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RGB marking with lentiviral vectors for multicolor clonal cell tracking

An Erratum to this article was published on 28 May 2015

This article has been updated


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.

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Figure 1: RGB marking of 293T cells with increasing amounts of RGB vectors.
Figure 2: Influence of exposure time on pictures of RGB-marked cells.
Figure 3: RGB-marked 293T cells.
Figure 4: Theoretical approach to RGB marking.

Change history

  • 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.


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We are indebted to many colleagues for their kind support with various cells and constructs. Particularly we want to thank R.Y. Tsien (Howard Hughes Medical Institute) for mCherry cDNA, A. Miyawaki (RIKEN) and T. Schroeder (Institute for Stem Cell Research) for Venus cDNA, D.W. Piston (Vanderbilt-Ingram Cancer Center) for Cerulean cDNA and W. Beyer (Heinrich-Pette-Institute) for VSV-G. Flow cytometry work was performed in the FACS Sorting Core Unit of the UMC Hamburg-Eppendorf. This work was supported by the Deutsche Forschungsgemeinschaft (SFB841 to B.F., D.B.; FE568/11-1 to B.F.). K.W. has received a young investigator grant within the Forschungsförderungsfonds der Medizinischen Fakultät (FFM) program of the UMC Hamburg-Eppendorf (NWF-12/09). M.T. was supported by the Integrated graduate school 'Inflammation and Regeneration' of the UMC Hamburg-Eppendorf.

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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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Correspondence to Boris Fehse.

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The authors declare no competing financial interests.

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Weber, K., Thomaschewski, M., Benten, D. et al. RGB marking with lentiviral vectors for multicolor clonal cell tracking. Nat Protoc 7, 839–849 (2012).

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