Highly Efficient Concentration of Lenti- and Retroviral Vector Preparations by Membrane Adsorbers and Ultrafiltration
- نوع فایل : کتاب
- زبان : انگلیسی
- مؤلف : Katrin Zimmermann (Katrin.Zimmermann@uni-bonn.de) Oliver Scheibe (Oliver.Scheibe@Sartorius-Stedim.com) Andreas Kocourek (Andreas.Kocourek@Sartorius-
- چاپ و سال / کشور: 2011
Description
Background Lentiviral 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 gammaretroviruses 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. Results Here, 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 5x109 infectious particles (IP)/ml). Lentiviral particles from small starting volumes (e.g. 40ml) as well as large volumes (up to 1,000ml) 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. Conclusions Taken together the method presented here offers an efficient alternative for the concentration of lenti- as well as retroviral vectors with different pseudotypes thatneeds no expensive equipment, is easy to handle and can be used to purify large quantities of viral vectors within a short time.
BMC Biotechnology 2011, 11:55 doi:10.1186/1472-6750-11-55 ISSN 1472-6750 Article type Methodology article Submission date 22 December 2010 Acceptance date 20 May 2011 Publication date 20 May 2011
