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X. Zhuo, M. Haeri, B. E. Knox; An Inducible System for Controlling Gene Expression in Transgenic Xenopus Rods. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1894.
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© ARVO (1962-2015); The Authors (2016-present)
To develop an inducible expression system in transgenic Xenopus rods.
Three expression systems were investigated for use in Xenopus rods: 1) TOPTK-i (Denayer et al., 2006, FEBS Lett. 580, 393), 2) LTRi (Tara et al.,2007 Cell, 130, 363) and 3) a novel modification of Gal4/UAS (Hartley et al., 2002, PNAS, 99, 1377). In the novel system, a driver, GAL4-GR-EGFP, was expressed under control of the Xenopus opsin promoter. This driver encodes the Gal4 transcriptional activation-DNA binding domains with the glucocorticoid receptor (GR) translocation domain at the carboxyl terminus. The GR domain enables dexamethasone-dependent nuclear translocation and thus inducible transcriptional activity through Gal4-UAS interactions. The reporter construct contained a UAS-hsp promoter controlling the gene of interest. Expression was measured quantitatively using live cell confocal microscopy.
Preliminary experiments indicated that our novel system was suitable for induction of gene expression in transgenic Xenopus rods. By contrast, TOPTK-iGFP expressed GFP without induction and LTRi-EGFP was not inducible in Xenopus. Using the novel system and a rhodopsin-mCherry fusion reporter, gene expression was recorded in the rod outer segment for at least a month, which is the time needed for disks to completely traverse the length of the outer segment. We observed fluorescent protein only after dexamethasone treatments. Multiple inductions produced bands of fluorescent protein that were spaced in the outer segment according to the treatment length, with expression returning to background levels quickly (~2 days) after drug removal. Kaplan banding of rhodopsin assisted the temporal analysis of gene expression with this reporter.
We have created an inducible system with robust and reliable control of gene expression in transgenic Xenopus. Although the mosaic expression, a common phenomenon associated with transgenes, still exists in this inducible system, quantitative analysis of fluorescence in individual cells permits a detailed analysis of gene expression. Moreover, the efficient induction and low background expression will permit future experiments using toxic or regulatory genes.
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