title: Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation.
abstract: BACKGROUND
  Viral vectors provide a method of stably introducing exogenous DNA into cells that are not easily transfectable allowing for the ectopic expression or silencing of genes for therapeutic or experimental purposes. However, some cell types, in particular bone marrow cells, dendritic cells and neurons are difficult to transduce with viral vectors. Successful transduction of such cells requires preparation of highly concentrated viral stocks, which permit a high virus concentration and multiplicity of infection (MOI) during transduction. Pseudotyping with the vesicular stomatitis virus G (VSV-G) envelope protein is common practice for both lentiviral and retroviral vectors. The VSV-G glycoprotein adds physical stability to retroviral particles, allowing concentration of virus by high-speed ultracentrifugation. Here we describe a method report for concentration of virus from large volumes of culture supernatant by means of successive rounds of ultracentrifugation into the same ultracentrifuge tube.
  
  
  METHODS
  Stable retrovirus producer cell lines were generated and large volumes of virus-containing supernatant were produced. We then tested the transduction ability of virus following varying rounds of concentration by ultra-centrifugation. In a second series of experiments lentivirus-containing supernatant was produced by transient transfection of 297T/17 cells and again we tested the transduction ability of virus following multiple rounds of ultra-centrifugation.
  
  
  RESULTS
  We report being able to centrifuge VSV-G coated retrovirus for as many as four rounds of ultracentrifugation while observing an additive increase in viral titer. Even after four rounds of ultracentrifugation we did not reach a plateau in viral titer relative to viral supernatant concentrated to indicate that we had reached the maximum tolerated centrifugation time, implying that it may be possible to centrifuge VSV-G coated retrovirus even further should it be necessary to achieve yet higher titers for specific applications. We further report that VSV-G coated lentiviral particles may also be concentrated by successive rounds of ultracentrifugation (in this case four rounds) with minimal loss of transduction efficiency.
  
  
  CONCLUSIONS
  This method of concentrating virus has allowed us to generate virus of sufficient titers to transduce bone marrow cells with both retrovirus and lentivirus, including virus carrying shRNA constructs.
authors.affiliation: Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 2M9, Canada.
authors.forename: Christine V
authors.initials: CV
authors.lastname: Ichim
authors.forename: Richard A
authors.initials: RA
authors.lastname: Wells
mesh_terms: D000818:Animals; D002460:Cell Line; D006801:Humans; D016086:Lentivirus / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D012190:Retroviridae / Q000378:metabolism*; D014161:Transduction, Genetic; D014461:Ultracentrifugation / Q000379:methods*; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism