May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
EGFP levels and toxicity in transgenic and virus injected animals.
Author Affiliations & Notes
  • T.S. Rex
    F.M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • J. Peet
    F.M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • E. Surace
    TIGEM, Naples, Italy
  • A. Auricchio
    TIGEM, Naples, Italy
  • E. Bendo
    F.M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • A.L. Lyubarsky
    F.M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • T.E. Hughes
    Cell Biology and Neuroscience, Montana State University, Bozeman, MT
  • A.M. Maguire
    F.M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • J. Bennett
    F.M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • E.N. Pugh Jr
    F.M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, PA
  • Footnotes
    Commercial Relationships  T.S. Rex, None; J. Peet, None; E. Surace, None; A. Auricchio, None; E. Bendo, None; A.L. Lyubarsky, None; T.E. Hughes, None; A.M. Maguire, None; J. Bennett, None; E.N. Pugh Jr, None.
  • Footnotes
    Support  NIH Vision Training Grant T32EY07035; NIH grants EY10820, EY12156, EY002660; FFB; RPB
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3707. doi:
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      T.S. Rex, J. Peet, E. Surace, A. Auricchio, E. Bendo, A.L. Lyubarsky, T.E. Hughes, A.M. Maguire, J. Bennett, E.N. Pugh Jr; EGFP levels and toxicity in transgenic and virus injected animals. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3707.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: Develop a quantitative assay of EGFP toxicity in the retina. Methods: EGFP was introduced by either transgenic approaches (human L–cone opsin or beta actin promoter) or viral subretinal injection (AAV.EGFP). EGFP was quantified by scanning frozen sections of retina on a quantitative confocal microscope and using calibrations with recombinant EGFP for conversion to concentration. Eyes were also examined by conventional histology. Results: Transgenic mice expressing EGFP under the human L– cone opsin promoter had normal histology, and their cones contained a spatially averaged EGFP concentration of 4.6µM. Transgenic mice expressing EGFP under the beta actin promoter had normal histology, and the rod photoreceptors contained a spatially averaged EGFP concentration of 1.4µM. Wild–type mice injected with AAV.EGFP in utero had normal histology, and the cone photoreceptors contained a spatially averaged EGFP concentration of 10µM. In contrast, the primate retinas had localized regions of photoreceptor degeneration. In the healthy areas, the RPE cells contained a spatially averaged EGFP concentration of 44µM EGFP. In all cases, the EGFP localization was cytoplasmic and, within the photoreceptors, was distributed from the synaptic terminal through the inner segments. In the case of the transgenic mouse expressing EGFP off of the beta actin promoter, EGFP was also localized to inner retinal neurons. Conclusions: EGFP is not toxic to the mouse retina even at levels of 10µM in photoreceptors. However, the primate retina appears to be sensitive to EGFP.

Keywords: gene transfer/gene therapy • microscopy: confocal/tunneling • retina 
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