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.
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References
Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–4451
Gossen M, Freundlieb S, Bender G, et al (1995) Transcriptional activation by tetracyclines in mammalian cells. Science 268:1766–1769
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 13:121–128
Berens C, Hillen W (2003) Gene regulation by tetracyclines: constraints of resistance regulation in bacteria shape TetR for application. Eur J Biochem 270:3109–3021
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 48:597–606
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 25:5183–5195
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 199:197–202
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Shiota, G., Yoshida, Y. (2012). “Tet-On” System Toward Hepatic Differentiation of Human Mesenchymal Stem Cells by Hepatocyte Nuclear Factor. In: Ochiya, T. (eds) Liver Stem Cells. Methods in Molecular Biology, vol 826. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-468-1_11
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DOI: https://doi.org/10.1007/978-1-61779-468-1_11
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