Single-step cloning-screening method: a new tool for developing and studying high-titer viral vector producer cells.
This article describes a novel method merging the cloning of viral vector producer cells with vector titer screening, allowing for screening 200-500 clones in 2 weeks. It makes use of a GFP separated into two fragments, S10 and S11 (Split GFP), fluorescing only upon transcomplementation. Producer cells carrying a S11 viral transgene are cloned in 96-well plates and co-cultured with target cells stably expressing S10. During the period of clone expansion, S11 viruses infect S10 target cells reconstituting the GFP signal. Transcomplemented fluorescence data provide direct estimation of the clone's productivity and can be analyzed in terms of density distribution, offering valuable information on the average productivity of the cell population and allowing the identification of high-producing clones. The method was validated by establishing a retrovirus producer from a nude cell line, in <3 months, inserting three vector constructs without clone selection or screening in between. Clones producing up to 10(8) infectious particles per ml were obtained, delivering optimal ratios of infectious-to-total particles (1 to 5). The method was additionally used to evaluate the production performance of HEK 293 and HEK 293T cell lines demonstrating that the latter sustains increased titers. Finally, it was used to study genetic manipulation of glutathione metabolism in retrovirus production showing that changing cell metabolism steers higher vector expression with titer increases of more than one order of magnitude.This method is a valuable tool not only for cell line development but also for genetic manipulation of viral vector and/or producer cells contributing to advancing the field of viral gene therapy.
Instituto de Biologia Experimental e Tecnológica (iBET), Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
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Rodrigues
Instituto de Biologia Experimental e Tecnológica (iBET), Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
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Formas-Oliveira
Instituto de Biologia Experimental e Tecnológica (iBET), Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
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Guerreiro
Instituto de Biologia Experimental e Tecnológica (iBET), Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
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Tomás
Instituto de Biologia Experimental e Tecnológica (iBET), Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
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Alves
Instituto de Biologia Experimental e Tecnológica (iBET), Oeiras, Portugal.|Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
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Coroadinha
D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D005820:Genetic Testing / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic