May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
High Efficiency Non-Viral Transfection of IPE and RPE Cells by Electroporation
Author Affiliations & Notes
  • R. Lindt
    Department of Ophthalmology, RWTH Aachen University, Aachen, Germany
  • S. Johnen
    Department of Ophthalmology, RWTH Aachen University, Aachen, Germany
  • S. Kaempf
    Department of Ophthalmology, RWTH Aachen University, Aachen, Germany
  • A. K. Salz
    Department of Ophthalmology, RWTH Aachen University, Aachen, Germany
  • P. Walter
    Department of Ophthalmology, RWTH Aachen University, Aachen, Germany
  • G. Thumann
    Department of Ophthalmology, RWTH Aachen University, Aachen, Germany
  • Footnotes
    Commercial Relationships  R. Lindt, None; S. Johnen, None; S. Kaempf, None; A.K. Salz, None; P. Walter, None; G. Thumann, None.
  • Footnotes
    Support  IZKF "BIOMAT."
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 3870. doi:https://doi.org/
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      R. Lindt, S. Johnen, S. Kaempf, A. K. Salz, P. Walter, G. Thumann; High Efficiency Non-Viral Transfection of IPE and RPE Cells by Electroporation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3870. doi: https://doi.org/.

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

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Abstract

Purpose: : Age-related macular degeneration is one of the most common causes for blindness of the elderly. The course of the disease can be affected positively by neurotrophic factors (e.g. PEDF) released by IPE cells that have been transplanted into the subretinal space where they have been shown to survive for at least 3 years. We therefore established methods for high efficiency transfection of pigment epithelial cells using electroporation.

Methods: : Electroporation using the Nucleofector system by Amaxa Biosystems and four different lipofection systems (LipofectamineTM 2000, LipofectamineTM LTX, MetafecteneTM and MetafecteneTM Pro) were used for transfection of ARPE-19 cells, freshly isolated and primary cultures of bovine and porcine IPE and RPE cells with DNA of green fluorescent protein (GFP). Transfection efficiencies and the progression of GFP protein expression were evaluated by fluorescence microscopy.

Results: : Transfection of ARPE-19 cells and primary cultured IPE and RPE cells by electroporation was highly efficient. Comparison of transfection efficiency showed that whereas electroporation routinely resulted in transfection efficiencies of about 80%, by lipofection showed transfection efficiencies below 5%. Approximately 100 days after transfection by electroporation IPE and RPE cells still showed good morphological characteristics and still expressed GFP protein albeit at a reduced level.

Conclusions: : Transfection by electroporation was highly efficient for pigment epithelial cells with efficiencies in the range of 80%. In addition expression of the transfected gene was expressed for over 3 months.

Keywords: age-related macular degeneration • gene transfer/gene therapy • retinal pigment epithelium 
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