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X. Wei, J. Zou, S. Bonaffini; Characterization of Four Distinct Transgenic Zebrafish Lines that Express GFP and Crb2b-Targeting siRNA Genes Under the Control of the RH2-2 Promoter. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1888.
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The inner segments of vertebrate photoreceptors are enriched with polarity scaffold proteins, such as Crumbs. Many of these polarity proteins are required to maintain the integrity of a variety of tissues. To investigate the functions of Crumbs 2b (Crb2b) in photoreceptors, the authors attempted to generate transgenic zebrafish lines to suppress Crb2b expression in green cones.
Four distinct hairpin siRNA-crb2b transgenes that target different regions of the crb2b mRNA were individually introduced into the zebrafish genome with an I-SceI meganuclease-based transgenic approach. The RH2-2 promoter was used to drive the expression of these transgenes. Each transgene construct also contains a RH2-2-driven GFP reporter. Positive transgenic lines were identified by both GFP expression and PCR confirmation of the siRNA-crb2b transgenes. Confocal immunomicroscopy was then used to analyze the expression patterns of Crb2b and GFP in the retina.
Eleven transgenic fish lines were identified in this study. However, none display apparent downregulation of Crb2b expression in the photoreceptor layer. The expression patterns of the GFP reporter vary among different transgenic lines. Here, the authors report detailed characterization of the GFP expression patterns in four different lines.
Our study suggests that different strategies are most likely needed for efficient RNAi-mediated gene silencing in transgenic animal models. Nevertheless, the four transgenic zebrafish lines are useful tools to study the development of the photoreceptors and brain in zebrafish.
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