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Research Articles: Targets

A retroviral expression system based on tetracycline-regulated tricistronic transactivator/repressor vectors for functional analyses of antiproliferative and toxic genes

¹ Molecular Biology Research Laboratory, Department of Pediatrics, Medical University Innsbruck; ² Division of Molecular Pathophysiology, Biocenter, Medical University Innsbruck; and ³ Tyrolean Cancer Research Institute, Innsbruck, Austria

Requests for reprints: Michael J. Ausserlechner, Molecular Biology Research Laboratory, Pediatric Department, Medical University Innsbruck, Innrain 66, A-6020 Innsbruck, Austria. Phone: 43-512-504-27748; Fax: 43-512-504-27750. E-mail: michael.j.ausserlechner{at}uibk.ac.at

Establishment of stably transfected mammalian cells with conditional expression of antiproliferative or proapoptotic proteins is often hampered by varying expression within bulk-selected cells and high background in the absence of the inducing drug. To overcome such limitations, we designed a gene expression system that transcribes the tetracycline-dependent rtTA2-M2-activator, TRSID-silencer, and selection marker as a tricistronic mRNA from a single retroviral vector. More than 92% of bulk-selected cells expressed enhanced green fluorescent protein or luciferase over more than three orders of magnitude in an almost linear, dose-dependent manner. To functionally test this system, we studied how dose-dependent expression of p27^Kip1 affects proliferation and viability of SH-EP neuroblastoma cells. Low to moderate p27^Kip1 expression caused transient G₀-G₁ accumulation without reduced viability, whereas high p27^Kip1 levels induced significant apoptosis after 72 hours. This proves that this expression system allows concentration-dependent analysis of gene function and implicates p27^Kip1 as a critical regulator of both proliferation and apoptosis in SH-EP neuroblastoma cells. [Mol Cancer Ther 2006;5(8):1927–34]

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: M.J. Ausserlechner and P. Obexer contributed equally to this work.

⁴ Unpublished data.

Received 11/30/05; revised 6/11/06; accepted 6/22/06.

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