In vitro-packaged SV40 pseudovirions as highly efficient vectors for gene transfer.
A procedure for in vitro packaging of plasmid DNA in recombinant SV40 capsid proteins was developed by Sandalon et al. (1997). Here, we report the highly efficient transduction into different human, murine and monkey cell lines using a scaled-up protocol for producing SV40 pseudovirions, packaged in vitro, carrying the human multidrug-resistance gene MDR1 encoding P-glycoprotein (P-gp) or the green fluorescent protein reporter gene (GFP) under control of SV40 and cytomegalovirus (CMV) promoters. The percentage of expressing cells was proportional to the number of transducing particles, with close to 100% of cells transduced at optimal ratios of transducing particles to cells. The ability to confer multidrug resistance was evaluated by measuring dye efflux and cell-surface expression in infected cells. The relative level of expression of P-gp driven by the different promoters varied among different cell lines. In human lymphoblastoid cells, which express high levels of major histocompatibility complex (MHC) class I (a surface receptor for SV40), constructs that carry an intron yield the highest expression. Our experiments further demonstrate that MDR1 and GFP expression driven by these promoters is transient; however, transduced cells remain MDR1-positive if selected in colchicine. Thus, the SV40 vectors are well suited to situations in which only short-term expression is required or expression is selected, such as for bone marrow protection during chemotherapy.
Kimchi-Sarfaty
Chava
C
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Ben-Nun-Shaul
Orly
O
Rund
Deborah
D
Oppenheim
Ariella
A
Gottesman
Michael M
MM
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003078:Colchicine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019450:Genes, MDR / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D013539:Simian virus 40 / Q000187:drug effects / Q000235:genetics*; D014158:Transcription, Genetic; D014161:Transduction, Genetic
D015395:Histocompatibility Antigens Class I; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; C012852:colchiceine; D003078:Colchicine