Inducible gene and shRNA expression in resident hematopoietic stem cells in vivo.
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.
Laurenti
Elisa
E
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.
Barde
Isabelle
I
Verp
Sonia
S
Offner
Sandra
S
Wilson
Anne
A
Quenneville
Simon
S
Wiznerowicz
Maciej
M
Macdonald
H Robson
HR
Trono
Didier
D
Trumpp
Andreas
A
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation; D002478:Cells, Cultured; D004318:Doxycycline / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D004318:Doxycycline