Name: genetic barcoding with fluorescent proteins for multiplexed applications
Uploaded: 2015-04-14T13:00:00
Description: Watch this Scientific Journal Video about Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications at JoVE.com

We would like to thank Dr. Garry Nolan from Stanford University for providing the Phoenix GP packaging cell line for the production of retroviral particles. We thank Dr. Roger Tsien at University of California San Diego for providing td Tomato. We would also like to thank the San Diego State University Flow Cytometry Core Facility for their service and help.

1. Preparation of Mammalian Cells, Viral Production and Transduction for Genetic Barcoding
<ol>
	<li>Plate 2.5 x 106 adherent cells in a 10 cm plate (or around 50-60% confluency) one day prior to transfection in Dulbecco&#8217;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 p...

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...

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 functions1-9, and through fluorescence/F&#246;rster resonance energy transfer for the detection of biomolecule interactions and conformational changes10-13. Since the isolation of the Aequorea victoria green fluorescent protein (GFP)14, the discovery of additional naturally occurring fluorescent proteins from other cnidarians, particularly corals, has largely increased the number of existing fluorescent proteins with distinguishable excitation/emission spectra. These, together with the introduction of mutations in their genes15-19, have further expanded the possibilities, obtaining a true palette of fluorescent proteins available to scientists that exploit microscopy, flow cytometry and other fluorescence-based technologies for their research.
In parallel, although independently, the development of retroviral technology has drastically facilitated the stable expression of ectopic genetic information in mammalian cells20-23. It is thus not surprising that this technology has been used to transfer genes of fluorescent proteins into a broad number of cell types and tissues24-28 or for production of transgenic animals29-31. Following the nature of retroviruses, the genetic information of the ectopic fluorescent protein is introduced within the genome of the cell32 and the cell becomes fluorescent `for ever&#180;. This property has allowed tracking of cell fate, or of a single cell within a population of cells. The now fluorescent cell has thus acquired its own biosignature and can be defined as barcoded. Its unique biosignature identifies it from other cells, and importantly, distinguishes it from cells genetically manipulated to express different fluorescent proteins with distinguishable absorption/emission spectra. Biological applications such as the tracking of reprogramming factors toward pluripotency33, the analysis of subnuclear factors for the elucidation of nucleolar localization34, the construction of fluorescent reporter plasmids for transcriptional studies35 or the genetic labeling of neurons for the study of neuronal network architecture36, are just four examples of the many that have exploited different fluorescent protein genes for the same experimental setup.
Flow cytometry has been broadly utilized for the analysis of biological processes at the single cell level, such as gene expression, cell cycle, apoptosis, and signaling through phosphorylation37-43.The stable expression of fluorescent protein genes in mammalian cells has further enhanced the utility of flow cytometry for cell analysis38,44 and ligand-receptor interactions45. Enhanced capabilities have allowed flow cytometry to become a widely utilized methodology for high-throughput and high-content screening46. Despite the now expanded number of fluorometers and robotics technologies that can couple plate reader systems, imaging and flow cytometry, there seems to be a lack in experimental design that can exploit and fit these enhanced technological capabilities.
Fast, reliable, simple and robust cell-based methodologies are drastically needed for multiplexed applications that further enhance high-throughput capacity. This is especially true in the field of drug discovery where engineering cell-based assays in a multiplexed format can enhance the power of high-throughput screening39,47-50. Multiplexing, as it allows simultaneous analyses in one sample, further enhances high-throughput capabilities51-54. 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 stains39,52,55, 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 activity56,57 with different clinically prevalent variants58. 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. Panels of cell populations distinguishable based on their fluorescent characteristics enhance multiplexed capabilities, which can be further exploited in combination with cell-based assays that tackle different biological questions. The protocol also describes how to engineer a panel of barcoded cells bearing one of the cell-based assays previously developed in the laboratory, as example59. This protocol is thus not intended to show the well-established retroviral/lentiviral technology for genetic transfer, the value of fluorescent proteins or the applications of flow cytometry60,48 but rather to show the enhancing power of combining the three for multiplexed applications.

<table border="0" cellpadding="0" cellspacing="0" width="1138">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Name of Material/ Equipment</td>
			<td style="width:160px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:515px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">10mL syringes</td>
			<td style="width:160px;">BD&#160;&#160;</td>
			<td>309604</td>
			<td>used for filtering the virus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45&#181;m plytetrafluoroethylen filter</td>
			<td style="width:160px;">pall corporation</td>
			<td>4219</td>
			<td>used for filtering the virus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td style="width:160px;">Corning</td>
			<td style="width:119px;">45000-304</td>
			<td>cell growth media for HEK 293T cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PEI (Polyethylenimine)</td>
			<td style="width:160px;">poly sciences</td>
			<td>23966-2</td>
			<td>&#160;2mg/mL concentration used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Hanging bucket centrifuge (refrigerated)</td>
			<td style="width:160px;">Eppendorf&#160;</td>
			<td style="width:119px;">5805 000.017</td>
			<td>used for spin infection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">PBS (phosphate buffered saline)</td>
			<td style="width:160px;">Corning</td>
			<td>21-040-CV</td>
			<td>used for washing of cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Polybrene (hexadimethreen bromide)</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td>107689</td>
			<td>Used to increase viral infection efficiency.&#160; Used at a 5&#181;g/mL concentration.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">FACSAria</td>
			<td style="width:160px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td>instrument used for sorting cell populations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">FACSCanto</td>
			<td style="width:160px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td>instrument used for cell analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Phoenix-GP</td>
			<td style="width:160px;">Gift from Gary Nolan</td>
			<td style="width:119px;"></td>
			<td>cell line used to produced retroviral particles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Fetal calf serum</td>
			<td style="width:160px;">Mediatech</td>
			<td>MT35015CV&#160;</td>
			<td>used for cell growth and sorting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">&#160;SupT1 cells</td>
			<td style="width:160px;">ATCC</td>
			<td>CRL-1942</td>
			<td>Human T lymphoblasts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">HEK 293T cells</td>
			<td style="width:160px;">ATCC</td>
			<td>CRL-11268</td>
			<td>Human Embryonic Kidney cells that also contain the SV40 large T-antigen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">RPMI 1640</td>
			<td style="width:160px;">Corning</td>
			<td>10-040-CV</td>
			<td>cell growth media for SupT1 cells</td>
		</tr>
	</tbody>
</table>

genetic barcoding with fluorescent proteins for multiplexed applications

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 &#180;indefinitely&#180;. 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.

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...

Watch this Scientific Journal Video about Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications at JoVE.com

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

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.

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

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