Viral Gene Delivery: Optimized Protocol for Production of High Titer Lentiviral Vectors
HIV-derived lentiviral vectors (LVV) are among the most commonly used gene delivery vehicles. Their production in high quantities, which enables concentration of viral particles to high titers, is important for their successful application in both biomedi
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Introduction Lentiviral vectors (LVV) are widely used for gene delivery in vitro and in vivo. They are non-immunogenic, fast to produce and offer a significant packaging capacity for complex expression cassettes (1). The most commonly used LVV are derived from HIV, and pseudotyped with the VSV-G protein from vesicular stomatitis virus. In the brain LVV can help to achieve high levels of transgene expression
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_5, © Springer Science+Business Media, LLC 2013
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making them suitable for experiments where this feature is critical. Recent introduction of optogenetic actuator proteins, such as Channelrhodopsin-2 (Chr2), which allow control of neuronal activity and signalling with light (2–6), has made this feature particularly important because these proteins need to be present in cells at fairly high levels. The titer of the vector has a direct impact on the level of expression and therefore the protocol used for LVV production is decisive for the success of many applications. Presently, LVV are prepared by co-transfection of three or four separate plasmids into a packaging cell line (7–9). In combination the plasmids encode the proteins sufficient to generate replicationdeficient, self-inactivating LVV particles (individually the plasmids do not encode the necessary proteins to generate a functional virus). Functional LVV are capable of transducing mammalian cells delivering genetic material for the expression of genes of interest. The packaging cells are induced to produce viral vector particles for a period of time, followed by several steps of vector concentration. Concentration in most cases is achieved using ultracentrifugation although affinity purification methods are also available. If the initial production step is not efficient the only way to compensate for that is to start with large quantities of packaging cells and media and then to concentrate the diluted LVV using complex purification protocols (7, 8). Approaches adopted by different laboratories for production as well as titration of LVV vary significantly. Since multiple parameters are usually different and not all relevant information is presented, it is difficult to directly compare the efficiency of published protocols. We sought to optimize LVV production, focusing on the stage of transfection of the packaging cell line. The absolute majority of studies currently use calcium phosphate (CaPO4) precipitation (7, 8) to introduce plasmids into packaging cell lines. Here we evaluate other transfection reagents and present the optimized protocol. In order to compare various methods and directly titer the purified LVV we used a construct, LVV EF1αPLAP, which encodes placental alkaline phosphatase (PLAP), under control of the human elongation factor-1α promoter (EF1α) as a model (9). When PLAP is expressed in LVV-transduced cells its enzymatic activity can be visualized by a simple and highly sensitive
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