RGB marking facilitates multicolor clonal cell tracking

Abstract

We simultaneously transduced cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. Individual cells were thereby marked by different combinations of inserted vectors, resulting in the generation of numerous mixed colors, a principle we named red-green-blue (RGB) marking. We show that lentiviral vector-mediated RGB marking remained stable after cell division, thus facilitating the analysis of clonal cell fates in vitro and in vivo. Particularly, we provide evidence that RGB marking allows assessment of clonality after regeneration of injured livers by transplanted primary hepatocytes. We also used RGB vectors to mark hematopoietic stem/progenitor cells that generated colored spleen colonies. Finally, based on limiting-dilution and serial transplantation assays with tumor cells, we found that clonal tumor cells retained their specific color-code over extensive periods of time. We conclude that RGB marking represents a useful tool for cell clonality studies in tissue regeneration and pathology.

Long-term in vivo single-cell tracking reveals the switch of migration patterns in adult-born juxtaglomerular cells of the mouse olfactory bulb

“…In fact, the deceleration of the cells in the course of migration ( Figures 4A and 7E) and existence of a number of additional criteria (e.g., the soma size, the direction of the leading process as indicator of migration direction, the ratio of the fluorescence recorded in different channels (proportional to the number of incorporated virus copies and the strength of FP expression) and the existence of stationary cells in the neighborhood) reduce the ambiguity in the system even further. It has to be noticed that the RGB-marking approach is not restricted to seven colors and, as shown previously [21,26], can provide a large number of different color hues. The precise readout of these hues, however, requires more sophisticated imaging and color-identification approaches, which seem unnecessary within the framework of the present study.…”

“…Specific multicolor labeling of individual adult-born neuroblasts was achieved using red-green-blue (RGB) cell-marking approach, utilizing simultaneous, viral vector-mediated expression of genes encoding fluorescent proteins (FPs) in the three basic colors mCherry (red), Venus (green) and Cerulean (blue) [21]. To enable monitoring of RGB-marked cells by means of in vivo two-photon imaging, we examined the excitation/emission spectra of each fluorophore.…”

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

“…8,9 The number of exploitable fluorescent colors can be significantly increased by applying combinatorial labeling strategies such as red-green-blue (RGB) marking. 10,11 The latter is based on the transduction of target cells with integrating retro-/lentiviral vectors that mediate highly variable expression of random combinations of these three basic colors (RGB), thus generating all possible color combinations. However, RGB marking does not permit precise quantification or recovery of single clones out of complex and heterogeneous subpopulations by fluorescent-activated cell sorting (FACS).…”

A Lentiviral Fluorescent Genetic Barcoding System for Flow Cytometry-Based Multiplex Tracking

“…12 In addition, marking approaches based on the combinatorial transduction of adherent cells with red (R), green (G), and blue (B) fluorescent protein encoding (RGB) vectors may produce a theoretical maximum of millions of colors. 13 Both systems depend on computer-assisted fluorescence microscopy for identifying clonal cell populations by their local proximity and the expression of common color codes, which prevents their utilization for the flow cytometric characterization of non-adherent cells. 13 Therefore, multiplex flow cytometry assays have been developed on the basis of "fluorescent cell barcoding" (FCB).…”

“…13 Both systems depend on computer-assisted fluorescence microscopy for identifying clonal cell populations by their local proximity and the expression of common color codes, which prevents their utilization for the flow cytometric characterization of non-adherent cells. 13 Therefore, multiplex flow cytometry assays have been developed on the basis of "fluorescent cell barcoding" (FCB). 14 This technique relies on the staining of individual samples with a unique color code prior to sample pooling and optional epitope staining before simultaneously acquiring all samples from a single tube.…”