- Top of page
- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
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. Developmental Dynamics 236:2808–2817, 2007. © 2007 Wiley-Liss, Inc.
- Top of page
- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
Sleeping Beauty (SB) is a DNA-based transposable element that belongs to the Tc1/mariner family and has been developed for transgenesis and insertional mutagenesis in a variety of vertebrate models (Ivics and Izsvak,2004). SB has been used to deliver genes into the germline of mice, fish, and Xenopus (Dupuy et al.,2002; Davidson et al.,2003; Grabher et al.,2003; Sinzelle et al.,2006). It has also been used as a gene therapy vector in the treatment of mouse models of human genetic diseases (Hackett et al.,2005). Insertional mutagenesis screens using SB have been carried out in mice and zebrafish and have identified genes involved in cancer as well as early development (Collier et al.,2005; Dupuy et al.,2005; Sivasubbu et al.,2006).
Sleeping Beauty was reconstructed from inactive Tc1 elements in teleost fish (Ivics et al.,1997) and its mechanism of transposition has been characterized by several laboratories (Izsvak and Ivics,2004). The SB transposase recognizes terminal invert/direct repeat sequences (IR/DR) in the transposon and mobilizes it from one DNA site to another through a cut-and-paste mechanism. The open reading frame of the transposase gene was removed from the transposon to generate a nonautonomous element that is unable to mobilize until the transposase is provided in trans. The transposase is highly specific for the SB IR/DR terminal repeat elements and is unable to mobilize inactive Tc1 elements in fish or other organisms. In human cell lines, integration of the SB transposon occurs randomly at TA dinucleotides with no obvious preference for individual chromosomes or chromosomal location. These characteristics have made SB an attractive system for genetic manipulations in vertebrates.
The SB system has been shown to function in a wide range of vertebrate cell lines from fish to humans, including Xenopus laevis A6 kidney cells, suggesting that host factors essential for transposition are conserved in vertebrates (Izsvak et al.,2000). Host factors, however, are thought to modify the transposition process as the efficiency of transposition varied significantly among the cell lines. The chromatin binding protein HMGB1 was identified as a host cofactor for the SB transposase and was shown to increase transposition in mammalian cell lines (Zayed et al.,2003). In addition, the transcription factor Miz-1 was shown to interact with the SB transposase (Walisko et al.,2006). These, and potentially other host factors, may influence the efficiency of SB transposition and should be considered when using SB in different cells and organisms.
Sinzelle and colleagues described a simple method for generating transgenic X. laevis using the SB system (Sinzelle et al.,2006). A β-actin promoter:GFP transgene flanked by IR/DR sequences was co-injected together with SB transposase RNA into fertilized eggs and resulted in animals with ubiquitous expression of the reporter. They demonstrate transposase-dependent integration of the transgene into the germline of founder animals and subsequent transmission of the transposon transgene to the F1 and F2 progeny. Although the SB transposase was required for transgenesis, integration site analysis revealed that transgene insertion was by a noncanonical transposition mechanism. Their work suggests that the SB system is an attractive alternative for generating transgenics in Xenopus.
Xenopus has many characteristics that make it a useful model to study vascular development and angiogenesis. The embryos develop externally and are transparent, which enables easy visualization of vascular structures. It has also been proposed that the vascular system of Xenopus shows more similarity to higher vertebrates than do other models, such as zebrafish (Levine et al.,2003). For example, Xenopus embryos have septated atria, defined heart valves, lungs, blood islands, and a vitelline network (Kau and Turpen,1983; Kolker et al.,2000; Mohun et al.,2000). Xenopus are tetrapods and allow study of the vascular system in developmental processes limited to higher vertebrates, such as limb formation, lung development, limb or tail regeneration, and tail regression during metamorphosis.
Levine and coworkers described a detailed atlas of vascular development in Xenopus by in vivo labeling of endothelial cells with circulating fluorescently labeled low-density lipoprotein (DiI-Ac-LDL; Levine et al.,2003). Vascular endothelial cells were labeled by injecting DiI-Ac-LDL into the beating heart of tail bud stage embryos. The fluorescent label is stable for several weeks and enabled the generation of a three-dimensional atlas of vascular development from stages 33 to 46. Their technique was ideal for examining early vascular development but has limitations. The early vascular development before the heart began beating and later stages of development, during limb formation or tail regression, were inaccessible using this technique.
The receptor tyrosine kinase flk-1/vegfr2/KDR-1 is expressed in the developing embryonic vasculature as well as bipotential hemangioblasts, cells that become either blood or endothelial cells (Millauer et al.,1993; Peters et al.,1993; Yamaguchi et al.,1993; Huber et al.,2004). Targeted deletion of flk-1 results in the failure of the vascular system to develop in mice, demonstrating an essential role for this receptor tyrosine kinase in this process (Shalaby et al.,1995). In Xenopus, the expression of flk-1 occurs in the developing vascular system (Cleaver et al.,1997). Xenopus flk-1 (xflk-1) is expressed in endothelial precursor cells that will form the major blood vessels of the embryo as well as in the primary vasculature and angiogenic-derived intersomitic vessels. These data suggest that xflk-1 is a useful marker for vascular development and angiogenesis in the frog.
We isolated the X. laevis flk-1 gene to generate a vascular endothelial-specific reporter transgene. An enhanced green fluorescent protein (eGFP) reporter was cloned in frame with the first coding exon of the xflk-1 gene and a DNA fragment containing a rabbit β-globin polyadenylation signal was cloned into the second exon. The xflk-1 transgene, including 2.5 kb of the 5′ promoter region and the entire first intron, was cloned into a nonautonomous SB transposon. Transgenic founders were generated by co-injecting the SB transposon transgene and synthetic messenger RNA encoding the SB transposase into fertilized eggs as described by Sinzelle and coworkers (Sinzelle et al.,2006). We demonstrate integration of the transgene into a single genomic locus and germline transmission of the transgene. Of interest, transgene mobilization occurred through noncanonical transposition, an observation that was also reported by Sinzelle and coworkers and may reflect Xenopus host factor interactions with the SB system. Transgenic animals express GFP in the same spatial and temporal pattern as the endogenous xflk-1 gene making this an ideal model to study vascular development and angiogenesis. Disruption of vascular architecture was readily visualized in transgenic animals that were injected with Xenopus vascular endothelial growth factor (xVEGF) RNA. These studies support and extend the work of Sinzelle et al. by demonstrating that the SB system efficiently integrates transgene DNA into the genome of X. laevis and that the animals maintain tissue-restricted expression of the transgene.