Abstract
We simultaneously transduced cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. Individual cells were thereby marked by different combinations of inserted vectors, resulting in the generation of numerous mixed colors, a principle we named red-green-blue (RGB) marking. We show that lentiviral vector–mediated RGB marking remained stable after cell division, thus facilitating the analysis of clonal cell fates in vitro and in vivo. Particularly, we provide evidence that RGB marking allows assessment of clonality after regeneration of injured livers by transplanted primary hepatocytes. We also used RGB vectors to mark hematopoietic stem/progenitor cells that generated colored spleen colonies. Finally, based on limiting-dilution and serial transplantation assays with tumor cells, we found that clonal tumor cells retained their specific color-code over extensive periods of time. We conclude that RGB marking represents a useful tool for cell clonality studies in tissue regeneration and pathology.
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Acknowledgements
We wish to thank to V. Matzat and U. Bergholz for expert technical assistance, R. Reusch for excellent mouse care and J. Petersen for continuous support with the uPA model and critical reading of the manuscript. Confocal imaging was performed with the kind help from O. Bruns in collaboration with the Nikon Application Center Northern Germany (Nikon). We are indebted to many colleagues for their kind support with various cells and constructs: H. Wege (University Medical Center Hamburg-Eppendorf) for FH-hTERT, D. Hösch (University Marburg) for BON cells, W. Beyer (Heinrich-Pette-Institute) for vesicular stomatitis virus G protein cDNA R.Y. Tsien (Howard Hughes Medical Institute) for mCherry cDNA, A. Miyawaki (RIKEN) and T. Schroeder (Institute for Stem Cell Research) for Venus cDNA and D.W. Piston (Vanderbilt-Ingram Cancer Center) for Cerulean cDNA. This work was supported by the Deutsche Forschungsgemeinschaft (SFB841 to B.F., D.B. and M.D. and FE568/11-1 to B.F.). M. Thomaschewski was supported by the integrated graduate school “Inflammation and Regeneration” of the University Medical Center Hamburg-Eppendorf.
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K.W. designed the study, produced LeGO vectors and performed and analyzed gene transfer experiments in target cells in vitro and in vivo. M. Thomaschewski and M.W. isolated, transduced and transplanted primary hepatocytes and analyzed mice. M. Thomaschewski also performed mouse studies with BON tumor cells. T.V. and M.L. performed experiments in uPA mice. K.C. identified vector insertions by LM-PCR and performed specific PCRs. B.N., M. Täger and C.S. designed and performed experiments with mouse HSCs. J.-M.P. and M.L. provided and prepared primary human hepatocytes. M.D. provided the uPA-SCID model and supervised in vivo experiments in that model. D.B. designed and performed mouse studies. B.F. designed the study, analyzed and evaluated results and wrote the manuscript. All authors read and approved the final version of the manuscript.
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Weber, K., Thomaschewski, M., Warlich, M. et al. RGB marking facilitates multicolor clonal cell tracking. Nat Med 17, 504–509 (2011). https://doi.org/10.1038/nm.2338
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DOI: https://doi.org/10.1038/nm.2338
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