First Author: M.Rudolf GERMANY
Co Author(s): M. Mandl Z. Aherrahrou M. Ranjbar S. Grisanti A. Mohi
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Several retinal diseases are marked by chronically increased local inflammation in which the retinal pigment epithelium (RPE) is a main responder. To make stressed RPE cells more resistant to local inflammation we want to transfect them with minicircles, which are small circular plasmid derivates free of all prokaryotic DNA elements. Encoded is an 18 amino acids long peptide which is able to scavenge pro-inflammatory oxidized phospholipids thereby regulating RPE environment. Here we present the RPE-transfection with minicircles and peptide detection.
In this study we use an in vitro RPE cell culture model in order to determine the molecular mechanisms of the peptide.
Primary human RPE- cells and ARPE-19 cells were cultured and were transfected with minicircles. Our first minicircle (MC1) encodes for the active peptide as well for green fluorescent protein (GFP). Both sequences are separated by an internal ribosome entry site (IRES) and translated independently from the same mRNA. Our control minicircle (MC2) encodes no peptide but only a red fluorescent protein (RFP) to monitor transfection efficiency. Finally, GFP and RFP expression is analyzed by fluorescence microscopy.
Both GFP and RFP are already detectable in RPE cells after 24h by fluorescence microscopy after MC transfer. The signal rose after 48h. Because GFP and the active scavenging peptide are transcribed on the same mRNA but translated as two independent molecules, the fluorescent marker is an indicator for expression efficiency.
The presented results show the feasibility to transfect human RPE cells with minicircle plasmids. A polyclonal antibody raised against the peptide will be used in future experiments for direct detection. In order to complement this study a very sensitive approach using mass spectrometry is planned.