May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
RNAi–Mediated Suppression of RDS/Peripherin and Rhodopsin in Photoreceptor Cells
Author Affiliations & Notes
  • M. Ader
    Genetics, Trinity College Dublin, Dublin, Ireland
  • A. Palfi
    Genetics, Trinity College Dublin, Dublin, Ireland
  • A.–S. Kiang
    Genetics, Trinity College Dublin, Dublin, Ireland
  • P. Humphries
    Genetics, Trinity College Dublin, Dublin, Ireland
  • J. Farrar
    Genetics, Trinity College Dublin, Dublin, Ireland
  • Footnotes
    Commercial Relationships  M. Ader, None; A. Palfi, None; A. Kiang, None; P. Humphries, None; J. Farrar, None.
  • Footnotes
    Support  Fighting Blindness Ireland
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 5203. doi:
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      M. Ader, A. Palfi, A.–S. Kiang, P. Humphries, J. Farrar; RNAi–Mediated Suppression of RDS/Peripherin and Rhodopsin in Photoreceptor Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5203.

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

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Abstract

Abstract: : Purpose: RNAi is a promising technology to silence the expression of genes of interest in specific tissues and may also be applied to suppress disease genes in human gene therapy approaches. To test the feasibility of RNAi in photorecepor cells, small hairpin RNAs (shRNAs) were used to knock–down mouse rds/peripherin (rds) and rhodopsin (rho) expression. Rds and rho are photoreceptor–specific genes in which a cumulative number of some 150 (mostly single base–) mutations have been described to be responsible for various forms of retinitis pigmentosa (RP), a hereditary and progressive retinal degeneration in humans. Methods: Potential shRNA sequences targeting rds or rho were tested and selected in COS7 cells. The most efficient sequences targeting each rds or rho, as well as a non–targeting construct as a negative control, were tagged with an enhanced green fluorescent protein (EGFP) expression cassette driven by a CMV promoter. One day–old mouse retinas were in vitro electroporated with the shRNA vectors and analysed after 14 days. Results were evaluated by immunofluorescence microscopy and RT–PCR. To extract RNA, transduced retinas were dissociated and EGFP–positive cells isolated by FACS sorting. Results: Our results indicate that the endogenously expressed shRNAs targeting rds or rho decreased the level of their corresponding mRNAs up to 80% when compared to non–targeting control shRNA. Furthermore, immunocytochemical analysis of rho–targeted photoreceptor cells revealed a strong decrease in the number of rhodopsin expressing cells at protein level. Conclusions: RNAi technology can significantly silence mouse rds and rho expression in photoreceptor cells and might therefore be a promising tool (i) to study development and function of photoreceptor cells, and (ii) for the development of therapeutic approaches targeting inherited diseases involving these cells. RNAi might be especially useful to knock–down mutant disease genes with dominant negative effects in a mutation–independent suppression and replacement strategy.

Keywords: gene transfer/gene therapy • retinal culture • immunohistochemistry 
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