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Silencing of mammalian genes by tetracycline-inducible shRNA expression

Nature Protocols volume 2, pages 3257–3269 (2007)Cite this article

Abstract

Conditional gene silencing in mammalian cells, via the controlled expression of short hairpin RNAs (shRNAs), is an effective method for studying gene function, particularly if the gene is essential for cell survival or development. Here we describe a simple and rapid protocol for the generation of tetracycline (Tet)-inducible vectors that express shRNAs in a time- and dosage-dependent manner. Tet-operator (TetO) sequences responsive to occupation by the Tet-repressor (TetR) were inserted at alternative positions within the wild-type H1 promoter and cloned into a eukaryotic expression vector. Additional cloning sites downstream of the promoter enable the insertion of shRNA sequences. This Tet-inducible shRNA expression system can be used for both transient and stable RNA interference (RNAi) approaches to control gene function in a spatiotemporal fashion. The entire protocol (preparation of constructs, generation of stable cell lines and functional analysis) can be completed in 3 months.

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Figure 1: Conditional expression of short hairpin RNA (shRNA) via tetracycline (Tet)-inducible H1 promoter constructs.
Figure 2: Map of H1 promoter variants and expression plasmid.
Figure 3: Coding sequence and structure of a typical short hairpin RNA (shRNA).
Figure 4: Comparison of plasmid DNA extracted by the modified phenol:chloroform:isoamylalcohol (PCI) or by standard methods.
Figure 5: Downregulation of Polo-like kinase 1 (Plk1) by tetracycline (Tet)-inducible expression of short hairpin RNA (shRNA).

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Acknowledgements

This work was supported by grants from the Nationales Genomforschungsnetz Deutschland (NGFN), Messer Stiftung, Sander Stiftung, Deutsche Krebshilfe, Schleussner Stiftung, Else Kröner-Fresenius-Stiftung and Carls-Stiftung.

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  1. Department of Gynaecology and Obstetrics, School of Medicine, J.W. Goethe-University, Theodor-Stern-Kai 7, Frankfurt/Main, D-60590, Germany

    Sven Kappel, Yves Matthess, Manfred Kaufmann & Klaus Strebhardt

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  1. Sven Kappel
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  2. Yves Matthess
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  3. Manfred Kaufmann
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  4. Klaus Strebhardt
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S.K. and Y.M. contributed equally to this work.

Corresponding author

Correspondence to Klaus Strebhardt.

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Kappel, S., Matthess, Y., Kaufmann, M. et al. Silencing of mammalian genes by tetracycline-inducible shRNA expression. Nat Protoc 2, 3257–3269 (2007). https://doi.org/10.1038/nprot.2007.458

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