A transient three-plasmid expression system for the production of high titer retroviral vectors.
We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants.
Soneoka
Y
Y
Department of Biochemistry, University of Oxford, UK.
Cannon
P M
PM
Ramsdale
E E
EE
Griffiths
J C
JC
Romano
G
G
Kingsman
S M
SM
Kingsman
A J
AJ
D016475:3T3 Cells; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000502:physiology; D001483:Base Sequence; D002087:Butyrates / Q000494:pharmacology; D020148:Butyric Acid; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D003587:Cytomegalovirus / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D017930:Genes, Reporter; D005814:Genes, Viral; D015752:Genes, env; D015750:Genes, gag; D015751:Genes, pol; D005822:Genetic Vectors / Q000235:genetics*; D006378:Helper Viruses / Q000502:physiology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D018741:Replication Origin; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D015678:Viral Structural Proteins / Q000235:genetics
D000952:Antigens, Polyomavirus Transforming; D002087:Butyrates; D015678:Viral Structural Proteins; D020148:Butyric Acid