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Tissue-specific and reversible RNA interference in transgenic mice

Nature Genetics volume 39, pages 914–921 (2007)Cite this article

Abstract

Genetically engineered mice provide powerful tools for understanding mammalian gene function. These models traditionally rely on gene overexpression from transgenes or targeted, irreversible gene mutation. By adapting the tetracycline (tet)-responsive system previously used for gene overexpression, we have developed a simple transgenic system to reversibly control endogenous gene expression using RNA interference (RNAi) in mice. Transgenic mice harboring a tet-responsive RNA polymerase II promoter driving a microRNA-based short hairpin RNA targeting the tumor suppressor Trp53 reversibly express short hairpin RNA when crossed with existing mouse strains expressing general or tissue-specific 'tet-on' or 'tet-off' transactivators. Reversible Trp53 knockdown can be achieved in several tissues, and restoring Trp53 expression in lymphomas whose development is promoted by Trp53 knockdown leads to tumor regression. By leaving the target gene unaltered, this approach permits tissue-specific, reversible regulation of endogenous gene expression in vivo, with potential broad application in basic biology and drug target validation.

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Figure 1: Germline transmission of a functional TRE-p53.1224 cassette.
Figure 2: Transgenic tet-transactivators can drive Trp53 knockdown in MEFs.
Figure 3: CMV-rtTA drives widespread and functional p53.1224 shRNA expression.
Figure 4: Trp53 knockdown protects thymocytes from radiation-induced apoptosis in vivo.
Figure 5: Liver-specific Trp53 knockdown in LAP-tTA/TRE-p53.1224 mice.
Figure 6: Tissue-specific Trp53 knockdown accelerates Eμ-Myc lymphomagenesis.
Figure 7: Restoring Trp53 expression in tumors causes apoptosis and regression.

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Acknowledgements

We thank H. Bujard (Heidelberg University) and D. Felsher (Stanford University) for LAP-tTA mice; D. Felsher for Eμ-tTA mice; R. Jaenisch (Whitehead Institute) for Rosa26-M2rtTA mice; H. Varmus, F. Cong and R. Sotillo (Memorial Sloan-Kettering Cancer Center) for CMV-rtTA mice and J. Adams (Walter and Eliza Hall Institute) for Eμ-Myc mice. Many thanks to L. Bianco, J. Coblentz and the Cold Spring Harbor Laboratory animal house staff; M. Lupu and C. Le at the Memorial Sloan-Kettering Cancer Center for MRI; M.S. Jiao, K. Manova and E. de Stanchina at Memorial Sloan-Kettering Cancer Center for histology and immunohistochemistry; members of the Hannon laboratory for advice on small RNA blotting and members of the Lowe laboratory for advice and discussions. This study was supported by a Mouse Models of Human Cancer Consortium grant, a program project grant from the National Cancer Institute and the Don Monti Memorial Research Foundation. K.M is a Robert and Theresa Lindsay Fellow and the Leeds scholar of the Watson School of Biological Sciences. D.J.B is an Engelhorn Scholar of the Watson School of Biological Sciences. V.K. is a Leukemia and Lymphoma Society Fellow.

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Authors and Affiliations

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

    Ross A Dickins, Gregory J Hannon & Scott W Lowe

  2. Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, 11724, New York, USA

    Katherine McJunkin, Darren J Burgess, Gregory J Hannon & Scott W Lowe

  3. Cold Spring Harbor Laboratory, Cold Spring Harbor, 11724, New York, USA

    Ross A Dickins, Katherine McJunkin, Prem K Premsrirut, Valery Krizhanovsky, Darren J Burgess, Sang Yong Kim, Lars Zender, Gregory J Hannon & Scott W Lowe

  4. Division of Molecular Pathology, Memorial Sloan-Kettering Cancer Center, New York, 10021, New York, USA

    Eva Hernando & Carlos Cordon-Cardo

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Contributions

R.A.D. and S.W.L. designed the study; R.A.D., K.M., E.H., P.K.P, V.K. and L.Z. performed experiments; S.Y.K. performed pronuclear injections; D.J.B. provided reagents; C.C.-C. supervised and interpreted histopathology; G.J.H. and S.W.L. supervised experiments and data interpretation and R.A.D. and S.W.L. wrote the paper.

Corresponding author

Correspondence to Scott W Lowe.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Germline transmission of a functional TRE-p16/p19.478 transgene. (PDF 364 kb)

Supplementary Fig. 2

Dose-response analysis of cultured thymocytes to radiation-induced apoptosis. (PDF 22 kb)

Supplementary Fig. 3

LAP-tTA drives reversible p53.1224 siRNA expression in the liver of different TRE-p53.1224 transgenic founder lines. (PDF 141 kb)

Supplementary Fig. 4

Accelerated lymphomagenesis is restricted to Eμ-myc; Eμ-tTA; TRE-p53.1224 mice. (PDF 30 kb)

Supplementary Fig. 5

Accelerated Eμ-myc lymphomagenesis using TRE-p53.1224 line D. (PDF 157 kb)

Supplementary Fig. 6

p53.1224 siRNA is undetectable in whole tissues of Eμ-tTA; TRE-p53.1224 mice. (PDF 164 kb)

Supplementary Fig. 7

Doxycycline restores p53 expression and causes apoptosis of Eμ-myc; Eμ-tTA; TREp53.1224 lymphoma cells. (PDF 423 kb)

Supplementary Table 1

Oligonucleotide sequences. (PDF 19 kb)

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Dickins, R., McJunkin, K., Hernando, E. et al. Tissue-specific and reversible RNA interference in transgenic mice. Nat Genet 39, 914–921 (2007). https://doi.org/10.1038/ng2045

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