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Nucleic Acids Res. 2014 Apr;42(7):e56. doi: 10.1093/nar/gku081. Epub 2014 Jan 29.

Multiplexing clonality: combining RGB marking and genetic barcoding.

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Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Martinistrasse 52, Hamburg 20246, Germany, Institute for Medical Informatics and Biometry, Faculty of Medicine, Technische Universität Dresden, Dresden 01307, Germany, ALS Automated Lab Solutions GmbH, Jena 07747, Germany, Department of Neuropathology, Hannover Medical School, Institute of Pathology, Hannover 30625, Germany, Department of Internal Medicine I, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany, Deep Sequencing Group SFB 655, Biotechnology Center, Technische Universität Dresden, Dresden 01307, Germany.


RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies.

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