RGB marking with lentiviral vectors for multicolor clonal cell tracking

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.