Construction and Optimization of Tetracycline-Responsive Gene Expression Systems

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Title: Construction and Optimization of Tetracycline-Responsive Gene Expression Systems
Authors: Roney, Ian James
Date: 2016
Abstract: Conditional gene expression systems that enable inducible and reversible transcriptional control are essential research tools and have broad applications in biomedicine and biotechnology. The reverse tetracycline transcriptional activator is a canonical system for engineered gene expression control that enables graded and gratuitous modulation of target gene transcription in eukaryotes from yeast to human cell lines and transgenic animals. However, the system has a tendency to activate transcription even in the absence of tetracycline and this leaky target gene expression impedes its use. Here, we identify single amino acid substitutions that greatly enhance the dynamic range of the system by reducing leaky transcription to undetectable levels while retaining high expression capacity in the presence of inducer. Furthermore, we show that these improved DNA binding domains can be fused to repression domains to create synthetic transcriptional repressors. The function of these transcriptional repressors is dependent on the location of their recruitment and their mechanisms of action.
URL: http://hdl.handle.net/10393/35058
http://dx.doi.org/10.20381/ruor-5142
CollectionThèses, 2011 - // Theses, 2011 -
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