September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
RT-qPCR quantification of mRNAs specific of Pigment Epithelium-Derived Factor (PEDF) and Vascular Endothelial Growth Factor (VEGF) in Rat eyes injected in the subretinal space with Primary Cells transfected with hPEDF.
Author Affiliations & Notes
  • Sergio Recalde
    Experimental Ophthalmology Laboratory, Clinica Universidad de Navarra, PAMPLONA, Navarra, Spain
    IdiSNA, Navarra Institute for Health Research, Pamplona, Navarra, Spain
  • Laurence Jeanson-LEH
    Genosafe, Evry, France
  • Patricia Fernandez
    Experimental Ophthalmology Laboratory, Clinica Universidad de Navarra, PAMPLONA, Navarra, Spain
    IdiSNA, Navarra Institute for Health Research, Pamplona, Navarra, Spain
  • Maria Hernandez
    Experimental Ophthalmology Laboratory, Clinica Universidad de Navarra, PAMPLONA, Navarra, Spain
    IdiSNA, Navarra Institute for Health Research, Pamplona, Navarra, Spain
  • Laura Garcia-Garcia
    Experimental Ophthalmology Laboratory, Clinica Universidad de Navarra, PAMPLONA, Navarra, Spain
    IdiSNA, Navarra Institute for Health Research, Pamplona, Navarra, Spain
  • Jaione Bezunartea-Bezunartea
    Experimental Ophthalmology Laboratory, Clinica Universidad de Navarra, PAMPLONA, Navarra, Spain
    IdiSNA, Navarra Institute for Health Research, Pamplona, Navarra, Spain
  • Daniel Scherman
    3Unité de Technologies Chimiques et Biologiques pour la Santé, INSERM U1022 – CNRS UMR8258, Paris, France
  • Séverine Pouillot
    Genosafe, Evry, France
  • Gabriele Thumann
    Département des neurosciences cliniques, Service d’ophtalmologie, Hôpitaux universitaires de Genève, Geneve, Switzerland
  • Alfredo Garcia-Layana
    Experimental Ophthalmology Laboratory, Clinica Universidad de Navarra, PAMPLONA, Navarra, Spain
    IdiSNA, Navarra Institute for Health Research, Pamplona, Navarra, Spain
  • Footnotes
    Commercial Relationships   Sergio Recalde, None; Laurence Jeanson-LEH, Genosafe (E); Patricia Fernandez, None; Maria Hernandez, None; Laura Garcia-Garcia, None; Jaione Bezunartea-Bezunartea, None; Daniel Scherman, None; Séverine Pouillot, Genosafe (E); Gabriele Thumann, None; Alfredo Garcia-Layana, Alcon (C), Allergan (C), Bayer (C), Novartis (C), Thea (C)
  • Footnotes
    Support  FP7 HEALTH 2012-305134. LGG received ADA predoctoral grant from University of Navarra
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 281. doi:
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      Sergio Recalde, Laurence Jeanson-LEH, Patricia Fernandez, Maria Hernandez, Laura Garcia-Garcia, Jaione Bezunartea-Bezunartea, Daniel Scherman, Séverine Pouillot, Gabriele Thumann, Alfredo Garcia-Layana; RT-qPCR quantification of mRNAs specific of Pigment Epithelium-Derived Factor (PEDF) and Vascular Endothelial Growth Factor (VEGF) in Rat eyes injected in the subretinal space with Primary Cells transfected with hPEDF.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):281.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : The aim of this study was to quantify human and rat PEDF, rat VEGF mRNAs in a model of laser-induced choroidal neovascularization (CNV) transfected with SB100X transposase with PEDF transposon encoding pFAR4 plasmids using real-time quantitative RT-PCR (RT-qPCR).

Methods : Retinal pigment epithelia cells (RPEs) and iris pigment epithelia cells (IPEs) were transfected with pFAR4-pigment epithelium-derived factor (PEDF) plasmid using the non-viral Sleeping Beauty (SB100X) transposon system (pFAR4-ITRs CMV PEDF BGH-pFAR4-CMV SB100x SV40) in the subretinal space of Brown Norway rats. The treatment groups were as follows: not injected, injected with PBS, injected with the Venus plasmid (GFP), injected with the human PEDF plasmid or injected with the human PEDF plasmid + the SB plasmid. Total RNA was extracted from the injected eyes and purified for reverse transcription. Human PEDF, rat VEGF and rat PEDF mRNAs were quantified in eyes of rats using real-time quantitative RTqPCR. Relative quantity from each RNA was calculated for human and rat PEDF or rVEGF Ct normalized to Ct of RPL30 and have been determined with the Relative Quantification software from Applied Biosystems v1.2 after analysis of each qPCR plate with the SDS v2.3 software.

Results : There were no significant differences in the mRNA expression in rat VEGF and PEDF for any group analyzed. Human PEDF was detected only in eyes injected with the hPEDF plasmid, with or without SB.

Conclusions : Human PEDF was detected in rat eyes injected with the hPEDF plasmid demonstrating the efficacy of this SB system to release the PEDF. This system could be useful as an antiangiogenic therapy for retinal diseases.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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