Abstract
Studying microRNA function in vivo requires genetic strategies to generate loss-of-function phenotypes. We used lentiviral vectors to stably and specifically knock down microRNA by overexpressing microRNA target sequences from polymerase II promoters. These vectors effectively inhibited regulation of reporter constructs and natural microRNA targets. We used bone marrow reconstitution with hematopoietic stem cells stably overexpressing miR-223 target sequence to phenocopy the genetic miR-223 knockout mouse, indicating robust interference of microRNA function in vivo.
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Acknowledgements
We thank A. Cantore, F. Benedicenti, L. Sergi Sergi and M. Rocchi for help with experiments. This work was supported by Telethon, the European Union (LSHB-CT-2004-005276 and LSHB-CT-2004-005242), and Fondazione Cariplo to L.N. B.G. is the recipient of a research fellowship from the German Research Foundation (Deutsche Forschungsgemeinschaft Forschungsstipendium).
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B.G. designed and performed research, analyzed data and wrote the paper. G.S., A.G. and M.P. performed research. M.A. and B.D.B. provided crucial reagents. L.N. coordinated the project, analyzed data and wrote the paper.
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Gentner, B., Schira, G., Giustacchini, A. et al. Stable knockdown of microRNA in vivo by lentiviral vectors. Nat Methods 6, 63–66 (2009). https://doi.org/10.1038/nmeth.1277
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DOI: https://doi.org/10.1038/nmeth.1277