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Probing tumor phenotypes using stable and regulated synthetic microRNA precursors

Nature Genetics volume 37, pages 1289–1295 (2005)Cite this article

A Corrigendum to this article was published on 01 March 2006

Abstract

RNA interference is a powerful method for suppressing gene expression in mammalian cells. Stable knock-down can be achieved by continuous expression of synthetic short hairpin RNAs, typically from RNA polymerase III promoters. But primary microRNA transcripts, which are endogenous triggers of RNA interference, are normally synthesized by RNA polymerase II. Here we show that RNA polymerase II promoters expressing rationally designed primary microRNA–based short hairpin RNAs produce potent, stable and regulatable gene knock-down in cultured cells and in animals, even when present at a single copy in the genome. Most notably, by tightly regulating Trp53 knock-down using tetracycline-based systems, we show that cultured mouse fibroblasts can be switched between proliferative and senescent states and that tumors induced by Trp53 suppression and cooperating oncogenes regress upon re-expression of Trp53. In practice, this primary microRNA–based short hairpin RNA vector system is markedly similar to cDNA overexpression systems and is a powerful tool for studying gene function in cells and animals.

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Figure 1: Effective knock-down by single-copy expression of miR-30–based shRNAs from a retroviral LTR promoter.
Figure 2: RNA Pol II–driven shRNAs can effectively promote tumorigenesis and chemotherapy resistance in vivo.
Figure 3: Stable and regulatable shRNA expression from a tetracycline-responsive RNA Pol II promoter.
Figure 4: Regulated shRNA expression using a tetracycline-on system.
Figure 5: Reversible Trp53 knock-down in primary MEFs.
Figure 6: Regulated Trp53 knock-down in tumors.

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Acknowledgements

We thank M. Narita for advice on generating vectors; R. Sachidanandam and N. Sheth for shRNA design; A. Malina for the pQTXIX vector; A. Denli, W. Keyes, D. Burgess and A. Bric for experimental assistance; F. Stegmeier and S. Elledge for communicating unpublished results; members of the laboratory of S.W.L. for advice and discussions; and L. Bianco and Cold Spring Harbor Laboratory animal house staff for their assistance. This study was supported by a Mouse Models of Human Cancer Consortium grant and a DNA Tumor Virus grant from the National Cancer Institute. This study was also supported by the Leukemia Research Foundation (R.A.D.), the Helen Hay Whitney Foundation (M.T.H.) and a Ruth L. Kirschstein NRSA (J.T.Z.). D.R.S. is a Beckman Foundation scholar of the Watson School of Biological Sciences. G.J.H. is supported by an Innovator Award from the US Army Breast Cancer Research Program. S.W.L. is an AACR-NCFR Research Professor.

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Authors and Affiliations

  1. Howard Hughes Medical Institute, 1 Bungtown Road, Cold Spring Harbor, New York, 11724, USA

    Ross A Dickins, Gregory J Hannon & Scott W Lowe

  2. Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York, 11724, USA

    Ross A Dickins, Michael T Hemann, Jack T Zilfou, Ingrid Ibarra, Gregory J Hannon & Scott W Lowe

  3. Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York, 11724, USA

    David R Simpson, Gregory J Hannon & Scott W Lowe

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Correspondence to Scott W Lowe.

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Dickins, R., Hemann, M., Zilfou, J. et al. Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat Genet 37, 1289–1295 (2005). https://doi.org/10.1038/ng1651

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