Abstract
RNA interference (RNAi) is an extremely effective tool for studying gene function in almost all metazoan and eukaryotic model systems. RNAi in mice, through the expression of short hairpin RNAs (shRNAs), offers something not easily achieved with traditional genetic approaches—inducible and reversible gene silencing. However, technical variability associated with the production of shRNA transgenic strains has so far limited their widespread use. Here we describe a pipeline for the generation of miR30-based shRNA transgenic mice that enables efficient and consistent targeting of doxycycline-regulated, fluorescence-linked shRNAs to the Col1a1 locus. Notably, the protocol details crucial steps in the design and testing of miR30-based shRNAs to maximize the potential for developing effective transgenic strains. In all, this 14-week procedure provides a fast and cost-effective way for any laboratory to investigate gene function in vivo in the mouse.
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Change history
11 October 2013
 In the version of this article initially published, the catalog number given for ESGRO (ESG1106) was incorrect. The correct catalog number should be ESG1107. The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We thank C. Beard and R. Jaenisch (Whitehead Institute) for pBS31 flp-in and pCAGs-FLPe-Puro vectors, Col1a1 3′ probe and KH2 ES cells. We thank J. Bolden, A. Lujambio and J. White for advice on technical procedures and critical review of the manuscript. Thanks to L. Bianco, J. Coblentz, E. Earl and the Cold Spring Harbor Laboratory animal house staff. We gratefully acknowledge J. Simon, D. Grace and J. Cappellani for technical assistance, and members of the Lowe laboratory for advice and discussions. This study was supported by a Mouse Models of Human Cancer Consortium grant and a program project grant from the National Cancer Institute. P.K.P. was a Medical Science Training Program Fellow of Stony Brook University, L.E.D. is supported by a National Health & Medical Research Council of Australia overseas Biomedical Training Fellowship, C.M. was supported by a fellowship from the Deutsche Forschungsgemeinschaft and an American Association for Cancer Research–Astellas USA Foundation Fellowship in Basic Cancer Research, J.Z. was the Andrew Seligson Memorial Fellow, R.A.D. is a Victorian Endowment for Science, Knowledge and Innovation (VESKI) Fellow and G.J.H. and S.W.L. are Howard Hughes Medical Institute investigators.
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L.E.D. designed and performed experiments, analyzed data and wrote the paper. P.K.P. and J.Z. designed and performed experiments and analyzed data. C.F., K.M., C.M. and Y.P. designed and performed experiments. R.A.D. and G.J.H. designed experiments. S.W.L. designed experiments and wrote the paper.
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P.K.P., C.F., G.J.H and S.W.L. are founders of Mirimus Inc., a company that has licensed technology related to this work.
Supplementary information
Supplementary Fig. 1
High copy retroviral transduction leads to overestimation of protein knockdown A. Flow cytometry analysis of NIH3T3 cells infected with pLMP-Ren.713 48hr post transduction (upper) and 5 days post puromycin selection (lower). Prior to and following selection, 'High copy' transduced cells show higher GFP expression than 'single copy' populations, reflecting higher levels of expression of the shRNAmir. B. Western blots for multiple individual shRNAmirs targeting two genes (APC and Bcl2), shows that high copy retroviral transduction leads to an overestimation of the potency of individual shRNAmirs. C. Graphs showing quantitation of APC mRNA transcript by quantitative PCR (top) and protein levels (bottom) measured by densitometry of the western blot shown in B. QPCR analysis indicates a reduction in mRNA levels but does not discriminate well between shRNAmirs that produce varying levels of protein depletion. In each case the values are normalized to a loading control (β2M for QPCR and β-actin for Western blot) and plotted relative to the 3T3 control. (EPS 8806 kb)
Supplementary Table 1
Strategies for developing transgenic mice strains. (PDF 31 kb)
Supplementary Table 2
Tissue specificities for dox-induced GFP-shRNAmir expression. (PDF 68 kb)
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Dow, L., Premsrirut, P., Zuber, J. et al. A pipeline for the generation of shRNA transgenic mice. Nat Protoc 7, 374–393 (2012). https://doi.org/10.1038/nprot.2011.446
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DOI: https://doi.org/10.1038/nprot.2011.446
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