Tuyển tập báo cáo các nghiên cứu khoa học quốc tế ngành hóa học dành cho các bạn yêu hóa học tham khảo đề tài: Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation | Ichim and Wells Journal of Translational Medicine 2011 9 137 http content 9 1 137 JOURNAL OF TRANSLATIONAL MEDICINE METHODOLOGY Open Access Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation Christine V Ichim1 2 and Richard A Wells1 2 3 4 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. Method 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 ultracentrifugation. 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 .
