RGB marking facilitates multicolor clonal cell tracking

Journal name:
Nature Medicine
Year published:
Published online


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.

At a glance


  1. The principle of vector-mediated RGB marking.
    Figure 1: The principle of vector-mediated RGB marking.

    (a) Based on color theories, mixing red, green and blue at one intensity should result in four mixed colors: yellow, violet, turquoise and white. (b) If the basic colors are mixed at all possible intensities, a full spectrum of rainbow colors is generated. (c) Concurrent vector-mediated introduction of three fluorescent proteins emitting red, green and blue, for example, mCherry (red), Venus (yellow-green) and Cerulean (blue) should in principle lead to seven types of transduced cells. According to the RGB principle, simultaneous expression of different basic colors in the cells could be expected to result in mixed colors (compare also Supplementary Fig. 2). (d) Lentiviral vector-mediated RGB marking of the indicated target cells using LeGO-C2, LeGO-V2 and LeGO-Cer2 (Supplementary Fig. 1). Shown are overlays of three color photographs taken consecutively with red, green and blue filters using a fluorescence microscope. All cells were cultured for approximately 72 h after plating; whereas 293T clones have stayed together during culture, FH-hTERT clones have spread over the tissue culture vessel. Primary hepatocytes have not replicated during culture.

  2. LeGO-mediated RGB marking facilitates analysis of polyclonal liver regeneration.
    Figure 2: LeGO-mediated RGB marking facilitates analysis of polyclonal liver regeneration.

    (a) In vitro analysis of primary hepatocytes confirming efficient RGB marking and generation of numerous different mixed colors. (b) Liver sections from uPA-SCID mice 1 month after transplantation with RGB-marked primary mouse hepatocytes indicate polyclonal engraftment. (c) A liver section regenerated by engrafted RGB-marked primary hepatocytes is depicted (same experiment as in b). The apparently one big blue clone (marked by an asterisk in the Cerulean image) in fact consists of three different clones that engrafted in the same area (overlay). These clones (indicated by arrows) are easily distinguishable on the basis of RGB marking, as they express one (blue middle clone), two (cyan top clone) or all three colors (white bottom clone) at the same time. All images represent overlays of three individual color photographs taken consecutively with red, green and blue filters using a fluorescence microscope.

  3. Efficient RGB marking of colony-forming hematopoietic stem and progenitor cells.
    Figure 3: Efficient RGB marking of colony-forming hematopoietic stem and progenitor cells.

    (a) RGB marking of HSCs and HPCs in vitro. (b) RGB-marked progenitors that generated different types of colonies in methyl cellulose (see also Supplementary Fig. 4). (c) Spleen of a C57BL/J6 mouse 10 d after transplantation of syngeneic RGB-marked HSCs and HPCs. CFU-S are easily distinguishable. (d) RGB marking of CFU-S in vivo. Images in a, b and d represent overlays of three individual color photographs taken consecutively with red, green and blue filters using a fluorescence microscope. The image in c was taken with a standard digital camera.

  4. Stable RGB marking of carcinogenic cell clones in vitro and in vivo.
    Figure 4: Stable RGB marking of carcinogenic cell clones in vitro and in vivo.

    (a) LeGO vector-mediated RGB marking of BON carcinoma cells in vitro. (b) Homogenous coloring of single-cell derived RGB-marked BON clones expanded in vitro. (c) Outgrowth of multiple liver tumors in NOD-SCID mice (shown are whole-liver sections) after transplantation of the two clones presented in b. Images in a and b were taken with a standard fluorescence microscope, image c with a confocal microscope. (d) Vector insertion site–specific PCR reactions confirming the identity of engrafted tumors with the ex vivo–expanded founder clones on the molecular level (for example, D3-specific PCR was positive on a tumor derived from clone D3, but negative on tumors derived from clones D5 and E3). M, marker ladder for DNA size.

  5. Serial transplantation of RGB marked tumors.
    Figure 5: Serial transplantation of RGB marked tumors.

    (a) Outgrowth of multiple RGB-marked liver tumors after transplantation of RGB-marked BON carcinoma cells (compare Fig. 4a). (b) Two examples of tumors (T1, T2) explanted from liver tissue. The insets show the same fields of view photographed with a phase-contrast microscope. (c) Cultured cells derived from explanted tumors displayed the same color as the initial tumors (T1 and T2). (d,e) Retransplantation of these tumor cells resulted in engraftment of uniformly colored tumors in secondary recipients resembling the color of the initial tumors T1 and T2. In d, single tumors are shown, whereas e shows whole-liver sections. (f) Infusion of mixed cells derived from tumors T1 and T2 resulted in three types of tumors: monochromatic violet (resembling T1) and yellow ones (resembling T2), as well as tumors containing cells of both violet and yellow colors. Images in a, e and f were taken with a confocal microscope, whereas images in b to d were generated with a standard fluorescence microscope. (g) Insertion site–specific PCR reactions confirming clonal identity of serially transplanted tumors.


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Author information


  1. Research Department Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

    • Kristoffer Weber,
    • Michael Thomaschewski,
    • Michael Warlich,
    • Kerstin Cornils &
    • Boris Fehse
  2. Internal Medicine I, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

    • Michael Warlich,
    • Tassilo Volz,
    • Marc Lütgehetmann,
    • Maura Dandri &
    • Daniel Benten
  3. Heinrich-Pette-Institute, Leibniz-Institute for Experimental Virology, Hamburg, Germany.

    • Birte Niebuhr,
    • Maike Täger &
    • Carol Stocking
  4. Department of Hepatobiliary and Transplant Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

    • Jörg-Matthias Pollok


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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