“Tet-On” System Toward Hepatic Differentiation of Human Mesenchymal Stem Cells by Hepatocyte Nuclear Factor

  • Goshi ShiotaEmail author
  • Yoko Yoshida
Part of the Methods in Molecular Biology book series (MIMB, volume 826)


“Tet-On” system requires two DNA constructs: the first one is a transcription regulatory unit, rtTA and the second construct is the responsive element Escherichia coli sequences (tetO) linked to Pcmv driven target gene. In the absence of inducing agent doxycycline (Dox), a tetracycline derivative, rtTA does not bind to or binds weakly to operator sequences of tetO; therefore, no target gene is transcribed. However, in the presence of Dox, tTA binds to tetO and pcmv, which in turn activates the target gene. In general, the induction of transgene by Dox is rapid and can occur within hours in some systems, offering an advantage over the original tTA system for studying acute effects of transgenes. Recently, we have established a Tet-regulated expression system for hepatocyte nuclear factor 3β (HNF3β) to investigate the potency of hepatic differentiation of human mesenchymal stem cells (MSC) by HNF3β.

Key words

Tetracycline-inducible system HNF3β Mesenchymal stem cell Hepatic differentiation Tet repressor Tetracycline operator 2 


  1. 1.
    Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–4451PubMedCrossRefGoogle Scholar
  2. 2.
    Gossen M, Freundlieb S, Bender G, et al (1995) Transcriptional activation by tetracyclines in mammalian cells. Science 268:1766–1769PubMedCrossRefGoogle Scholar
  3. 3.
    Zhu Z, Zheng T, Lee CG et al (2002) Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling. Semin Cell Dev Biol 13:121–128PubMedCrossRefGoogle Scholar
  4. 4.
    Berens C, Hillen W (2003) Gene regulation by tetracyclines: constraints of resistance regulation in bacteria shape TetR for application. Eur J Biochem 270:3109–3021PubMedCrossRefGoogle Scholar
  5. 5.
    Ishii K, Yoshida Y, Akechi Y et al (2008) Hepatic differentiation of human bone marrow-derived mesenchymal stem cells by tetracycline-regulated hepatocyte nuclear factor 3beta. Hepatology 48:597–606PubMedCrossRefGoogle Scholar
  6. 6.
    Mori T, Kiyono T, Imabayashi H et al (2005) Combination of hTERT and bmi-1, E6, or E7 induces prolongation of the life span of bone marrow stromal cells from an elderly donor without affecting their neurogenic potential. Mol Cell Biol 25:5183–5195PubMedCrossRefGoogle Scholar
  7. 7.
    Takahashi M, Degenkolb J, Hillen W (1991) Determination of the equilibrium constant between Tet repressor and tetracycline at limiting Mg2+ concentrartion: a generally applicable method for effector-dependent high-affinity complexes. Anal Biochem 199:197–202PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Division of Molecular and Genetic Medicine, Department of Genetic Medicine and Regenerative Therapeutics, Graduate School of MedicineTottori UniversityYonagoJapan
  2. 2.Department of Molecular NeuropathologyTokyo Metroporitan Institute for NeuroscienceTokyoJapan

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