TY - JOUR
T1 - An inducible Tet-Off-H2B-GFP lentiviral reporter vector for detection and in vivo isolation of label-retaining cells
AU - Falkowska-Hansen, Berit
AU - Kollar, Jasmin
AU - Grüner, Barbara Maria
AU - Schanz, Merle
AU - Boukamp, Petra
AU - Siveke, Jens
AU - Rethwilm, Axel
AU - Kirschner, Marc
N1 - Funding Information:
We thank Steffen Schmitt, Klaus Hexel and Oana Butto for excellent assistance in flow cytometry, FACSorting, immunoblot analysis and recombinant DNA techniques. We also thank Elisa Opitz for developing the lentiviral infection protocol. We thankfully acknowledge Prof. C. Beltinger (University Children's Hospital, Ulm, Germany), Prof. T. Pietsch (Universitätsklinikum Bonn, Germany) and Prof. L. Rudolph (Department of Molecular Medicine and Max-Planck-Research Group on Stem Cell Aging, Ulm, Germany) for providing cell lines for this study. This work is part of project 7 of the Research Consortium Tumor Stem Cells funded by the Deutsche Krebshilfe (German Cancer Aid to A.R. and P.B.).
PY - 2010/7
Y1 - 2010/7
N2 - Many regenerative cells are label-retaining cells (LRCs) due to their ability to keep a DNA label over a prolonged time. Until recently, isolation of vital LRCs was hampered due to the necessary use of fixation methods. To circumvent this, we generated a lentiviral-(HIV-1) based vector expressing a Tet-Off controlled histone 2B-GFP (Tet-Off-H2B-GFP) reporter gene for the detection and isolation of viable LRCs. In initial experiments, the vector was successfully used to infect 2- and 3-dimensional tissue culture models. Infected cultures from skin and pancreatic cells showed a very tight regulation of H2B-GFP, were sensitive to minimal amounts of doxycycline (Dox) and had a stable transgenic expression over the time of this study. Our lentiviral vector represents a reliable and easy to handle system for the successful infection, detection and isolation of LRCs from various tissues in vitro, in vivo and ex vivo.
AB - Many regenerative cells are label-retaining cells (LRCs) due to their ability to keep a DNA label over a prolonged time. Until recently, isolation of vital LRCs was hampered due to the necessary use of fixation methods. To circumvent this, we generated a lentiviral-(HIV-1) based vector expressing a Tet-Off controlled histone 2B-GFP (Tet-Off-H2B-GFP) reporter gene for the detection and isolation of viable LRCs. In initial experiments, the vector was successfully used to infect 2- and 3-dimensional tissue culture models. Infected cultures from skin and pancreatic cells showed a very tight regulation of H2B-GFP, were sensitive to minimal amounts of doxycycline (Dox) and had a stable transgenic expression over the time of this study. Our lentiviral vector represents a reliable and easy to handle system for the successful infection, detection and isolation of LRCs from various tissues in vitro, in vivo and ex vivo.
KW - H2B-GFP
KW - Isolation of LRCs
KW - Label-retaining cells
KW - Lentiviral
KW - Tet-Off
UR - http://www.scopus.com/inward/record.url?scp=77953232124&partnerID=8YFLogxK
U2 - 10.1016/j.yexcr.2010.02.015
DO - 10.1016/j.yexcr.2010.02.015
M3 - Article
AN - SCOPUS:77953232124
SN - 0014-4827
VL - 316
SP - 1885
EP - 1895
JO - Experimental Cell Research
JF - Experimental Cell Research
IS - 11
ER -
