An enhanced autogene-based dual-promoter cytoplasmic expression system yields increased gene expression.
The relatively low levels of transfection that can be achieved by current gene-delivery systems have limited the therapeutic utility of gene transfer. This is especially true for nonviral gene-delivery systems, where the levels of gene expression achieved are usually below the levels achieved by viral gene transfer systems. One strategy for increasing gene expression is to design a cytoplasmic expression system that does not require nuclear delivery for gene expression to occur. This can be achieved through the use of an autocatalytic cytoplasmic expression system using phage RNA polymerases. Here we describe cytoplasmic expression systems that yield increased levels of gene expression following in vitro transfection. We demonstrate direct evidence for an exponential, autocatalytic increase in gene expression using autogenes, as well as levels of reporter gene expression that are 20-fold higher than standard CMV-based nuclear expression systems. The development of a high-efficiency plasmid-based expression system could significantly improve the gene expression properties of nonviral gene-delivery systems, thereby increasing their clinical utility.
Finn
J
J
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada.
Lee
A C H
AC
MacLachlan
I
I
Cullis
P
P
D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006207:Half-Life; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins
D012333:RNA, Messenger; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases