Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry

Abstract

The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/ emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis. V C 2013 International Society for Advancement of Cytometry

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

“…Recently, fluorescent genetic barcoding, 14 an alternative approach allowing flow cytometry-mediated clonal tracking, was suggested. That approach nicely demonstrated that hepatocytic and T cell lines can be fluorescently labeled with three colors and can subsequently be single-cell sorted to generate individual clones barcoded by different fluorescence intensities.…”

“…13 A recent advancement of the FCB approach, termed fluorescent genetic barcoding (FGB), harnesses the retroviral expression of up to three fluorescent proteins additionally stratified by two different fluorescence intensities. 14 The FGB technique permits clonal cell behavior monitoring over time in vitro for up to 12 different clones, as long as fluorescence signals of analyzed cells are bright enough to facilitate clear-cut identification of two stacked intensity levels. Furthermore, the development of the Brainbow mice was based on stochastic recombination of fluorescent proteins exploiting the Cre/ lox system, thereby creating a mosaic gene expression that allows for in vivo analysis of neuronal connections.…”

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

“…As a consequence, the 1G-FGB system appears most suitable for applications that permit the enrichment of pure color-coded populations for longitudinal multiplexing studies, while circumventing the need for FCB-mediated staining procedures. 14,15 To accelerate the generation of double and triple marker expressing color-coded cells, we next developed a silencing-resistant coexpression system based on a chimeric CBX3-SFFV promoter and 2A cleavage sites (Figure 2A). Owing to cargo size constrains of the lentiviral vector system, 27 we utilized a minimal 700 bp CBX3 fragment with proven anti-silencing function in P19 and induced pluripotent stem cells.…”

“…For this purpose, a "fluorescent genetic barcoding" (FGB) approach has been developed, which relies on stable gammaretroviral gene transfer of fluorescent marker cassettes into target cell populations, and yielded up to 12 color-coded populations derived from 1-3 fluorescent markers. 15 Despite these encouraging results, the currently available FGB system can mainly be applied to multiplexing experiments with cell lines, due to the requirement for extended cultivation times facilitating cell expansion and sorting of pure color-coded populations. Furthermore, the requirement for multiple vector copies per cell for producing color codes composed of different fluorescent markers and staggered expression intensities offers the risk for longitudinal changes in population identity caused by vector silencing or variegation of vector expression.…”

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

“…Fluorescent genetic barcoding not only allows for elegant multiplexing, but also, once engineered, circumvents the need of time consuming protocols, reduces costs accompanied with antibodies, beads and stains 39,52,55 , and can reduce the number of screens required for high-throughput applications. We have recently described how retroviral technology can enhance multiplexing through fluorescent genetic barcoding for biological applications, by expressing an assay previously developed to monitor HIV-1 protease activity 56,57 with different clinically prevalent variants 58 . The methodology is explained in a more descriptive manner focusing on how to select and amplify genetically fluorescent barcoded cells and how to produce panels of clonal populations expressing distinct fluorescent proteins and/or different fluorescence intensities.…”