tropicalis and passed through the germline via a simple breeding strategy of crossing transposase expressing and transposon lines. This remobilization system provides another tool to exploit transgenesis and opens new opportunities for gene trap and enhancer trap strategies.
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24116167 ???displayArticle.pmcLink???PMC3792888 ???displayArticle.link???PLoS One ???displayArticle.grants???[+]
Figure 2. Mating scheme for generation and analysis of Ef1αGFPTol2 remobilized transposons.The diagram is a schematic of the crosses to test transposon mobilization in each of three different lines. We crossed three F1 heterozygous ZP3T2γMN transposase frogs (one from each injected tranposase animal U1946♂, U1984♀ and U1985♀) with three F1 homozygous Ef1αGFPTol2 frogs to generate three F2 double transgenic (Ef1αGFPTol2/ZP3T2γGMN) offspring clutches. Double transgenic animals from each clutch were outcrossed, and F3 ubiquitous GFP+ embryos of each phenotype observed were collected and tested for remobilization. High intensity F3 embryos, found only in the U1984♀ line, were raised to adulthood, tested for remobilization, and outcrossed to test for germline transmission.
Figure 3. Remobilization Data.A. Number of remobilizations mapped to donor scaffold 8 compared to those mapped to other scaffolds. Proximity (Mb) of remobilized transposons on scaffold 8 to the donor locus is also shown. B. Number of intragenic versus intergenic integrations for remobilizations on both donor and other scaffolds, including proximity (kb) of intergenic integrations to the nearest flanking gene. The size of the pie charts indicates the relative number of remobilizations on donor versus other scaffolds. Number of samples in each category are shown within the pie slices for both A and B, and total n numbers are indicated beneath each graph.
Figure 1. Transgenic lines.A. Diagram of ZP3T2γGMN construct used to produce transgenic frogs expressing Tol2 transposase. Not to scale. I-SceI: Meganuclease site necessary for transgenesis. Zebrafish zona pellucida glycoprotein 3 (zp3) promoter: drives egg specific expression of Tol2 transposase. Gamma crystallin promoter (γCry): drives expression of eGFP in lens of the eye as a reporter for transgene insertion. B. RT-PCR for transposase expression in transgenic ZP3T2γGMN F1 offspring arising from outcrosses of transposase transgene injected animals U1946♂, U1984♀ and U1985♀. Pools of 10 egg, stage 30 gfp+ and gfp- embryos were tested. In the U1946♂ and U1985♀ lines, one testis from adult male frogs was also tested. OCD primers were used as positive controls (- RT). reactions using GFP+ embryos, and water (data not shown) were also used as negative controls. C. Diagram of Ef1αGFPTol2 construct. Tol2 left (L) and right (R) arms, EF1α enhancer driving eGFP transgene. Arrows: indicate specified LM-PCR and sequencing primer binding sites on transposon arms. D. Ubiquitous GFP+ phenotypes. Low, Medium, and High Intensity phenotypes were seen in F3 and F4 embryos.
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