Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
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.
???displayArticle.pubmedLink???
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 U1946â, U1984â and U1985â) 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 U1984â 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.
Balciunas,
Trapping fish genes with transposons.
2005, Pubmed
Balciunas,
Trapping fish genes with transposons.
2005,
Pubmed
Balciunas,
Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.
2006,
Pubmed
Bellen,
P-element-mediated enhancer detection: a versatile method to study development in Drosophila.
1989,
Pubmed
Bonin,
A piggyBac transposon gene trap for the analysis of gene expression and function in Drosophila.
2004,
Pubmed
Calvi,
Evidence for a common evolutionary origin of inverted repeat transposons in Drosophila and plants: hobo, Activator, and Tam3.
1991,
Pubmed
Clark,
Transposon vectors for gene-trap insertional mutagenesis in vertebrates.
2004,
Pubmed
Clark,
In vivo protein trapping produces a functional expression codex of the vertebrate proteome.
2011,
Pubmed
Doherty,
A flk-1 promoter/enhancer reporter transgenic Xenopus laevis generated using the Sleeping Beauty transposon system: an in vivo model for vascular studies.
2007,
Pubmed
,
Xenbase
Grabundzija,
Comparative analysis of transposable element vector systems in human cells.
2010,
Pubmed
Hamlet,
Tol2 transposon-mediated transgenesis in Xenopus tropicalis.
2006,
Pubmed
,
Xenbase
Harland,
Xenopus research: metamorphosed by genetics and genomics.
2011,
Pubmed
,
Xenbase
Hirsch,
Xenopus, the next generation: X. tropicalis genetics and genomics.
2002,
Pubmed
,
Xenbase
Hirsch,
Xenopus tropicalis transgenic lines and their use in the study of embryonic induction.
2002,
Pubmed
,
Xenbase
Kawakami,
Transposon tools and methods in zebrafish.
2005,
Pubmed
Kawakami,
Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage.
2000,
Pubmed
Kawakami,
Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio.
1999,
Pubmed
Koga,
Transposition mechanisms and biotechnology applications of the medaka fish Tol2 transposable element.
2004,
Pubmed
Koga,
Transposable element in fish.
1996,
Pubmed
Kondrychyn,
Genome-wide analysis of Tol2 transposon reintegration in zebrafish.
2009,
Pubmed
Korzh,
Transposons as tools for enhancer trap screens in vertebrates.
2007,
Pubmed
Liao,
Tol2 gene trap integrations in the zebrafish amyloid precursor protein genes appa and aplp2 reveal accumulation of secreted APP at the embryonic veins.
2012,
Pubmed
Liu,
Tandem-repeated Zebrafish zp3 genes possess oocyte-specific promoters and are insensitive to estrogen induction.
2006,
Pubmed
Mátés,
Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates.
2009,
Pubmed
Miskey,
DNA transposons in vertebrate functional genomics.
2005,
Pubmed
Muñoz-López,
DNA transposons: nature and applications in genomics.
2010,
Pubmed
Offield,
The development of Xenopus tropicalis transgenic lines and their use in studying lens developmental timing in living embryos.
2000,
Pubmed
,
Xenbase
Ogino,
Highly efficient transgenesis in Xenopus tropicalis using I-SceI meganuclease.
2006,
Pubmed
,
Xenbase
O'Kane,
Detection in situ of genomic regulatory elements in Drosophila.
1987,
Pubmed
Quiñones-Coello,
Exploring strategies for protein trapping in Drosophila.
2007,
Pubmed
Urasaki,
Efficient transposition of the Tol2 transposable element from a single-copy donor in zebrafish.
2008,
Pubmed
Wallingford,
Xenopus.
2010,
Pubmed
,
Xenbase
Yergeau,
Remobilization of Tol2 transposons in Xenopus tropicalis.
2010,
Pubmed
,
Xenbase
Yergeau,
Manipulating the Xenopus genome with transposable elements.
2007,
Pubmed
,
Xenbase
Yergeau,
Forward genetic screens in Xenopus using transposon-mediated insertional mutagenesis.
2012,
Pubmed
,
Xenbase
Yergeau,
Remobilization of Sleeping Beauty transposons in the germline of Xenopus tropicalis.
2011,
Pubmed
,
Xenbase
Yergeau,
Injection-mediated transposon transgenesis in Xenopus tropicalis and the identification of integration sites by modified extension primer tag selection (EPTS) linker-mediated PCR.
2007,
Pubmed
,
Xenbase
Yergeau,
Transposon transgenesis in Xenopus.
2010,
Pubmed
,
Xenbase
