A flk‐1 promoter/enhancer reporter transgenic Xenopus laevis generated using the Sleeping Beauty transposon system: An in vivo model for vascular studies

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. Developmental Dynamics 236: 2808 -2817, 2007.

Krüppel-like factor 2 cooperates with the ETS family protein ERG to activateFlk1expression during vascular development

“…Reporter expression persists during subsequent development, when GFP fluorescence is observed throughout the smallest vessels of the trunk and tail (Fig. 2B,C) (Doherty et al, 2007).…”

“…A construction containing 2.5 kb of Xenopus Flk1 5Ј flanking sequences plus first intron sequences is sufficient to drive GFP expression throughout the blood vessels of transgenic embryos ( Fig. 2A-C) (Warkman et al, 2004;Doherty et al, 2007). Expression is evident in major vessels including the posterior cardinal veins, intersomitic vessels and the vascular plexus, and is equivalent to endogenous Flk1 expression at a comparable stage (compare Fig.…”

Notch signaling, wt1 and foxc2 are key regulators of the podocyte gene regulatory network in Xenopus

“…wt1, hey1, mafb, lmx1b, tcf21 and nphs1 have been studied previously in Xenopus wholemounts (Carroll and Vize, 1996;Ishibashi and Yasuda, 2001;Haldin et al, 2003;Coolen et al, 2005;Gerth et al, 2005;Simrick et al, 2005;Taelman et al, 2006;Haldin et al, 2008), but to precisely compare the glomus expression domains it was important to perform a side-by-side analysis of all ten genes. We initially analyzed embryos at stage 35 because Xenopus podocytes are functional at stage 38 and vascularization of the glomus occurs around stage 32 (Nieuwkoop and Faber, 1994;Vize et al, 2003;Doherty et al, 2007). Both whole-mount in situ hybridizations and Paraplast-embedded sections thereof revealed distinct expression…”

Transposon tools hopping in vertebrates

“…In a series of yet-unexplained observations in Xenopus, two research groups have noted a positive gene transfer rate for SB that did not appear to be due to standard transposition reactions, instead displaying non-canonical footprints [43,44]. Given the abundance of related Tc1/mariner transposons in frog genomes, this may reflect interference by endogenous transposable elements, or this may be due to some other aspect of SB biology that is limited in frogs.…”