Improved Tet-responsive promoters with minimized background expression

BMC Biotechnology

Methodology Article

Niels Heinz¹  Rainer Loew²  Mathias Hampf³  Manfred Gossen⁴  Hermann Bujard⁵ 

[1] EUFETS GmbH, 55743, Idar-Oberstein, Germany;Hannover Medical School, 30625, Hannover, Germany;EUFETS GmbH, 55743, Idar-Oberstein, Germany;Zentrum für Molekulare Biologie (ZMBH), Universität Heidelberg, 69120, Heidelberg, Germany;Max Delbrück Center for Molecular Medicine (MDC), 13125, Berlin, Germany;Max Delbrück Center for Molecular Medicine (MDC), 13125, Berlin, Germany;Berlin-Brandenburg Center for Regenerative Therapies (BCRT), 13353, Berlin, Germany;Zentrum für Molekulare Biologie (ZMBH), Universität Heidelberg, 69120, Heidelberg, Germany;

关键词: Luciferase Activity;    Minimal Promoter;    Fluorescence Activate Cell Sorting Analysis;    Transient Transfection Assay;    Background Expression;   

DOI  :  10.1186/1472-6750-10-81

 received in 2010-04-23, accepted in 2010-11-24,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundThe performance of the tetracycline controlled transcriptional activation system (Tet system) depends critically on the choice of minimal promoters. They are indispensable to warrant low expression levels with the system turned "off". On the other hand, they must support high level of gene expression in the "on"-state.ResultsIn this study, we systematically modified the widely used Cytomegalovirus (CMV) minimal promoter to further minimize background expression, resulting in an improved dynamic expression range. Using both plasmid-based and retroviral gene delivery, our analysis revealed that especially background expression levels could be significantly reduced when compared to previously established "standard" promoter designs. Our results also demonstrate the possibility to fine-tune expression levels in non-clonal cell populations. They also imply differences regarding the requirements for tight regulation and high level induction between transient and stable gene transfer systems.ConclusionsUntil now, our understanding of mammalian transcriptional regulation including promoter architecture is limited. Nevertheless, the partly empirical modification of cis-elements as shown in this study can lead to the specific improvement of the performance of minimal promoters. The novel composite Ptet promoters introduced here will further expand the utility of the Tet system.

【 授权许可】

CC BY   
© Loew et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.