Format

Send to

Choose Destination
J Gene Med. 2006 Aug;8(8):1037-47.

A gene regulation system with four distinct expression levels.

Author information

1
Department of Microbiology, Institute for Biology, Friedrich-Alexander-University Erlangen-Nuremberg, Staudtstr. 5, 91058 Erlangen, Germany.

Abstract

BACKGROUND:

The amount of a particular protein, and not just its presence or absence, frequently determines the outcome of a developmental process or disease progression. These dosage effects can be studied by conditionally expressing such proteins at different levels. With typical gene regulation systems like the Tet-On system, intermediate expression levels can be obtained by varying the effector concentration. However, this strategy is limited to situations in which these concentrations can be precisely controlled and, thus, not suited for animal models or gene therapy approaches. Here, we present a Tet transregulator setup that allows establishment of four levels of promoter activity largely independent of effector concentration.

METHODS:

A newly introduced transsilencer is combined with a reverse transactivator. As the regulators respond differentially to tetracycline derivatives, four expression levels are obtained by adding different effectors. To facilitate integration of the components, we generated versatile all-in-one vectors. Apart from a cassette expressing the transregulators and a selection marker, these vectors encode a bidirectional, regulated promoter driving expression of GFP and the gene of interest. The features of this stepwise regulation system were analyzed by transient and stable transfections of human cell lines.

RESULTS:

We demonstrate in a variety of experimental settings that coexpression of these transregulators leads to robust stepwise regulation. Depending on the respective effectors, four expression levels are achieved with different responsive promoters, cell lines and target genes.

CONCLUSIONS:

This system shows that a promoter can be adjusted to different activities and provides an excellent strategy to investigate protein dosage effects.

PMID:
16779863
DOI:
10.1002/jgm.932
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Wiley
Loading ...
Support Center