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Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi

Nature Biotechnology volume 29, pages 79–83 (2011)Cite this article

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Abstract

Short hairpin RNAs (shRNAs) are versatile tools for analyzing loss-of-function phenotypes in vitro and in vivo1. However, their use for studying genes involved in proliferation and survival, which are potential therapeutic targets in cancer and other diseases, is confounded by the strong selective advantage of cells in which shRNA expression is inefficient. We therefore developed a toolkit that combines Tet-regulated miR30-shRNA technology, robust transactivator expression and two fluorescent reporters to track and isolate cells with potent target knockdown. We demonstrated that this system improves the study of essential genes and was sufficiently robust to eradicate aggressive cancer in mice by suppressing a single gene. Further, we applied this system for in vivo negative-selection screening with pooled shRNAs and propose a streamlined, inexpensive workflow that will facilitate the use of RNA interference (RNAi) for the identification and evaluation of essential therapeutic targets.

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Figure 1: Dual-color TRMPV vectors enable Tet-regulated shRNA expression for suppression of genes involved in cell proliferation and survival.
Figure 2: TRMPV enables RNAi-based evaluation of genes involved in tumor maintenance in vivo.
Figure 3: TRMPV-induced suppression of Rpa3 cures clonal MLL-AF9;NrasG12D AML.
Figure 4: Pooled negative selection RNAi screening in vivo detects shRpa3 depletion in MLL-AF9;NrasG12D AML.

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Acknowledgements

We thank R. Dickins and C. Miething for discussions in setting up this system, as well as A. Lujambio, A. Rappaport, M. Saborowski and C. Vakoc for testing TRMPV in other models. We also thank Y. Dou (Univ. of Michigan) for providing human MLL-AF9. We gratefully acknowledge B. Ma and S. Muller for excellent technical assistance. We also thank E. Hodges, K. Chang, M. Rooks and the McCombie laboratory for help with Solexa sequencing as well as A. Gordon for bioinformatics support. P. Moody and T. Spencer were of great assistance in flow cytometry. Finally, we thank S. Kogan for histology. This work was supported by the Howard Hughes Medical Institute, the Starr Foundation and the Don Monti Memorial Research Foundation. J.Z. is the Andrew Seligson Memorial Fellow at Cold Spring Harbor Laboratory, and K.M. is the Robert and Teresa Lindsay Fellow of the Watson School of Biological Sciences. L.E.D. is supported by an Overseas Biomedical Research Fellowship of the National Health and Medical Research Council of Australia.

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Author notes

  1. Johannes Zuber and Katherine McJunkin: These authors contributed equally to this work.

Authors and Affiliations

  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Johannes Zuber, Katherine McJunkin, Christof Fellmann, Lukas E Dow, Meredith J Taylor, Gregory J Hannon & Scott W Lowe

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

    Katherine McJunkin, Gregory J Hannon & Scott W Lowe

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

    Gregory J Hannon & Scott W Lowe

  4. Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland

    Christof Fellmann

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  1. Johannes Zuber
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Contributions

J.Z. and K.M. designed and performed experiments. C.F. and L.E.D. contributed new reagents and performed experiments. M.J.T. managed mouse monitoring and husbandry. G.J.H. and S.W.L. supervised this project. J.Z., K.M. and S.W.L. wrote the paper.

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Correspondence to Scott W Lowe.

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The authors declare no competing financial interests.

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Zuber, J., McJunkin, K., Fellmann, C. et al. Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi. Nat Biotechnol 29, 79–83 (2011). https://doi.org/10.1038/nbt.1720

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