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A 221-bp fragment of the mouse opsin promoter directs expression specifically to the rod photoreceptors of transgenic mice

Published online by Cambridge University Press:  02 June 2009

Alexander B. Quiambao
Affiliation:
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago
Neal S. Peachey
Affiliation:
Hines VA Hospital and Department of Neurology, Stritch School of Medicine, Loyola University, Chicago
Nancy J. Mangini
Affiliation:
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago
Pal Röhlich
Affiliation:
Department of Human Morphology and Developmental Biology, Laboratory I. of Electron Microscopy, Semmelweis University of Medicine, Budapest, Hungary
Joe G. Hollyfield
Affiliation:
Research Institute FFb, Cleveland Clinic Foundation, Cleveland
Muayyad R. Al-Ubaidi
Affiliation:
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago

Abstract

Mutations in the human rod opsin gene have been shown to segregate with autosomal dominant retinitis pigmentosa (ADRP) and photoreceptor degeneration in transgenic mice. While these degenerations are characterized by the primary degeneration of rods, cones eventually die as well. To determine whether this subsequent cone degeneration is the result of expression of mutant rod opsin in the cones, the retinal cell-type specificity of a 221-bp fragment of the mouse rod opsin promoter was evaluated. Two transgenic mouse lines generated by injecting a fusion gene comprised of a 221-bp fragment of the mouse rod opsin promoter and the simian virus 40 large tumor antigen gene (Tag) were examined. The expression of Tag causes photoreceptor cell degeneration in members of both transgenic lines. However, the two lines differed with respect to the level of Tag expression and the rate and extent of photoreceptor cell degeneration. Immunocytochemical localization of opsin and Tag in surviving photoreceptor cells was determined and the results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Rod- and cone-mediated function was evaluated by electroretinography (ERG). In the higher Tag-expressing transgenic line only one row of nuclei remained in the outer nuclear layer at postnatal day (P) 150. While these nuclei showed no antigenicity for rod opsin or Tag, they did stain with an antibody that reacts with both rod and cone S-antigens (arrestins), indicating that these cells were surviving photoreceptor nuclei. Positive staining with peanut agglutinin, which uniquely decorates matrix domains surrounding cones in the normal retina, confirmed that the surviving photoreceptor nuclei were of cone origin. RT-PCR substantiated the results from immunostaining; amplification product was obtained using blue cone opsin transcripts but not from either Tag or rod opsin transcripts. The second transgenic mouse line exhibited a much slower photoreceptor cell death that was associated with low levels of Tag transgene transcript. At P120, ~50% of photoreceptors remained and an ~45% reduction in the rod ERG a-wave was observed. Cone-mediated ERGs, however, were normal. The results demonstrate the rod-specific expression of Tag as directed by the 221-bp fragment of the mouse rod opsin promoter and suggest that the cone degeneration in ADRP or transgenic mice associated with mutations in the rod opsin gene is a secondary effect of rod degeneration.

Type
Research Articles
Copyright
Copyright © Cambridge University Press 1997

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