Concentration of viral vectors by co-precipitation with calcium phosphate

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 inf ectivity. This study describes a gentle and simple method to concentrate ML V 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 su pernatant with calcium phosphate, low-speed centrifugation, dialysis, and i nfection assays with Lac-Z transducing vectors. Results Murine leukemia virus vectors and HIV vectors pseudotyped with vesi cular stomatitis virus glycoprotein (VSV.G) or MLV envelopes were concentra ted successfully using the calcium phosphate co-precipitation method. Param eters 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 dialysi s against saline to remove EDTA. Volumes were decreased from 300 mi to 10 m i, with 50-100% recovery, and titers can be concentrated up to 1000-fold. Conclusions The calcium phosphate co-precipitation method to concentrate vi rus 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 (C) 2001 John Wile y & Sons, Ltd.