RGB marking with lentiviral vectors for multicolor clonal cell tracking

Journal name:
Nature Protocols
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Published online
Corrected online


Cells transduced with lentiviral vectors are individually marked by a highly characteristic pattern of insertion sites inherited by all their progeny. We have recently extended this principle of clonal cell marking by introducing the method of RGB marking, which makes use of the simultaneous transduction of target cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. In accordance with the additive color model, individual RGB-marked cells display a large variety of unique and highly specific colors. Color codes remain stable after cell division and can thus be used for clonal tracking in vivo and in vitro. Our protocol for efficient RGB marking is based on established methods of lentiviral vector production (3–4 d) and titration (3 d). The final RGB-marking step requires concurrent transduction with the three RGB vectors at equalized multiplicities of infection (1–12 h). The initial efficiency of RGB marking can be assessed after 2–4 d by flow cytometry and/or fluorescence microscopy.

At a glance


  1. RGB marking of 293T cells with increasing amounts of RGB vectors.
    Figure 1: RGB marking of 293T cells with increasing amounts of RGB vectors.

    To identify optimal concentrations of the three RGB vectors, 293T cells were seeded at identical densities (50,000 cells per well of a 24-well plate), whereas amounts of vectors used for simultaneous transduction were increased stepwise by a factor of two starting from '0.5×' (compare Table 3). '1×' corresponds to a multiplicity of infection (MOI) of 0.7 per vector, which theoretically should result in a transduction rate of ~50% per vector17. (ae) The actual transduction efficiencies per RGB vector as estimated by FACS, as well as the numbers of nontransduced cells, are indicated below the pictures. (a) As is evident, a too-low MOI does not facilitate RGB marking. In our example, the majority of cells (more than two-thirds) is not gene marked, which is reflected by the large black areas. Those cells that were gene-modified mostly express only one of the basic colors. (b,c) Gene transfer rates between ~40 and 70% per vector result in efficient RGB marking. With increasing transduction efficiencies, numbers of nonmarked cells and cells displaying only basic colors rapidly decrease. (d,e) Further increases in gene transfer have deleterious effects. Although the number of nonmarked cells still becomes lower, the diversity of colors decreases again and a shift toward pastel and gray/white shades is observable. Moreover, very high transduction rates are associated with a significant impact on cell viability as indicated by the large 'holes' in the cell layer in e.

  2. Influence of exposure time on pictures of RGB-marked cells.
    Figure 2: Influence of exposure time on pictures of RGB-marked cells.

    Microscopic photographs of RGB-marked 293T cells. (ad) The multicolored images on the right were generated by overlaying three pictures obtained for the basic colors red, green and blue. Notably, the effect of instrument settings such as exposure times, white balance and camera gain on the eventual colors needs to be considered. Here, an example of the effect of exposure times for the three basic pictures is provided; all other parameters were identical. As is evident, independently of the exposure time, overlay of the three pictures taken with red, green and blue filters always result in efficient RGB coloring. (bd) However, shorter exposure times for red (b), green (c) and blue (d) result in lower intensity and, respectively, lower contribution of the named colors to RGB marking. Although this may not be relevant for one-time analysis, it becomes important for time kinetics when you need to follow up on given clones on the basis of their individual color codes.

  3. RGB-marked 293T cells.
    Figure 3: RGB-marked 293T cells.

    Microscopic photograph of RGB-marked 293T cells. The image was generated by overlaying three pictures obtained for the basic colors red, green and blue. After 4 d in culture, clones can easily be distinguished on the basis of their color.

  4. Theoretical approach to RGB marking.
    Figure 4: Theoretical approach to RGB marking.

    Distribution of color groups after RGB marking of cells. The mathematical set theory allows calculating the size of all color groups depending on the initial gene transfer rate for each of the three vectors. The mathematical model shown underlines the crucial impact of transduction rates on the efficiency of RGB marking. It is based on the assumption that all cells of the targeted population have the same probability to become transduced. Our own preliminary empirical data are in full accordance with this model (not shown). At low gene transfer rates, e.g., at 20% per color (x axis), a large proportion of cells express a single color ('single', about 38%), few cells express two colors ('double', about 10%) and very few cells express all three colors ('triple', about 1%). In this case, the overall gene transfer rate is about 50% ('sum'). At higher gene transfer rates, e.g., at 50% per color, almost the same number of cells express only a single color (37.5%), but the relative numbers of cells expressing two (37.5%) or three colors (12.5%) have increased markedly. The overall gene transfer rate, in a transduction rate of 50% per color, is 87.5%. With higher transduction rates for each single vector, the proportion of cells simultaneously expressing all three colors will rapidly increase, whereas the rate of nontransduced cells approaches zero. However, color diversity will decrease in favor of pastel, gray and white color shades. Single: sum of all cells expressing a single color only (red, green or blue); Double: the sum of all cells expressing two colors (red and green, red and blue or blue and green); triple: the sum of all cells expressing three colors (red, green and blue); sum: the sum of all cells expressing any color, which represents the overall transduction rate.

