Research Department Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Germany.
Nat Protoc. 2012 Apr 5;7(5):839-49. doi: 10.1038/nprot.2012.026.
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.
用慢病毒载体转导的细胞通过其所有后代继承的高度特征性插入位点模式被单独标记。我们最近通过引入 RGB 标记方法扩展了克隆细胞标记的这一原则，该方法利用同时转导靶细胞与三个编码红色、绿色或蓝色荧光蛋白的慢病毒基因本体论 (LeGO) 载体。根据加色模型，单个 RGB 标记的细胞显示出大量独特且高度特异性的颜色。颜色代码在细胞分裂后保持稳定，因此可用于体内和体外的克隆跟踪。我们用于有效 RGB 标记的方案基于已建立的慢病毒载体生产（3-4 天）和滴定（3 天）方法。最后一步的 RGB 标记需要以相等的感染复数（1-12 小时）同时转导三个 RGB 载体。通过流式细胞术和/或荧光显微镜可以在 2-4 天后评估初始的 RGB 标记效率。
