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P.R. Helguera, O.L. Moritz, D.S. Papermaster; Antibiotic (G418) Selection Enchances Uniformity of Expression of Transgenic Dynamin I-GFP in Xenopus laevis Rods . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2840.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Dynamin I is a GTPase protein involved in recycling of synaptic vesicles. It has been previously shown (Xu et al., ARVO 2001) that frog rod photoreceptors expressing human Dynamin I K44A (a dominant mutation that suppresses GTPase activity) undergo cell death, and that apoptosis can be prevented in these cells by exposure to constant light. We observed differences in the expression levels between cells, a recurrent phenomenon in transgenic frogs (Moritz et al., 2001), due to gene silencing artifacts. In order to progress in studies of apoptotic pathways underlying these observations, we are developing strategies to decrease transgene silencing. We are also trying to establish whether the same effect can be achieved in cones to evaluate the contribution of synaptic disorders as potential models of neuro-degenerative disease in the eye. Methods: Transgenic tadpoles expressing mutant and wild-type Dynamin GFP-fusion proteins under control of the X. laevis rod or red cone opsin promoters were generated as described (Moritz et al., 1999), and exposed to constant light for 28 days. Tadpoles were then sacrificed and analyzed by fluorescence microscopy. In order to improve expression homogeneity throughout the photoreceptor layer of the retina, or at least assure a minimal threshold, we used G418 tadpole selection protocols (Moritz et al., 2002), which take advantage of the neomycin resistance gene also present on our plasmids. Results: Using rod opsin promoter plasmids, we obtained a population of transgenic X. laevis with uniform high level expression of GFP fusion protein and reduced transgene silencing. Despite G418 selection, transgenic tadpoles with cone promoter plasmids exhibited a low signal from the GFP fusion protein. Our previous results showed that in constant darkness transgenic tadpoles expressing GFP-Dynamin I K44A developed progressive rod degeneration: slight after 10 days and complete at 14 days. We are establishing if this is an accumulative effect due to darkness or if it is a darkness-triggered phenomena and its progress is only time-dependent by analysis of tadpoles sacrificed after progressively longer dark exposure prior to being returned to constant light. Conclusions: G418 selection successfully improved the homogeneity of gene expression in transgenic tadpoles expressing GFP-Dynamin I. The red cone opsin promoter is not as robust as the rod opsin promoter.
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