Nucleic Acids Research Advance Access published online on February 11, 2008
Nucleic Acids Research, doi:10.1093/nar/gkm1119
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Methods Online |
Improvement of reporter activity by IRES-mediated polycistronic reporter system
1Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, 80336 Munich and 2Max Planck Institute of Biochemistry, Department of Molecular Medicine, 82152, Martinsried, Germany
*To whom correspondence should be addressed. Tel: +49 89 51605437; Fax: +49 89 51605223; Email: bouabe{at}mvp.uni-muenchen.de
Received July 14, 2007. Revised November 27, 2007. Accepted November 30, 2007.
Many in vitro and in vivo applications for transgenesis require co-expression of heterologous genes. The use of internal ribosome entry sites (IRESs) in dicistronic expression vectors enables the expression of two genes controlled by one promoter in target cells or whole organisms. Here we describe the expansion of IRES exploitation to generate multicistronic vectors capable of expressing multiple reporter genes, especially to improve the fluorescence yield of autofluorescent reporter gene products such as green fluorescent protein (GFP). We found that the increase in fluorescence output of GFP is proportional to the number of IRES-GFP repeats in the multicistronic vector. At least four genes can be expressed from a multicistonic vector by using tandem IRES elements, with no significant alteration of the expression level of the cap-dependent translated gene. Moreover, gene expression under the control of multiple IRES element has no effect on the posttranscriptional regulation through 3'-untranslated regions (3'UTR). Thus, endogenous gene expression and regulation, especially those controlled by weak promoters, can be analyzed with our IRES-dependent polycistronic reporter gene expression system.