Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Abstract

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded ´indefinitely´. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing. Video LinkThe video component of this article can be found at

α-MHC MitoTimer mouse: In vivo mitochondrial turnover model reveals remarkable mitochondrial heterogeneity in the heart

“…C2C12 skeletal myoblasts (ATCC, #CRL-1772) were maintained in growth media consisting of DMEM (Gibco, #11995-073) containing 10% fetal bovine serum (Life Technologies #16010-159) and antibiotic/antimycotic (Life Technologies, #15240-062). The C2C12-mitoTimer cells were established utilizing self-inactivating retroviral particles [ 1 ]. Transduced cells were sorted by flow cytometry on a BD FACSAria II.…”

A Cell Double-Barcoding System for Quantitative Evaluation of Primary Tumors and Metastasis in Animals That Uncovers Clonal-Specific Anti-Cancer Drug Effects

“…Cell DNA barcoding allows dissecting many aspects of tumor dynamics, metastasis and drug effects [4][5][6][7][8][9][10][11][12][13][14]. Systems for cell barcoding include direct introduction of barcodes via lentiviral infection, CRISPR-based barcoding, or a combination of barcoding with fluorescent markers [9][10][11][14][15][16][17]. A very important advantage of these systems is high sensitivity and the ability to simultaneously monitor the fate of thousands and even millions of cells [5,6,18].…”

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“…Furthermore, this allows us to ensure stable, nondeteriorating expression of different fluorescent proteins at different intensity levels. 61,62 The utility of such formats was demonstrated by Stolp et al who used different cell-based platforms genetically barcoded for high-throughput screening purposes. 29,30 Here, we show additional multiplexing capabilities by combining two biological systems, each one interrogating an independent process: one that monitors cleavage by the viralencoded protease and the other that accurately detects cleavage by the host enzyme furin.…”

“…Furthermore, this allows us to ensure stable, nondeteriorating expression of different fluorescent proteins at different intensity levels. 61,62 The utility of such formats was demonstrated by Stolp et al who used different cell-based platforms genetically barcoded for high-throughput screening purposes. 29,30…”