A flk-1 promoter/enhancer reporter transgenicXenopus laevis generated using theSleeping Beauty transposon system: An in vivo model for vascular studies

Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Developmental Dynamics (Impact Factor: 2.38). 10/2007; 236(10):2808-17. DOI: 10.1002/dvdy.21321
Source: PubMed


We have used the Sleeping Beauty (SB) transposable element to generate transgenic Xenopus laevis with expression of green fluorescent protein (GFP) in vascular endothelial cells using the frog flk-1 promoter. This is the first characterization of a SB-generated transgenic Xenopus that has tissue-restricted expression. We demonstrate that the transgene integrated into single genomic loci in two independent founder lines and is transmitted through the germline at the expected Mendelian frequencies. Transgene integration occurred through a noncanonical transposition process possibly reflecting Xenopus-specific interactions with the SB system. The transgenic animals express GFP in the same spatial and temporal pattern as the endogenous flk-1 gene throughout development and into adulthood. Overexpression of xVEGF122 in the transgenic animals disrupts vascular development that is visualized by fluorescent microscopy. These studies demonstrate the convenience of the SB system for generating transgenic animals and the utility of the xflk-1:GFP transgenic line for in vivo studies of vascular development.

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Available from: Joanne Doherty, Nov 04, 2014
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    • "Shortly after that however, when the tadpoles have become more fully transparent, reporter signal is easily monitored. Previously, a transgenic Xenopus laevis reporter line was generated using a similar xflk-1:GFP construct, but no reporter expression was described in the lymphatics (Doherty et al., 2007). Possibly, the use of slightly different promoter or intron fragments yielded BEC-restricted versus our BEC/LEC GFP expression. "
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    ABSTRACT: The importance of the blood- and lymph vessels in the transport of essential fluids, gases, macromolecules and cells in vertebrates warrants optimal insight into the regulatory mechanisms underlying their development. Mouse and zebrafish models of lymphatic development are instrumental for gene discovery and gene characterization but are challenging for certain aspects, e.g. no direct accessibility of embryonic stages, or non-straightforward visualization of early lymphatic sprouting, respectively. We previously demonstrated that the Xenopus tadpole is a valuable model to study the processes of lymphatic development. However, a fluorescent Xenopus reporter directly visualizing the lymph vessels was lacking. Here, we created transgenic Tg(Flk1:eGFP) Xenopus laevis reporter lines expressing green fluorescent protein (GFP) in blood- and lymph vessels driven by the Flk1 (VEGFR-2) promoter. We also established a high-resolution fluorescent dye labeling technique selectively and persistently visualizing lymphatic endothelial cells, even in conditions of impaired lymph vessel formation or drainage function upon silencing of lymphangiogenic factors. Next, we applied the model to dynamically document blood and lymphatic sprouting and patterning of the initially avascular tadpole fin. Furthermore, quantifiable models of spontaneous or induced lymphatic sprouting into the tadpole fin were developed for dynamic analysis of loss-of-function and gain-of-function phenotypes using pharmacologic or genetic manipulation. Together with angiography and lymphangiography to assess functionality, Tg(Flk1:eGFP) reporter tadpoles readily allowed detailed lymphatic phenotyping of live tadpoles by fluorescence microscopy. The Tg(Flk1:eGFP) tadpoles represent a versatile model for functional lymph/angiogenomics and drug screening.
    Full-text · Article · Sep 2013 · Biology Open
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    • "Our studies have focused on using the class II DNA 'cut-and-paste' transposable elements to modify the frog genome for gene- and enhancer-trapping and for insertional mutagenesis [4-9]. Transposable elements have been used for many years to experimentally modify the genomes of plants and invertebrates and, more recently, have been applied to vertebrate model systems [10,11]. "
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    ABSTRACT: The Sleeping Beauty (SB) transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization. Transgenic frogs that express the SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'. In this study, we demonstrate that SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.
    Full-text · Article · Nov 2011 · Mobile DNA
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    • "Furthermore, the full sequencing of the X. laevis genome using the homozygous inbred strain J from our resource center is now ongoing and the first assembly is already available (R. Harland, personal communication). With these new available resources, it will become possible to identify and isolate regulatory regions of immunologically relevant genes and produce transgenic reporter animals expressing fluorescent reporter genes (such as GFP) under the control of these regulatory regions as in mouse and zebra fish (Smith, Ataliotis et al. 2005; Doherty, Johnson Hamlet et al. 2007; Hall, Flores et al. 2009). X. laevis transgenic lines expressing, for example, GFP under the transcriptional control of the CD4 (CD4 T cells) or the Foxp3 (T regulatory cells) promoter regions (i.e., homologs of these genes have already been identified in the X. tropicalis genome), would permit to localize and follow the fate of these cells in transplanted skin tissues during rejection and tolerance induction. "

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