May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Lentivirus-Mediated Gene Transfer in Retinal Stem Cells - Effects of Long-Term Expression
Author Affiliations & Notes
  • W. Li
    Ophthalmology, University of Miami Miller School of Medicine, Miami, Florida
  • B. Janic
    Ophthalmology, University of Miami Miller School of Medicine, Miami, Florida
  • X. Zhang
    Ophthalmology, University of Miami Miller School of Medicine, Miami, Florida
  • Footnotes
    Commercial Relationships W. Li, None; B. Janic, None; X. Zhang, None.
  • Footnotes
    Support NIH R01EY016211, P30EY014801 and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 4090. doi:
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      W. Li, B. Janic, X. Zhang; Lentivirus-Mediated Gene Transfer in Retinal Stem Cells - Effects of Long-Term Expression. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4090.

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

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Abstract

Purpose:: Retinal stem cells (RSCs)/progenitors are critical for the development of therapeutic approaches for degenerative retinal diseases. Lentivirus-mediated gene transfer is an important strategy for RSC ex vivo modulation. This study is to investigate long-term gene expression mediated by lentiviral vector in RSCs.

Methods:: RSCs were isolated from mouse retina at postnatal day 1 and cultured in serum-free culture medium supplemented with EGF and FGF. Retinal spheres were transduced with GFP-expressing feline immunodeficiency virus (FIV). GFP expression was analyzed by flow cytometry and fluorescent microscope. RSCs were differentiated in mitogen-free culture medium supplemented with 5% FBS. Expression of cell-specific markers was analyzed by immunocytochemistry in both undifferentiated and differentiated RSCs. Differentiated postmitotic cells were revealed by BrdU incorporation assay.

Results:: Stem cell marker nestin was detected in the cultured retinal spheres. FIV transduced more than 97% of retinal spheres with sustainable GFP expression for at least two months in culture. Upon differentiation, cell-specific markers, including MAP-2, opsin and GFAP, were detected in FIV-transduced or control RSCs at similar levels, suggesting that FIV transduction did not affect cell differentiation. However, GFP expression substantially decreased in BrdU-negative differentiated cells, suggesting the downregulation of the transgene expression in differentiated postmitotic cells.

Conclusions:: FIV-mediated gene transfer induces sustainable gene expression in undifferentiated RSCs. However, the transgene expression is down-regulated in differentiated RSCs.

Keywords: regeneration • gene transfer/gene therapy • differentiation 
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