Abstract
Tetracycline-regulated integrating vectors allow pharmacologically controlled genetic modification of murine and human hematopoietic stem cells (HSCs). This approach combines the stable transgene insertion into a host genome with the opportunity for time- and dose-controlled reversible transgene expression in HSCs. Here, we describe the step-by-step protocol for transduction of murine stem-cell enriched populations of bone marrow cells, such as lineage negative cells (Lin−), with a lentiviral vector expressing the enhanced green fluorescent protein (EGFP) under the control of the tetracycline-regulated promoter. This chapter explains how to establish in vitro and in vivo systems to study transgene dose-dependent mechanisms affecting cell fate decisions of genetically modified hematopoietic cells.
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Acknowledgements
This work was supported by the German Research Foundation (DFG, Research priority program SPP 1230 Ba1837/7-2, cluster of excellence REBIRTH Exc 62/1, SFB7308) and the European Union (grants PERSIST FP7-HEALTH-2007-B-222878 and CellPID FP7-HEALTH-2010-261387). We thank Christopher Baum for his support. We thank Niels Heinz and Bernhard Schiedlmeier for useful advice on Tet-regulated vectors. We thank Michael Morgan for carefully reading the final draft.
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Stahlhut, M., Schambach, A., Kustikova, O.S. (2016). Transduction of Murine Hematopoietic Stem Cells with Tetracycline-regulated Lentiviral Vectors. In: Federico, M. (eds) Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools. Methods in Molecular Biology, vol 1448. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3753-0_5
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DOI: https://doi.org/10.1007/978-1-4939-3753-0_5
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