title: Improved single-chain transactivators of the Tet-On gene expression system.
abstract: BACKGROUND
  The Tet-Off (tTA) and Tet-On (rtTA) regulatory systems are widely applied to control gene expression in eukaryotes. Both systems are based on the Tet repressor (TetR) from transposon Tn10, a dimeric DNA-binding protein that binds to specific operator sequences (tetO). To allow the independent regulation of multiple genes, novel Tet systems are being developed that respond to different effectors and bind to different tetO sites. To prevent heterodimerization when multiple Tet systems are expressed in the same cell, single-chain variants of the transactivators have been constructed. Unfortunately, the activity of the single-chain rtTA (sc-rtTA) is reduced when compared with the regular rtTA, which might limit its application.
  
  
  RESULTS
  We recently identified amino acid substitutions in rtTA that greatly improved the transcriptional activity and doxycycline-sensitivity of the protein. To test whether we can similarly improve other TetR-based gene regulation systems, we introduced these mutations into tTA and sc-rtTA. Whereas none of the tested mutations improved tTA activity, they did significantly enhance sc-rtTA activity. We thus generated a novel sc-rtTA variant that is almost as active and dox-sensitive as the regular dimeric rtTA. This variant was also less sensitive to interference by co-expressed TetR-based tTS repressor protein and may therefore be more suitable for applications where multiple TetR-based regulatory systems are used.
  
  
  CONCLUSIONS
  We developed an improved sc-rtTA variant that may replace regular rtTA in applications where multiple TetR-based regulatory systems are used.
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands. x.zhou@amc.uva.nl <x.zhou@amc.uva.nl>
authors.forename: Xue
authors.initials: X
authors.lastname: Zhou
authors.forename: Jori
authors.initials: J
authors.lastname: Symons
authors.forename: Rieuwert
authors.initials: R
authors.lastname: Hoppes
authors.forename: Christel
authors.initials: C
authors.lastname: Krueger
authors.forename: Christian
authors.initials: C
authors.lastname: Berens
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hillen
authors.forename: Ben
authors.initials: B
authors.lastname: Berkhout
authors.forename: Atze T
authors.initials: AT
authors.lastname: Das
mesh_terms: D015870:Gene Expression / Q000502:physiology*; D005786:Gene Expression Regulation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics*; D015854:Up-Regulation
chemical_list: D011994:Recombinant Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein