Factors limiting autogene-based cytoplasmic expression systems.
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 non-viral gene delivery systems, where the levels of gene expression achieved are usually well below the levels achieved by viral gene transfer systems. Previous work from our laboratory describes an enhanced dual promoter autogene-based cytoplasmic expression system that gives rise to levels of gene expression 20-fold higher than that of a CMV nuclear expression plasmid control. Here various strategies are described to increase the levels of autogene-based gene expression by changing variables such as the type of nuclear promoter, phage RNAP gene, and IRES element. Although insights into the function of various IRES elements were gained, none of these changes demonstrated a significant increase in gene expression. However, determination of the mRNA levels achieved using quantitative RNase protection assays and immunofluorescence experiments revealed that transgene mRNA levels were saturated at up to 10 times higher than all other mRNA in the transfected cell combined. It follows that mRNA production, as well as translation, are important factors limiting autogene-based cytoplasmic expression.
Finn
Jonathan
J
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada. jdfinn@interchange.ubc.ca
MacLachlan
Ian
I
Cullis
Pieter
P
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics; D002460:Cell Line; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D015138:RNA, Nuclear / Q000235:genetics; D012270:Ribosomes / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics; D014780:Viruses / Q000235:genetics
D012333:RNA, Messenger; D015138:RNA, Nuclear; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases