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.
1549-4918/asset/olbannerleft.jpg?v=1&s=699114e871887e6b838f6a1c657fe256cfe127a6)
1549-4918/asset/olbannerright.gif?v=1&s=603f8f2ab5cd9d4f783c231915608956af51aeea)
Tissue-Specific Stem Cells
You have full text access to this OnlineOpen article
Inducible Gene and shRNA Expression in Resident Hematopoietic Stem Cells In Vivo†‡§
Article first published online: 16 JUL 2010
DOI: 10.1002/stem.460
Copyright © 2010 AlphaMed Press
Additional Information
How to Cite
Laurenti, E., Barde, I., Verp, S., Offner, S., Wilson, A., Quenneville, S., Wiznerowicz, M., MacDonald, H. R., Trono, D. and Trumpp, A. (2010), Inducible Gene and shRNA Expression in Resident Hematopoietic Stem Cells In Vivo. STEM CELLS, 28: 1390–1398. doi: 10.1002/stem.460
- †
- ‡
Disclosure of potential conflicts of interest is found at the end of this article.
- §
First published online in STEM CELLS EXPRESS July 16, 2010.
Publication History
- Issue published online: 18 AUG 2010
- Article first published online: 16 JUL 2010
- Manuscript Accepted: 24 MAY 2010
- Manuscript Received: 25 FEB 2010
Funded by
- Swiss National Science Foundation
- Swiss Cancer League
- EU- FP6 Programs “CONSERT”
- EU-FP7 Programs “EuroSyStem”
- PERSIST
Keywords:
- Hematopoietic stem cells;
- In vivo inducible genetic manipulation;
- Doxycycline;
- tTR-KRAB;
- Hematopoiesis;
- Lentiviral vectors
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