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

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

Abstract

“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 

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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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