科研之友
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摘要
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 and situations in which these concentrations can be precisely controlled 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 four expression levels are obtained by adding different effectors. derivatives, 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.
- 出版日期2006-8
