Abstract
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 2000g, 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.
Original language | English (US) |
---|---|
Pages (from-to) | 188-194 |
Number of pages | 7 |
Journal | Journal of Gene Medicine |
Volume | 3 |
Issue number | 2 |
DOIs | |
State | Published - Mar 2001 |
Keywords
- Co-precipitation
- Concentration
- Inexpensive production
- MLV pseudotyped lentivirus
- Retrovirus
ASJC Scopus subject areas
- Molecular Medicine
- Molecular Biology
- Genetics
- Drug Discovery
- Genetics(clinical)