Abstract
Cell-type-specific gene silencing is critical to understand cell functions in normal and pathological conditions, in particular in the brain where strong cellular heterogeneity exists. Molecular engineering of lentiviral vectors has been widely used to express genes of interest specifically in neurons or astrocytes. However, we show that these strategies are not suitable for astrocyte-specific gene silencing due to the processing of small hairpin RNA (shRNA) in a cell. Here we develop an indirect method based on a tetracycline-regulated system to fully restrict shRNA expression to astrocytes. The combination of Mokola-G envelope pseudotyping, glutamine synthetase promoter and two distinct microRNA target sequences provides a powerful tool for efficient and cell-type-specific gene silencing in the central nervous system. We anticipate our vector will be a potent and versatile system to improve the targeting of cell populations for fundamental as well as therapeutic applications.
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Acknowledgements
This work was partially supported by the CEA and the European Community’s Seventh Framework Program FP7/2007–2013 under the grant agreement number HEALTH-F5-2008-222925 (NEUGENE) and by the Swiss National Science Foundation 31003A-140945. We thank Carole Malgorn (CEA, France) and Lydie Boussicault (CEA, France) for their help in genotyping mice; Fanny Petit (CEA, France), Martine Guillermier (CEA, France), Diane Houitte (CEA, France), Virginie Zimmer (LCMN, Lausanne) and Alexia Spoerl (LCMN, Lausanne) for help with animal experiments; and Dr Emmanuel Brouillet (CEA, France) and the Cellular Imaging Facility (CIF, Lausanne) for their advice about microscopic acquisitions.
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Merienne, N., Delzor, A., Viret, A. et al. Gene transfer engineering for astrocyte-specific silencing in the CNS. Gene Ther 22, 830–839 (2015). https://doi.org/10.1038/gt.2015.54
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DOI: https://doi.org/10.1038/gt.2015.54
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