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A. R. Murray, M. R. Al-Ubaidi; Is the Rhodopsin Glycosylated N-Terminus Sufficient for Outer Segment Morphogenesis and Maintenance?. Invest. Ophthalmol. Vis. Sci. 2007;48(13):592.
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Rhodopsin is glycosylated on two asparagines at its N-terminus. Non-glycosylated opsin results in the malformation of rod outer segments (ROS). We have generated a transgenic mouse model to determine if the glycosylated N-terminus of rhodopsin is adequate for proper ROS formation.
A PCR approach, using overlapping primers, was used to generate a vector (NBC) that consisted of the opsin promoter and the body of the human Beta-adrenergic receptor with its N- and C- termini replaced with those of mouse rhodopsin. PCR genotyping was used to identify potential founders as well as all resulting litters. NBC animals were characterized using Southern and Western Blot analyses to establish individual transgenic lines. Transgenic animals were mated to opsin-/- animals to analyze the NBC proteins in the presence of varying copies of endogenous opsin. Electroretinography (ERG) was used to assess retinal function. Histological and immunohistochemical analyses will be performed at both the light and electron microscopic levels.
Three NBC founders were identified by PCR genotyping and each has produced several litters from matings with opsin-/- mice. ERG analysis revealed that heterozygous NBC mice in the opsin+/- background are functionally indistinguishable from their non-transgenic littermates. NBC mice in the opsin-/- background are being analyzed by ERG and for levels of NBC protein, cellular localization and structure of ROS.
NBC expression in opsin+/- retinas did not produce deleterious effects. Besides enhancing our understanding of the role of opsin glycosylation in ROS formation, the NBC mice will be instrumental in our understanding of the link between a mutation in opsin and its deleterious effects.
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