Generation and Analysis of Crx/ODAG Transgenic Mice

Abstract

Abstract: : Purpose: Previously, we cloned a novel gene from mouse retina by differential display and named it as ocular development–associated gene (ODAG). To analyze its biological role in the retinal development, we generated and analyzed transgenic mice overexpressing ODAG under the control of a photoreceptor –specific promoter. Methods: A DNA fragment encompassing CRX (cone rod homeobox) promoter, ODAG cDNA, IRES (internal ribosomal entry site), GFP (green fluorescent protein), and SV40 splicing and polyA signals was microinjected into pronuclei of BDF2 eggs. Transgenic mice were identified by Southern blot using ODAG cDNA as a probe. Expression of ODAG protein was examined by Western blot with an anti–ODAG polyclonal antibody. Parafin–embedded sections were subjected to pathological and immunohistochemical analyses. Results: Transgenic retina showed about ten times higher expression of ODAG compared to non–transgenic retina. Macroscopically, eyeballs of transgenic mice were gradually enlarged with age, occasionally associated with corneal opacity and neovascularization in the ciliary body. Pathological examination revealed that the architecture of retinal layers was disorganized with marked reduction of ganglion cells and rod and cone cells were poorly developed and detached from the pigmental layer. Conclusions: These results strongly suggest that regulated expression of ODAG is essential for normal retinal development. We are currently investigating the changes in gene expression profile and intraocular pressure caused by ODAG overexpression. These mice can be used as a model of glaucoma and so on if their intraocular pressure is high.

Keywords: transgenics/knock-outs • gene/expression • retinal development