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1 Department of Neuroscience, University of Florida McKnight Brain Institute and College of Medicine
2 Department of Physiology and Functional Genomics, University of Florida McKnight Brain Institute and College of Medicine
3 Department of Pharmacodynamics, University of Florida College of Pharmacy, Gainesville, Florida 32610
4 Department of Physiology, University of Bristol School of Medical Sciences, Bristol BS8 1TD, United Kingdom
The aim of this study was to develop an efficient method for packaging and concentrating lentiviral vectors that consistently yields high-titer virus on a scale suitable for in vivo applications. Transient cotransfection of 293T packaging cells with DNA plasmids encoding lentiviral vector components was optimized using SuperFect, an activated dendrimer-based transfection reagent. The use of SuperFect allowed reproducible and efficient production of high-titer lentiviral vector at concentrations greater than 1 x 107 transducing units per ml (TU/ml) and required less than one-third of the total amount of DNA used in traditional calcium phosphate transfection methods. Viral titers were further increased using a novel concentration protocol that yielded an average final titer of 1.4 x 1010 TU/ml. Lentiviruses produced using these methods exhibited efficient transduction of central nervous system and peripheral tissues in vivo. The method is reproducible and can be scaled up to facilitate the use of these vectors in animal studies.
cardiomyocytes; in vivo gene delivery; neurons; retina; transfection
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