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Cytotechnology

, Volume 63, Issue 3, pp 211–216 | Cite as

Development of a tightly-regulated tetracycline-dependent transcriptional activator and repressor co-expression system for the strong induction of transgene expression

  • Hiroshi Hosoda
  • Takahisa Miyao
  • Shusaku Uchida
  • Shinsuke Sakai
  • Satoshi KidaEmail author
Brief Report

Abstract

The teteracycline (Tc)-dependent and -inducible transcriptional activator (rtTA) system has been used to express regulated transgene expression in vitro and in vivo. However, previous reports have demonstrated that, even in the absence of Tc, the rtTA binds weakly to the tetracycline response element (TRE), leading to a low level of background activity. In order to reduce the leaky gene expression induced by rtTA, we previously established a tightly regulated system (A-IRES-R system) that makes use of both the rtTA (A) and a Tc-dependent repressor (TetR-Kruppel-associated box; KRAB) (R). In addition, others have described a transactivator rtTA2-M2 (M2) that displays higher sensitivity to Dox than rtTA. In this study, to further develop the A-IRES-R system, we generated a derivative Tc system (M2-IRES-R system) that co-expresses both rtTA-M2 and TetR-KRAB from a single vector. We show that compared to the A-IRES-R system, the M2-IRES-R system leads to a greater level of induced TRE-mediated transcription in the presence of doxycycline (Dox) and yet displays a similar level of basal TRE-mediated transcription in the absence of Dox. Furthermore, the M2-IRES-R system also displays less leaky gene expression in the absence of Dox compared to rtTA-M2 and rtTA systems. Taken together, our results suggest that the M2-IRES-R system enables to tightly regulate and highly induce the expression of transgene compared to other systems.

Keywords

rtTA-M2 Tight regulation Transgenic technique Basal transcription 

Notes

Acknowledgment

This work was supported by a Grant-in Aid for High Technology Research and Priority Areas—Molecular Brain Science—from the ministry of Education, Science and Culture, Japan and the Research Grant for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare, Japan.

Supplementary material

10616_2011_9335_MOESM1_ESM.pdf (148 kb)
Supplementary material 1 (PDF 148 kb)

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Hiroshi Hosoda
    • 1
  • Takahisa Miyao
    • 1
  • Shusaku Uchida
    • 2
  • Shinsuke Sakai
    • 2
  • Satoshi Kida
    • 1
    • 2
    Email author
  1. 1.Department of Bioscience, Faculty of Applied BioscienceTokyo University of AgricultureTokyoJapan
  2. 2.Department of Agricultural Chemistry, Faculty of Applied BioscienceTokyo University of AgricultureTokyoJapan

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