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A.L. Lyubarsky, K.L. Feathers, K. Teofilo, D.S. Williams, A. Swaroop, E.N. Pugh, Jr., D.A. Thompson; Nrl Knockout Mice Lacking Rpe65 Do Not Synthesize 11–cis Retinal . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2966.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize the retinal phenotype of mice homozygous for Rpe65 and Nrl loss–of–function alleles and to test the hypothesis that mouse cones have an Rpe65–independent mechanism of 11–cis retinal synthesis.
Nrl–/– mice, whose retinas contain a single class of photoreceptor cells previously identified as cones (Daniele et al. IOVS 2005;46:2156–67), were mated with Rpe65–/– mice (T.M. Redmond, NEI) to generate Rpe65–/–;Nrl–/– mice. All results were obtained from mice of age 34–40 days. Histochemical and immunocytochemical analysis were performed on eyes embedded in sucrose/OCT or plastic. Retinoid content was determined by extracting eyes from dark–adapted mice with organic solvents and hydroxylamine, and subsequent normal phase HPLC analysis. Protein expression was evaluated by western analysis of whole eye extracts.
Eyes of Rpe65–/–;Nrl–/– double knockout mice had outer segments similar to, albeit shorter than, those of Nrl–/– mice, and apparent accumulation of lipid droplets in the RPE typical of Rpe65–/– mice. Western analysis confirmed loss of Rpe65, Nrl, and Rhodopsin. HPLC analysis of retinoid content yielded the quantities shown below (pmol/eye).
'nd' indicates not detected (less than 2 pmol/eye)
Rpe65–/–;Nrl–/– double knockout mice are unable to synthesize 11–cis retinal in the absence of Rpe65; thus, Rpe65 is required for synthesis of 11–cis retinal for the cone cells of Nrl–/– mice. If this conclusion also applies to the cones of wild type mice, then the normally rod–dominant mouse does not have a cone–specific isomerase that can support pigment regeneration. Such mechanisms may represent specialized adaptations present only in cone–dominant species. The first two authors contributed equally to this study.
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