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 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.
Pham
L
L
Molecular Medicine Program, Mayo Foundation, Rochester, MN 55905, USA.
Ye
H
H
Cosset
F L
FL
Russell
S J
SJ
Peng
K W
KW
D000818:Animals; D002130:Calcium Phosphates / Q000737:chemistry*; D002460:Cell Line; D005822:Genetic Vectors / Q000648:ultrastructure; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D011233:Precipitin Tests; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic
D002130:Calcium Phosphates; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous