title: Preparation of vesicular stomatitis virus-G (VSV-G) conjugate and its use in gene transfer.
abstract: The fusiogenic envelope G glycoprotein of the vesicular stomatitis virus (VSV-G) that has been used to pseudotype retrovirus and lentivirus vectors can be used alone as an efficient vehicle for gene transfer. VSV-G protein is secreted into the culture medium as sendimentable vesicles from cells transfected with a VSV-G expression plasmid in the absence of other viral components. The VSV-G vesicles in the conditioned medium can be partially purified by pelleting through sucrose cushion ultracentrifugation. Protein-DNA complexes are formed by mixing the VSV-G vesicles with naked plasmid DNA. Such complexes show markedly enhanced transfection efficiency when added to the culture medium of recipient cells. The cell tropism of VSV-G-DNA complex-mediated gene transfer resembles that of VSV-G-pseudotyped retrovirus and lentivirus vectors, and the complex is therefore particularly useful for transfection of cells that are refractory to other methods. Still, some cells are refractory to VSV-G-mediated transfection. It should also be noted that overdose of VSV-G can be quite toxic to the recipient cells. The primitive complexes formed by mixing a viral fusiogenic envelope protein with naked DNA may represent a step toward fusing useful features of viral and nonviral vectors for safer and more efficient gene transfer. This protocol describes simple methods for preparation of VSV-G and for gene transfer with DNA-VSV-G complexes.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyanohara
mesh_terms: D002460:Cell Line; D003470:Culture Media / Q000737:chemistry; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D014162:Transfection / Q000379:methods*; D018116:Vesiculovirus / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D003470:Culture Media; D004268:DNA-Binding Proteins; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D004247:DNA