Abstract
BackgroundLentiviral vectors (LVs) can efficiently transduce a broad spectrum of cells and tissues, including dividing and non-dividing cells. So far the most widely used method for concentration of lentiviral particles is ultracentrifugation (UC).An important feature of vectors derived from lentiviruses and prototypic gamma-retroviruses is that the host range can be altered by pseudotypisation. The most commonly used envelope protein for pseudotyping is the glycoprotein of the Vesicular Stomatitis Virus (VSV.G), which is also essential for successful concentration using UC.ResultsHere, we describe a purification method that is based on membrane adsorbers (MAs). Viral particles are efficiently retained by the anionic exchange MAs and can be eluted with a high-salt buffer. Buffer exchange and concentration is then performed by utilizing ultrafiltration (UF) units of distinct molecular weight cut off (MWCO). With this combined approach similar biological titers as UC can be achieved (2 to 5 × 109 infectious particles (IP)/ml). Lentiviral particles from small starting volumes (e.g. 40 ml) as well as large volumes (up to 1,000 ml) cell culture supernatant (SN) can be purified. Apart from LVs, vectors derived from oncoretroviruses can be efficiently concentrated as well. Importantly, the use of the system is not confined to VSV.G pseudotyped lenti- and retroviral particles and other pseudotypes can also be purified.ConclusionsTaken together the method presented here offers an efficient alternative for the concentration of lenti- as well as retroviral vectors with different pseudotypes that needs no expensive equipment, is easy to handle and can be used to purify large quantities of viral vectors within a short time.
Highlights
Lentiviral vectors (LVs) can efficiently transduce a broad spectrum of cells and tissues, including dividing and non-dividing cells
Purification of lentiviral particles by using membrane adsorbers For purification of LVs, we initially focused on VSV.G pseudotyped lentiviral particles, because they are the most widely applied type of lentivectors
After loading of the lentiviral particles to the membrane adsorbers (MAs) and washing, retained viral particles were eluted with high salt elution buffer (1 M sodium chloride)
Summary
Lentiviral vectors (LVs) can efficiently transduce a broad spectrum of cells and tissues, including dividing and non-dividing cells. An important feature of vectors derived from lentiviruses and prototypic gamma-retroviruses is that the host range can be altered by pseudotypisation. Lentiviral vectors (LVs) are versatile tools for molecular medicine and gene therapy [1]. They are able to integrate their viral genome into both dividing and nondividing cells [2,3]. Retroviruses are besides adenoviruses - the most widely used vectors in range by enabling them to enter the cells via receptor mediated endocytosis [12]. VSV.G-pseudotyped viral vectors were shown to be more stable and can be concentrated by ultracentrifugation (UC) without a significant loss in titer [13,14]
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