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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: 2.67). 10/2007; 236(10):2808-17. DOI: 10.1002/dvdy.21321
Source: PubMed

ABSTRACT 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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    • "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. "
    Skin Grafts - Indications, Applications and Current Research, 08/2011; , ISBN: 978-953-307-509-9
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    • "). Our results with X. laevis SB founder lines are consistent with findings from Sinzelle et al. and our own work with SB in X. tropicalis (Sinzelle et al., 2006; Doherty et al., 2007). The Southern blot data presented in Figure 3b,d indicates that integration in Xenopus is by means of noncanonical transposition . "
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    ABSTRACT: Transposon-based integration systems have been widely used for genetic manipulation of invertebrate and plant model systems. In the past decade, these powerful tools have begun to be used in vertebrates for transgenesis, insertional mutagenesis, and gene therapy applications. Sleeping Beauty (SB) is a member of Tc1/mariner class of transposases and is derived from an inactive form of the gene isolated from Atlantic salmon. SB has been used extensively in human cell lines and in whole animal vertebrate model systems such as the mouse, rat, and zebrafish. In this study, we describe the use of SB in the diploid frog Xenopus tropicalis to generate stable transgenic lines. SB transposon transgenes integrate into the X. tropicalis genome by a noncanonical process and are passed through the germline. We compare the activity of SB in this model organism with that of Tol2, a hAT (hobo, Ac1, TAM)-like transposon system.
    Developmental Dynamics 07/2009; 238(7):1727-43. DOI:10.1002/dvdy.21994 · 2.67 Impact Factor
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    • "Southern blot analysis showed that the transgene cassette was integrated into the host genome with the plasmid sequence, indicating that transgene integration occurred by a non-canonical transposition process. Independently of this study, similar non-canonical transposition events, mediated by the SB transposon, were reported in both X. laevis and X. tropicalis (Hamlet and Mead 2003; Doherty et al. 2007). "
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    ABSTRACT: Recent developments in genomic resources and high-throughput transgenesis techniques have allowed Xenopus to 'metamorphose' from a classic model for embryology to a leading-edge experimental system for functional genomics. This process has incorporated the fast-breeding diploid frog, Xenopus tropicalis, as a new model-system for vertebrate genomics and genetics. Sequencing of the X. tropicalis genome is nearly complete, and its comparison with mammalian sequences offers a reliable guide for the genome-wide prediction of cis-regulatory elements. Unique cDNA sets have been generated for both X. tropicalis and X. laevis, which have facilitated non-redundant, systematic gene expression screening and comprehensive gene expression analysis. A variety of transgenesis techniques are available for both X. laevis and X. tropicalis, and the appropriate procedure may be chosen depending on the purpose for which it is required. Effective use of these resources and techniques will help to reveal the overall picture of the complex wiring of gene regulatory networks that control vertebrate development.
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