Production of high-titer helper-free retroviruses by transient transfection.
The generation of high-titer, helper-free retroviruses by transient transfection has been achieved by using the highly transfectable 293T cell line into which are stably introduced constructs that express retroviral packaging functions. The resulting ecotropic virus packaging cell line BOSC 23 produces infectious retrovirus at > 10(6) infectious units/ml of supernatant within 72 hr after CaPO4-mediated transfection. A stringent assay for replication-competent virus showed that no helper virus was present. The system can produce high titers of retroviral vectors expressing genes that are extremely difficult to propagate at high titer in stable producer lines. This method should facilitate and extend the use of helper-free retroviral gene transfer, as well as be useful for gene therapy.
Pear
W S
WS
Rockefeller University, New York, NY 10021.
Nolan
G P
GP
Scott
M L
ML
Baltimore
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D016475:3T3 Cells; D000818:Animals; D016026:Bone Marrow Transplantation / Q000502:physiology; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002738:Chloroquine / Q000494:pharmacology; D005260:Female; D016313:Genes, abl; D006378:Helper Viruses / Q000502:physiology*; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010960:Plasminogen Activators / Q000096:biosynthesis; D012194:RNA-Directed DNA Polymerase / Q000032:analysis; D012091:Repetitive Sequences, Nucleic Acid; D012190:Retroviridae / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D014779:Virus Replication / Q000187:drug effects; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism
D002738:Chloroquine; D012194:RNA-Directed DNA Polymerase; D001616:beta-Galactosidase; D010960:Plasminogen Activators