Improved Tet-responsive promoters with minimized background expression.

BACKGROUND: The 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. RESULTS: In 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. CONCLUSIONS: Until 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.

Promoter crosstalk effects on gene expression.

Closely spaced transcription signals formally assigned to different, neighboring genes can functionally interact both in their authentic genomic context as well as in engineered transgene constellations. To describe these promoter crosstalk effects quantitatively and qualitatively, we used various combinations of inducible and constitutive expression signals linked in cis. Our results demonstrate that such interactions can be bidirectional, making it difficult to unambiguously assign a particular promoter element exclusively to a specific gene. We show that especially for inducible promoters, crosstalk effects can cause a substantial distortion in the expression of proximal genes, challenging established strategies in generating transgenic animal models and tissue culture systems. Furthermore, these findings provide guidelines for the design of transgenic transcription units that, while refractory to interactions with their chromosomal environment, leave the expression programs of neighboring genes largely untouched.

A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays.

We established a quantitative reporter gene protocol, the P/Rluc assay system, allowing the sequential measurement of Photinus and Renilla luciferase activities from the same extract. Other than comparable commercial reporter assay systems and their noncommercial counterparts, the P/Rluc assay system was formulated under the aspect of full compatibility with standard methods for protein assays. This feature greatly expands the range of applications for assay systems quantifying the expression of multiple luciferase reporters.