Inducible Gene and shRNA Expression in Resident Hematopoietic Stem Cells In Vivo§

Authors

  • Elisa Laurenti,

    1. Ecole Polytechnique Fédérale de Lausanne (EPFL), ISREC - Swiss Institute for Experimental Cancer Research, School of Life Science, and “Frontiers in Genetics” National Center for Competence in Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
    2. Ludwig Institute for Cancer Research Ltd., Lausanne Branch, University of Lausanne, Switzerland
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  • Isabelle Barde,

    1. Ecole Polytechnique Fédérale de Lausanne (EPFL), ISREC - Swiss Institute for Experimental Cancer Research, School of Life Science, and “Frontiers in Genetics” National Center for Competence in Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
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  • Sonia Verp,

    1. Ecole Polytechnique Fédérale de Lausanne (EPFL), ISREC - Swiss Institute for Experimental Cancer Research, School of Life Science, and “Frontiers in Genetics” National Center for Competence in Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
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  • Sandra Offner,

    1. Ecole Polytechnique Fédérale de Lausanne (EPFL), ISREC - Swiss Institute for Experimental Cancer Research, School of Life Science, and “Frontiers in Genetics” National Center for Competence in Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
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  • Anne Wilson,

    1. Ludwig Institute for Cancer Research Ltd., Lausanne Branch, University of Lausanne, Switzerland
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  • Simon Quenneville,

    1. Ecole Polytechnique Fédérale de Lausanne (EPFL), ISREC - Swiss Institute for Experimental Cancer Research, School of Life Science, and “Frontiers in Genetics” National Center for Competence in Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
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  • Maciej Wiznerowicz,

    1. Ecole Polytechnique Fédérale de Lausanne (EPFL), ISREC - Swiss Institute for Experimental Cancer Research, School of Life Science, and “Frontiers in Genetics” National Center for Competence in Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
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  • H. Robson MacDonald,

    1. Ludwig Institute for Cancer Research Ltd., Lausanne Branch, University of Lausanne, Switzerland
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  • Didier Trono,

    1. Ecole Polytechnique Fédérale de Lausanne (EPFL), ISREC - Swiss Institute for Experimental Cancer Research, School of Life Science, and “Frontiers in Genetics” National Center for Competence in Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
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  • Andreas Trumpp

    Corresponding author
    1. Ecole Polytechnique Fédérale de Lausanne (EPFL), ISREC - Swiss Institute for Experimental Cancer Research, School of Life Science, and “Frontiers in Genetics” National Center for Competence in Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
    2. Division of Stem Cells and Cancer, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
    3. Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
    • Ecole Polytechnique Fédérale de Lausanne (EPFL), ISREC - Swiss Institute for Experimental Cancer Research, School of Life Science, and “Frontiers in Genetics” National Center for Competence in Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
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  • Author contributions: E.L. designed and performed most experiments, analyzed data and wrote the paper, I.B. designed and performed experiments and produced lentiviral vectors, S.V. and S.O. performed experiments and produced lentiviral vectors, A.W. conducted the cell cycle analysis after Dox treatment, S.Q. designed and cloned the shRNA vector, M.W. built several constructs including tTR-KRAB expression vectors, H.R. McD. was involved in planning of experiments and interpretation of data; D.T. designed the strategy, supervised the work, and wrote the paper. A.T coordinated the entire project, designed the strategy, supervised the work, and wrote the paper.

  • Disclosure of potential conflicts of interest is found at the end of this article.

  • §

    First published online in STEM CELLS EXPRESS July 16, 2010.

Abstract

Hematopoietic stem cells (HSC) are probably the best understood somatic stem cells and often serve as a paradigm for other stem cells. Nevertheless, most current techniques to genetically manipulate them in vivo are either constitutive and/or induced in settings of hematopoietic stress such as after irradiation. Here, we present a conditional expression system that allows for externally controllable transgenesis and knockdown in resident HSCs, based on a lentiviral vector containing a tet-O sequence and a transgenic mouse line expressing a doxycyclin-regulated tTR-KRAB repressor protein. HSCs harvested from tTR-KRAB mice are transduced with the lentiviral vector containing a cDNA (i.e., Green Fluorescent Protein (GFP)) and/or shRNA (i.e., p53) of interest and then transplanted into lethally irradiated recipients. While the vector is effectively repressed by tTR-KRAB during homing and engraftment, robust GFP/shp53 expression is induced on doxycyclin treatment in HSCs and their progeny. Doxycylin-controllable transcription is maintained on serial transplantation, indicating that repopulating HSCs are stably modified by this approach. In summary, this easy to implement conditional system provides inducible and reversible overexpression or knock down of genes in resident HSCs in vivo using a drug devoid of toxic or activating effects. STEM CELLS 2010;28:1390–1398

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