Concentration of viral vectors by co-precipitation with calcium phosphate
✍ Scribed by Linh Pham; Hong Ye; Francois-Loic Cosset; Stephen J. Russell; Kah-Whye Peng
- Publisher
- John Wiley and Sons
- Year
- 2001
- Tongue
- English
- Weight
- 161 KB
- Volume
- 3
- Category
- Article
- ISSN
- 1099-498X
No coin nor oath required. For personal study only.
✦ Synopsis
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 60mm calcium chloride, pelleting at 2,000 g, resuspending the pellet in a small volume of 0.1m 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 1,000-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.