William Y Go1, Steffan N Ho. 1. Departments of Pathology and Cellular and Molecular Medicine, School of Medicine, University of California - San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0644, USA.
Abstract
BACKGROUND: Exogenously controlled gene expression systems are essential for both the in vivo analysis of gene function and the regulated delivery of therapeutic gene products. However, differences in experimental methods used to characterize the various systems prohibit informative comparisons. The purpose of this study was to identify an optimal system for regulated gene expression studies through a rigorous direct comparison of the dimerizer and the reverse tet transactivator (rtTA) transcriptional switch systems. METHODS: An optimized bicistronic rapamycin-dependent dimerizer construct and an optimized rtTA construct (based on rtTA(s)-M2) were developed that utilize a chimeric mammalian activation domain with a flexible interdomain linker. These constructs were reconstituted in identical eukaryotic expression vectors and compared in transient transfection assays employing target gene reporter constructs that differ only in the relevant DNA binding sites. RESULTS: The optimized rtTA(s)-M2 construct, designated rtTAM2.2, exhibited a twofold increase in the magnitude of doxycycline-dependent reporter gene induction and an eightfold increase in sensitivity as compared to the rtTA(s)-M2 construct. This correlated with a significantly higher level of expression of the rtTAM2.2 protein. In direct comparisons the rtTAM2.2 system mediated inducible expression to a level tenfold greater than the bicistronic dimerizer system. However, while the dimerizer system exhibited no detectable rapamycin-independent expression, a low level of doxycycline-independent target gene expression was detectable. CONCLUSIONS: The improved rtTAM2.2 rtTA system described here may prove optimal when the overall magnitude of target gene induction is critical, while the dimerizer system may be advantageous when the complete absence of ligand-independent target gene expression is essential. Copyright 2002 John Wiley & Sons, Ltd.
BACKGROUND: Exogenously controlled gene expression systems are essential for both the in vivo analysis of gene function and the regulated delivery of therapeutic gene products. However, differences in experimental methods used to characterize the various systems prohibit informative comparisons. The purpose of this study was to identify an optimal system for regulated gene expression studies through a rigorous direct comparison of the dimerizer and the reverse tet transactivator (rtTA) transcriptional switch systems. METHODS: An optimized bicistronic rapamycin-dependent dimerizer construct and an optimized rtTA construct (based on rtTA(s)-M2) were developed that utilize a chimeric mammalian activation domain with a flexible interdomain linker. These constructs were reconstituted in identical eukaryotic expression vectors and compared in transient transfection assays employing target gene reporter constructs that differ only in the relevant DNA binding sites. RESULTS: The optimized rtTA(s)-M2 construct, designated rtTAM2.2, exhibited a twofold increase in the magnitude of doxycycline-dependent reporter gene induction and an eightfold increase in sensitivity as compared to the rtTA(s)-M2 construct. This correlated with a significantly higher level of expression of the rtTAM2.2 protein. In direct comparisons the rtTAM2.2 system mediated inducible expression to a level tenfold greater than the bicistronic dimerizer system. However, while the dimerizer system exhibited no detectable rapamycin-independent expression, a low level of doxycycline-independent target gene expression was detectable. CONCLUSIONS: The improved rtTAM2.2 rtTA system described here may prove optimal when the overall magnitude of target gene induction is critical, while the dimerizer system may be advantageous when the complete absence of ligand-independent target gene expression is essential. Copyright 2002 John Wiley & Sons, Ltd.
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