title: A gene regulation system with four distinct expression levels.
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.
authors.affiliation: Department of Microbiology, Institute for Biology, Friedrich-Alexander-University Erlangen-Nuremberg, Staudtstr. 5, 91058 Erlangen, Germany.
authors.forename: Christel
authors.initials: C
authors.lastname: Krueger
authors.forename: Christina
authors.initials: C
authors.lastname: Danke
authors.forename: Klaus
authors.initials: K
authors.lastname: Pfleiderer
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schuh
authors.forename: Hans-Martin
authors.initials: HM
authors.lastname: Jäck
authors.forename: Susanne
authors.initials: S
authors.lastname: Lochner
authors.forename: Peter
authors.initials: P
authors.lastname: Gmeiner
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hillen
authors.forename: Christian
authors.initials: C
authors.lastname: Berens
mesh_terms: D002460:Cell Line; D004318:Doxycycline / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008156:Luciferases / Q000378:metabolism; D008911:Minocycline / Q000494:pharmacology; D008954:Models, Biological; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity; D013752:Tetracycline / Q000494:pharmacology*; D015534:Trans-Activators / Q000235:genetics; D014162:Transfection; D019076:Transgenes
chemical_list: D005819:Genetic Markers; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D008156:Luciferases; D013752:Tetracycline; D008911:Minocycline; D004318:Doxycycline