Change history

Corrected online 03 April 2015

In the version of this article initially published, the text 'RGB-marked 293T cells.' was missing from the legend of Figure 3 in the HTML version of the article. It originally read 'Microscopic photograph of The image was generated…' It should have read ‘Microscopic photograph of RGB-marked 293T cells. The image was generated…' The error has been corrected in the HTML version of the article.


  1. Barese, C.N. & Dunbar, C.E. Contributions of gene marking to cell and gene therapies. Hum. Gene Ther. 22, 659668 (2011).
  2. Wang, G.P. et al. Dynamics of gene-modified progenitor cells analyzed by tracking retroviral integration sites in a human SCID-X1 gene therapy trial. Blood 115, 43564366 (2010).
  3. Schumacher, T.N., Gerlach, C. & van Heijst, J.W. Mapping the life histories of T cells. Nat. Rev. Immunol. 10, 621631 (2010).
  4. Giepmans, B.N., Adams, S.R., Ellisman, M.H. & Tsien, R.Y. The fluorescent toolbox for assessing protein location and function. Science 312, 1724 (2006).
  5. Weber, K. et al. RGB marking facilitates multicolor clonal cell tracking. Nat. Med. 17, 504509 (2011).
  6. Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 5662 (2007).
  7. 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, 698706 (2008).
  8. Weber, K., Mock, U., Petrowitz, B., Bartsch, U. & Fehse, B. Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis. Gene Ther. 17, 511520 (2010).
  9. Tiscornia, G., Singer, O. & Verma, I.M. Production and purification of lentiviral vectors. Nat. Protoc. 1, 241245 (2006).
  10. Kutner, R.H., Zhang, X.Y. & Reiser, J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat. Protoc. 4, 495505 (2009).
  11. Geraerts, M., Willems, S., Baekelandt, V., Debyser, Z. & Gijsbers, R. Comparison of lentiviral vector titration methods. BMC Biotechnol. 6, 34 (2006).
  12. Strang, B.L., Ikeda, Y., Cosset, F.L., Collins, M.K. & Takeuchi, Y. Characterization of HIV-1 vectors with gammaretrovirus envelope glycoproteins produced from stable packaging cells. Gene Ther. 11, 591598 (2004).
  13. Schambach, A. et al. Lentiviral vectors pseudotyped with murine ecotropic envelope: increased biosafety and convenience in preclinical research. Exp. Hematol. 34, 588592 (2006).
  14. Rothe, M. et al. Epidermal growth factor improves lentivirus vector gene transfer into primary mouse hepatocytes. Gene Ther. doi:10.1038/gt.2011.117 (2011).
  15. Santoni de Sio, F. & Naldini, L. Short-term culture of human CD34+ cells for lentiviral gene transfer. Methods Mol. Biol. 506, 5970 (2009).
  16. Müller, L.U. et al. Rapid lentiviral transduction preserves the engraftment potential of Fanca−/− hematopoietic stem cells. Mol. Ther. 16, 11541160 (2008).
  17. Fehse, B., Kustikova, O.S., Bubenheim, M. & Baum, C. Pois(s)on—it's a question of dose.... Gene Ther. 11, 879881 (2004).
  18. Modlich, U. et al. Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis. Blood 105, 42354246 (2005).
  19. O'Doherty, U., Swiggard, W.J. & Malim, M.H. Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. J. Virol. 74, 1007410080 (2000).
  20. Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905909 (2005).
  21. Rizzo, M., Springer, G., Granada, B. & Piston, D. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445449 (2004).
  22. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 8790 (2002).
  23. Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 15671572 (2004).

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

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

    • Daniel Benten


K.W. and B.F. developed the idea of RGB marking. K.W. and M.T. produced LeGO vectors and performed and analyzed gene transfer experiments in different target cells. K.W. and B.F. designed the study and wrote the manuscript. D.B. and B.F. supervised experiments, analyzed and evaluated results. All authors read and approved the final version of the manuscript.

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