Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Summary

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal 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.

Introduction

Technologies such as fluorescence spectroscopy, fluorescence microscopy and flow cytometry, all rely on fluorescence, a property widely exploited in biochemical, biomedical, and chemical applications. Fluorescence, whether intrinsic or through labeling, has been exploited for the analysis of protein expression patterns and profiles, cell fate, protein interactions and biological functions^1-9, and through fluorescence/Förster resonance energy transfer for the detection of biomolecule interactions and conformational changes^10-13. Since the isolation of the Aequorea victoria green fluorescent protein (GFP)¹⁴, the discovery o....

Protocol

1. Preparation of Mammalian Cells, Viral Production and Transduction for Genetic Barcoding

1. Plate 2.5 x 10⁶ adherent cells in a 10 cm plate (or around 50-60% confluency) one day prior to transfection in Dulbecco’s modified eagle medium (DMEM) with 10% fetal calf serum (FCS). For retroviral production use packaging cell-line of choice such as Phoenix-GP (a kind gift from Gary Nolan, Stanford University).
   NOTE: The packaging cell line stably expresses Gag and Pol proteins for retroviral particle formation in trans. Make sure cells are healthy and adhered to the plate in a monolayer, prior to transfection.
2. 24 hr later....

Results

Multiplexing fluorescent genetically barcoded cells for the purpose of biological applications can only be achieved once individual clonal populations have been generated. Multiplexing is most effective when barcoded populations have clear distinct fluorescent characteristics with minimal spectral overlap. The example shown in Figure 1 with clonal populations of mammalian SupT1 cells illustrates that barcoded cells with mCherry and cyano fluorescent protein (CFP) can be easily analyzed simultaneously wit.......

Discussion

Here two well-established procedures have been combined; genetic engineering through retroviral technology and detection of fluorescent proteins utilizing flow cytometry. Fluorescent protein-based genetic barcoding for the production of unique cell lines provides a robust and simple way for multiplexed applications. Generating genetically engineered barcoded cells through retroviral technology, is initially a lengthy process, but allows one to obtain, once established, a reliable and stable source of cell material. The n.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 ml syringes	BD	309604	used for filtering the virus
0.45 µm ploytetrafluoroethylene filter	Pall Corporation	4219	used for filtering the virus
DMEM (Dulbecco's Modified Eagle Medium)	Corning	45000-304	cell growth media for HEK 293T cells
PEI (Polyethylenimine)	poly sciences	23966-2	2 mg/ml concentration used
Hanging bucket centrifuge (refrigerated)	Eppendorf	5805 000.017	used for spin infection
PBS (phosphate buffered saline)	Corning	21-040-CV	used for washing of cells
Polybrene (hexadimethreen bromide)	Sigma-Aldrich	107689	Used to increase viral infection efficiency. Used at a 5 µg/ml concentration.
FACSAria	BD Biosciences		instrument used for sorting cell populations
FACSCanto	BD Biosciences		instrument used for cell analysis
Phoenix-GP	Gift from Gary Nolan		cell line used to produced retroviral particles
Fetal calf serum	Mediatech	MT35015CV	used for cell growth and sorting
SupT1 cells	ATCC	CRL-1942	Human T lymphoblasts
HEK 293T cells	ATCC	CRL-11268	Human Embryonic Kidney cells that also contain the SV40 large T-antigen
RPMI 1640	Corning	10-040-CV	cell growth media for SupT1 cells