May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Tetracycline-Inducible Expression of Mutant (P347S) Human Rhodopsin in Mice
R.C. Strom; A. Hackam; D.J. Zack; R. Adler
Author Affiliations & Notes
  • R.C. Strom
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, United States
  • A. Hackam
    Wilmer Eye Institute, Johns Hopkins University at The Guerrieri Center for Genetic Engineering and Molecular Ophthalmology, Baltimore, MD, United States
  • D.J. Zack
    Wilmer Eye Institute, Johns Hopkins University at The Guerrieri Center for Genetic Engineering and Molecular Ophthalmology, Baltimore, MD, United States
  • R. Adler
    Wilmer Eye Institute, Johns Hopkins University at The Guerrieri Center for Genetic Engineering and Molecular Ophthalmology, Baltimore, MD, United States
  • Footnotes
    Commercial Relationships  R.C. Strom, None; A. Hackam, None; D.J. Zack, None; R. Adler, None.
  • Footnotes
    Support  NIH core grant EY1765; Research to Prevent Blindness; (Hackam) Canadian Instit. of Health
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 2823. doi:
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      R.C. Strom, A. Hackam, D.J. Zack, R. Adler; Tetracycline-Inducible Expression of Mutant (P347S) Human Rhodopsin in Mice . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2823.

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Abstract

Abstract: : Purpose: In retinitis pigmentosa (RP) and its animal models, gene mutations expressed in rods lead not only to rod degeneration, but also to non-cell autonomous secondary cone death. The underlying mechanisms, however, remain unknown. The goal of this study was to develop an RP animal model, in which the timing and expression level of a rod mutation can be experimentally controlled in order to facilitate the analysis of the sequence of events leading to cone loss. To accomplish this goal we have developed tetracycline-inducible transgenic mice expressing the dominant rhodopsin mutation P347S, which causes a fairly severe form of RP. Methods:The transgene construct contains human rhodopsin-P347S with tetracycline response elements (TRE) upstream and enhanced green fluorescent protein (EGFP) downstream, separated by an IRES, for reporter gene expression. Photoreceptors expressing the rhodopsin transgene should therefore also be GFP positive. Constructs were injected into B6SJLF2 embryos and the resulting pups were screened by PCR for transgene integration. Transgenic founders were crossed with transgenic mice expressing the tetracycline-dependent transactivator factor (rtTA) under the control of rod photoreceptor-specific bovine opsin promoter (Chang et al., 2000). Four mg/ml doxycycline was added to the animals’ drinking water to induce transgene expression. Results: The expression of the human rhodopsin transgene in photoreceptors was observed at the mRNA level by RT-PCR and real-time PCR. The expression of transgene protein was confirmed by GFP fluorescence in photoreceptors and by labeling with anti-human-specific rhodopsin antibody. The effects of mutant rhodopsin expression on photoreceptor survival are currently being investigated. Conclusions: We have developed a tetracycline-inducible mutant rhodopsin mouse and have begun to characterize the expression parameters. With the ability to regulate the timing and level of mutant gene expression we can better study the sequence of cellular and molecular events causing non-cell autonomous degeneration.

Keywords: retinal degenerations: cell biology • transgenics/knock-outs • cell death/apoptosis 
© 2003, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Permission to republish any abstract or part of an abstract in any form must be obtained in writing from the ARVO Office prior to publication.
Copyright © 2015 Association for Research in Vision and Ophthalmology.
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