Inducible gene expression in transgenic Xenopus embryos

Abstract

The amphibian Xenopus laevis has been successfully used for many years as a model system for studying vertebrate development. Because of technical limitations, however, molecular investigations have mainly concentrated on early stages. We have developed a straightforward method for stage-specific induction of gene expression in transgenic Xenopus embryos [1] [2]. This method is based on the Xenopus heat shock protein 70 (Xhsp70 [3]) promoter driving the expression of desired gene products. We found that ubiquitous expression of the transgene is induced upon relatively mild heat treatment. Green fluorescent protein (GFP) was used as a marker to monitor successful induction of gene expression in transgenic embryos. We used this method to study the stage specificity of Wnt signalling function. Transient ectopic Wnt-8 expression during early neurulation was sufficient to repress anterior head development and this capacity was restricted to early stages of neurulation. By transient over-expression at different stages of development, we show that frizzled-7 disrupted morphogenesis sequentially from anterior to posterior along the dorsal axis as development proceeds. These results demonstrate that this method for inducible gene expression in transgenic Xenopus embryos will be a very powerful tool for temporal analysis of gene function and for studying molecular mechanisms of vertebrate organogenesis.

Inverse drug screens: a rapid and inexpensive method for implicating molecular targets

“…It reveals maternal effects, since the drugs down-regulate activity of maternal proteins that would remain untouched by morpholino or siRNA-based approaches. By adding reagents at defined time-points, one can obtain timing (stage-specific) information on the POI; inducible expression technologies provide a similar functionality (Adam et al, 2000;Blau and Rossi, 1999;Wheeler et al, 2000), but drug experiments are often faster and cheaper than the molecular genetics necessary to create a clean inducible effect, and work even in model systems where no satisfactory inducible technique is available.…”

Transgenic Xenopus laevis strain expressing cre recombinase in muscle cells

“…Fusion proteins of the Cre or FLP recombinase with the ligand binding domain of a mutated estrogen or progesterone receptor, allowing a tamoxifen-or RU486-dependent activation, respectively, of the recombinase have been used successfully (Branda and Dymecki, 2004). Another approach involves the heat-shock promoter (Wheeler et al, 2000) to drive expression of the recombinase and has already been applied in transgenic zebrafish (Thummel et al, 2005). As the heat-shock promoter has been used most successfully for conditional transgenesis in Xenopus without showing any basal activity (Buchholz et al, 2004;Fu et al, 2005), a heat-shock promoter-controlled Cre strain would be the most promising approach to ubiquitously activate silent reporter genes in transgenic reporter strains.…”

“…However, investigating transgenes interfering with normal development, one can hardly obtain sexually mature founder animals; therefore, the use of an inducible system is essential. So far, different inducible systems have been applied in Xenopus laevis, including heat shock controlled transgenes (Wheeler et al, 2000;Buchholz et al, 2004), as well as RU486 or doxycycline inducible systems (Das and Brown, 2004). Also, binary systems such as the GAL4/UAS system (Chae et al, 2002;Hartley et al, 2002) and the Cre/loxP system (Ryffel et al, 2003) have been adapted successfully to Xenopus for the conditional expression of transgenes.…”

Xenopus

“…Transgenic techniques are now becoming more refined, allowing more targeted gene expression. Four different approaches have been taken: The use of the binary Gal4-UAS system, the development of modified progesterone RU-486 and Tetracycline-on inducible systems, the use of heat shock-inducible transgenes, and most recently the development of a Cre-Lox recombinase system (42)(43)(44)(45).…”