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Concentration of viral vectors by co‐precipitation with calcium phosphate
Author(s) -
Pham Linh,
Ye Hong,
Cosset FrancoisLoic,
Russell Stephen J.,
Peng KahWhye
Publication year - 2001
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/1521-2254(2000)9999:9999<::aid-jgm159>3.0.co;2-9
Subject(s) - centrifugation , vesicular stomatitis virus , calcium , titer , chemistry , chromatography , virus , retrovirus , virology , precipitation , murine leukemia virus , in vivo , infectivity , biology , physics , microbiology and biotechnology , organic chemistry , meteorology
Background 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. Methods 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. Results 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 60 mM calcium chloride, pelleting at 2000 g , resuspending the pellet in a small volume of 0.1 M EDTA‐saline, and dialysis against saline to remove EDTA. Volumes were decreased from 300 ml to 10 ml, with 50–100% recovery, and titers can be concentrated up to 1000‐fold. Conclusions The calcium phosphate co‐precipitation method to concentrate virus is applicable to retrovirus and lentivirus preparations. It uses simple techniques and does not require expensive equipment. Multiple rounds of co‐precipitation can be carried out if required. Copyright © 2001 John Wiley & Sons, Ltd.