Concentration of viral vectors by co-precipitation with calcium phosphate

The Journal of Gene Medicine
L PhamK W Peng

Abstract

The envelope glycoproteins, surface unit (SU) and transmembrane (TM) of the murine leukemia virus (MLV) are not covalently linked and tend to dissociate upon high-speed centrifugation, leading to loss of vector infectivity. This study describes a gentle and simple method to concentrate MLV vectors or HIV vectors pseudotyped with MLV envelopes. Having a fast and inexpensive method to concentrate large volumes of vector supernatant will facilitate in vivo experiments and clinical trials that require high titer vector stocks. The methods employed in the study were co-precipitation of viral supernatant with calcium phosphate, low-speed centrifugation, dialysis, and infection assays with Lac-Z transducing vectors. Murine leukemia virus vectors and HIV vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV.G) or MLV envelopes were concentrated successfully using the calcium phosphate co-precipitation method. Parameters that influence virus yield and the reproducibility of the method were investigated. The optimized protocol involves virus harvest in serum-free media, co-precipitation using 60mM calcium chloride, pelleting at 2,000 g, resuspending the pellet in a small volume of 0.1M EDTA-saline, and dialysis against sa...Continue Reading

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Citations

Mar 7, 2014·Cytotechnology·Masoud NasriMehdi Allahbakhshian Farsani
Jul 6, 2010·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Rongjun ChenNigel K H Slater
Nov 16, 2004·Journal of Virological Methods·Miguel Sena-EstevesAlan W Flake
Sep 15, 2006·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·María de las Mercedes SeguraAlain Garnier
Feb 2, 2006·Biotechnology Advances·María de Las Mercedes SeguraAlain Garnier
Mar 18, 2015·Biotechnology Journal·Nikolaus HammerschmidtAlois Jungbauer
Apr 18, 2012·The Biochemical Journal·Toshie SakumaYasuhiro Ikeda
Nov 22, 2005·Molecular Therapy : the Journal of the American Society of Gene Therapy·Darren NesbethDavid Darling
Sep 8, 2001·Molecular Therapy : the Journal of the American Society of Gene Therapy·S Thaler, B S Schnierle
Aug 13, 2013·International Journal of Molecular Medicine·Yun ZhouZhansheng Jia
Jul 17, 2018·Journal of Chemical Technology and Biotechnology·Daniel BurgstallerPeter Satzer
Mar 7, 2020·Journal of Chemical Information and Modeling·Hristina R ZhekovaSergei Noskov

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