Abstract
The ability to control transgene expression within specific tissues is an important tool for studying the molecular and cellular mechanisms of development, physiology, and disease. We developed a Tet-On system for spatial and temporal control of transgene expression in zebrafish rod photoreceptors. We generated two transgenic lines using the Xenopus rhodopsin promoter to drive the reverse tetracycline-controlled transcriptional transactivator (rtTA), one with self-reporting GFP activity and one with an epitope tagged rtTA. The self-reporting line includes a tetracycline response element (TRE)-driven GFP and, in the presence of doxycycline, expresses GFP in larval and adult rods. A time-course of doxycycline treatment demonstrates that maximal induction of GFP expression, as determined by the number of GFP-positive rods, is reached within approximately 24 hours of drug treatment. The epitope-tagged transgenic line eliminates the need for the self-reporting GFP activity by expressing a FLAG-tagged rtTA protein. Both lines demonstrate strong induction of TRE-driven transgenes from plasmids microinjected into one-cell embryos. These results show that spatial and temporal control of transgene expression can be achieved in rod photoreceptors. Additionally, system components are constructed in Gateway compatible vectors for the rapid cloning of doxycycline-inducible transgenes and use in other areas of zebrafish research.
Highlights
Zebrafish have emerged as a powerful model organism for studying molecular and cellular mechanisms regulating embryogenesis and organ formation
Retinal sections of Dox-treated 6 dpf Tg(Xla.rho:reverse tetracycline-controlled transcriptional transactivator (rtTA), tetracycline response element (TRE):green fluorescent protein (GFP)); alb2/2 larvae, showed strong GFP fluorescence in rod photoreceptors that co-labeled with the anti-Rhodopsin antibody (Fig. 1C)
Retinal sections from adult Tg(Xla.rho:rtTA; TRE:GFP) fish treated with Dox for 72 h before fixation were positive for anti-GFP immunofluorescence in rod photoreceptors, which co-labeled with anti-Rhodopsin antibody (Fig. 1E)
Summary
Zebrafish have emerged as a powerful model organism for studying molecular and cellular mechanisms regulating embryogenesis and organ formation. Heat-shock induction using the hsp70l promoter combined with CreER has improved the conditional temporal control of Cre recombinase-based gene expression [4]. Another approach uses an ecdysone receptor-Gal chimeric protein to control Gal activity in the Gal4/UAS system [6]. The tetracycline (Tet)- or doxycycline (Dox)-inducible Tet-On system [10,11] has been used in zebrafish to conditionally control Tet-responsive transgene expression in specific cell types This system was first used to drive Dox-induced, heart-specific transgenes where expression was limited to the heart but leaky transgene expression was observed in the absence of drug in one of the two transgenic lines produced [12]. The leakiness of the transgene was improved by fusing the Tet activator to either a modified glucocorticoid receptor or the ecdysone receptor; in addition, the expression was reversible [13]
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