Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

RGB marking facilitates multicolor clonal cell tracking


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: The principle of vector-mediated RGB marking.
Figure 2: LeGO-mediated RGB marking facilitates analysis of polyclonal liver regeneration.
Figure 3: Efficient RGB marking of colony-forming hematopoietic stem and progenitor cells.
Figure 4: Stable RGB marking of carcinogenic cell clones in vitro and in vivo.
Figure 5: Serial transplantation of RGB marked tumors.


  1. Shaner, N.C., Steinbach, P. & Tsien, R. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909 (2005).

    CAS  Article  Google Scholar 

  2. Giepmans, B.N., Adams, S.R., Ellisman, M.H. & Tsien, R.Y. The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 (2006).

    CAS  Article  Google Scholar 

  3. Vafaizadeh, V. et al. Mammary epithelial reconstitution with gene-modified stem cells assigns roles to Stat5 in luminal alveolar cell fate decisions, differentiation, involution and mammary tumor formation. Stem Cells 28, 928–938 (2010).

    CAS  PubMed  Google Scholar 

  4. Yamauchi, K. et al. Development of real-time subcellular dynamic multicolor imaging of cancer-cell trafficking in live mice with a variable-magnification whole-mouse imaging system. Cancer Res. 66, 4208–4214 (2006).

    CAS  Article  Google Scholar 

  5. Tysnes, B.B. Tumor-initiating and -propagating cells: cells that we would like to identify and control. Neoplasia 12, 506–515 (2010).

    CAS  Article  Google Scholar 

  6. Dick, J.E. Stem cell concepts renew cancer research. Blood 112, 4793–4807 (2008).

    CAS  Article  Google Scholar 

  7. Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).

    CAS  Article  Google Scholar 

  8. Weber, K., Bartsch, U., Stocking, C. & Fehse, B. A multi-color panel of novel lentiviral “gene ontology” (LeGO) vectors for functional gene analysis. Mol. Ther. 16, 698–706 (2008).

    CAS  Article  Google Scholar 

  9. Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    CAS  Article  Google Scholar 

  10. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

    CAS  Article  Google Scholar 

  11. Rizzo, M.A., Springer, G., Granada, B. & Piston, D. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).

    CAS  Article  Google Scholar 

  12. Kustikova, O.S. et al. Dose finding with retroviral vectors: Correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population. Blood 102, 3934–3937 (2003).

    CAS  Article  Google Scholar 

  13. Fehse, B., Kustikova, O.S., Bubenheim, M. & Baum, C. Pois(s)on—it's a question of dose.... Gene Ther. 11, 879–881 (2004).

    CAS  Article  Google Scholar 

  14. Wege, H. et al. Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential. Gastroenterology 124, 432–444 (2003).

    CAS  Article  Google Scholar 

  15. Dandri, M. et al. Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus. Hepatology 33, 981–988 (2001).

    CAS  Article  Google Scholar 

  16. Petersen, J. et al. Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein. Nat. Biotechnol. 26, 335–341 (2008).

    CAS  Article  Google Scholar 

  17. Hock, H. Some hematopoietic stem cells are more equal than others. J. Exp. Med. 207, 1127–1130 (2010).

    CAS  Article  Google Scholar 

  18. Metcalf, D. et al. Two distinct types of murine blast colony-forming cells are multipotential hematopoietic precursors. Proc. Natl. Acad. Sci. USA 105, 18501–18506 (2008).

    CAS  Article  Google Scholar 

  19. Townsend, C.M. Jr., Ishizuka, J. & Thompson, J.C. Studies of growth regulation in a neuroendocrine cell line. Acta Oncol. 32, 125–130 (1993).

    Article  Google Scholar 

  20. Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).

    CAS  Article  Google Scholar 

  21. Rieger, M.A., Hoppe, P.S., Smejkal, B.M., Eitelhuber, A.C. & Schroeder, T. Hematopoietic cytokines can instruct lineage choice. Science 325, 217–218 (2009).

    CAS  Article  Google Scholar 

  22. Cui, K. et al. Chromatin signatures in multipotent human hematopoietic stem cells indicate the fate of bivalent genes during differentiation. Cell Stem Cell 4, 80–93 (2009).

