Rapid concentration and purification of retrovirus by flocculation with Polybrene.
We have previously shown that the combined addition of Polybrene (PB) and chondroitin sulfate C (CSC) to retrovirus stocks leads to the formation of retrovirus-polymer complexes (i.e., flocs) that rapidly sediment onto cells, increases the efficiency of gene transfer, and can be used to rapidly concentrate and purify retrovirus stocks. The viruses remain associated with the polyelectrolyte complexes, however, which may complicate their use in downstream applications. In this study we determined if retrovirus could be flocculated using only one polymer (PB). We found that when retrovirus stocks were incubated with 320 microg/ml of PB, more than 70% of the viruses, and fewer than 0.3% of all other proteins, were pelleted by low-speed centrifugation. In contrast to retrovirus complexes formed with two polymers, retrovirus flocculated with PB disaggregated when they were resuspended in fresh medium. We conclude that flocculation of retroviruses with a single cationic polymer (PB) is a useful method for rapidly concentrating and purifying retroviruses, and may prove particularly useful when it is desirable to generate purified virus that is not part of a polymer complex.
Landázuri
Natalia
N
The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Emory University, Atlanta, GA 30322, USA.
Gupta
Monique
M
Le Doux
Joseph M
JM
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D005427:Flocculation; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000378:metabolism; D006583:Hexadimethrine Bromide / Q000494:pharmacology* / Q000633:toxicity; D051379:Mice; D041681:NIH 3T3 Cells; D012190:Retroviridae / Q000187:drug effects* / Q000302:isolation & purification*; D014161:Transduction, Genetic / Q000379:methods; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D006583:Hexadimethrine Bromide; D001616:beta-Galactosidase