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A Comparative Analysis of Novel Fluorescent Proteins as Reporters for Gene Transfer Studies

Peter Bell, Luk H. Vandenberghe, Di Wu, Julie Johnston, Maria Limberis and James M. Wilson

Gene Therapy Program, Division of Medical Genetics, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (PB,LHV,DW,JJ,ML,JMW), and Molecular Medicine, Katholieke Universiteit Leuven, Leuven, Belgium (LHV)

Correspondence to: James M. Wilson, MD, PhD, 125 S. 31st Street, Translational Research Laboratories Suite 2000, Philadelphia, PA 19104-3403. E-mail: wilsonjm{at}mail.med.upenn.edu

We evaluated novel fluorescent proteins (FPs) as reporters for gene transfer in animals and cells with the aim to develop more-sensitive assays for vector development and the optimization of gene transfer strategies in gene therapy. Adeno-associated virus serotype 5 vectors carrying an expression cassette with a chicken ß-actin promoter encoding the green FPs ZsGreen1, AcGFP1, hMGFP (with and without intron), and EGFP and the red FPs DsRed2 and TurboRFP were administered to mice at identical doses for each organ to target liver, lung, and muscle. Despite the fact that all FPs were expressed from an identical vector backbone, the observed number of fluorescent cells and fluorescence intensities varied between, but was consistent within, each combination of a specific protein and organ. The highest number of fluorescent cells was observed in liver with EGFP and in lung with ZsGreen1 and EGFP. In muscle, AcGFP1 and ZsGreen1 produced the most-intense fluorescence in fibers. In contrast, in culture cells, ZsGreen1 showed substantially stronger fluorescence than all other proteins. Our data demonstrate that each FP has tissue-specific expression profiles that need to be taken into consideration when comparing the performance of vectors in different organs. (J Histochem Cytochem 55:931–939, 2007)

Key Words: fluorescent proteins • AAV • gene transfer • liver • lung • muscle

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