    CAS  Article  Google Scholar 

  23. Stein, S. et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat. Med. 16, 198–204 (2010).

    CAS  Article  Google Scholar 

  24. Kim, Y.J. et al. Sustained high-level polyclonal hematopoietic marking and transgene expression 4 years after autologous transplantation of rhesus macaques with SIV lentiviral vector–transduced CD34+ cells. Blood 113, 5434–5443 (2009).

    CAS  Article  Google Scholar 

  25. Aiuti, A. et al. Gene therapy for immunodeficiency due to adenosine deaminase deficiency. N. Engl. J. Med. 360, 447–458 (2009).

    CAS  Article  Google Scholar 

  26. Lütgehetmann, M. et al. In vivo proliferation of hepadnavirus-infected hepatocytes induces loss of covalently closed circular DNA in mice. Hepatology 52, 16–24 (2010).

    Article  Google Scholar 

  27. Novelli, M. et al. X-inactivation patch size in human female tissue confounds the assessment of tumor clonality. Proc. Natl. Acad. Sci. USA 100, 3311–3314 (2003).

    CAS  Article  Google Scholar 

  28. Leedham, S.J. & Wright, N.A. Human tumour clonality assessment—flawed but necessary. J. Pathol. 215, 351–354 (2008).

    CAS  Article  Google Scholar 

  29. Weber, K., Mock, U., Petrowitz, B., Bartsch, U. & Fehse, B. LeGO vectors equipped with novel drug-selectable fluorescent proteins—new building blocks for cell marking and multi-gene analysis. Gene Ther. 17, 511–520 (2010).

    CAS  Article  Google Scholar 

  30. Beyer, W.R., Westphal, M., Ostertag, W. & von Laer, D. Oncoretrovirus and lentivirus vectors pseudotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concentration and broad host range. J. Virol. 76, 1488–1495 (2002).

    CAS  Article  Google Scholar 

  31. Li, W.C., Ralphs, K.L. & Tosh, D. Isolation and culture of adult mouse hepatocytes. Methods Mol. Biol. 633, 185–196 (2010).

    CAS  Article  Google Scholar 

  32. Benten, D. et al. Hepatic targeting of transplanted liver sinusoidal endothelial cells in intact mice. Hepatology 42, 140–148 (2005).

    CAS  Article  Google Scholar 

  33. Schüler, A. et al. The MADS transcription factor Mef2c is a pivotal modulator of myeloid cell fate. Blood 111, 4532–4541 (2008).

    Article  Google Scholar 

  34. Schwieger, M. et al. Homing and invasiveness of MLL/ENL leukemic cells is regulated by MEF2C. Blood 114, 2476–2488 (2009).

    CAS  Article  Google Scholar 

  35. Weber, K & Fehse, B. Diva-Fit: a step-by-step manual for generating high-resolution graphs and histogram overlays of flow cytometry data obtained with FACSDiva software. Cell. Ther. Transplant. 1, e.000045.01 (2009).

    Google Scholar 

  36. Kustikova, O.S., Baum, C. & Fehse, B. Retroviral integration site analysis in hematopoietic stem cells. Methods Mol. Biol. 430, 255–267 (2008).

    CAS  Article  Google Scholar 

  37. Kustikova, O.S., Modlich, U. & Fehse, B. Retroviral insertion site analysis in dominant haematopoietic clones. Methods Mol. Biol. 506, 373–390 (2009).

    CAS  Article  Google Scholar 

  38. Cornils, K. et al. Stem cell marking with promotor-deprived self-inactivating retroviral vectors does not lead to induced clonal imbalance. Mol. Ther. 17, 131–143 (2009).

    CAS  Article  Google Scholar 

  39. Appelt, J.U. et al. QuickMap: a public tool for large-scale gene therapy vector insertion site mapping and analysis. Gene Ther. 16, 885–893 (2009).

    CAS  Article  Google Scholar 

  40. Rozen, S. & Skaletsky, H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365–386 (2000).

    CAS  Google Scholar 

Download references


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.

Author information

Authors and Affiliations



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.

Corresponding author

Correspondence to Boris Fehse.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Tables 1 and 2 (PDF 662 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Weber, K., Thomaschewski, M., Warlich, M. et al. RGB marking facilitates multicolor clonal cell tracking. Nat Med 17, 504–509 (2011).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing