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 particle formation in trans. Make sure cells are healthy and adhered to the plate in a monolayer, prior to transfection.</li>
	<li>24 hr later, prepare the DNA transfection mixture.
	<ol>
		<li>To 125 &#956;l of serum-free DMEM add 3 &#956;g Vesicular Stomatitis Virus Envelope glycoprotein vector (pCI-VSVg) and 3 &#956;g of transfer vector carrying the fluorescent protein gene of interest. Mix and add 15 &#956;l of polyethylimine (2 mg/ml).</li>
		<li>Incubate mixture at room temperature for 15 min and add to cells drop-wise. 
		NOTE: Polyethylimine is added to the homogenous DNA mixture as transfection reagent for the formation of polyplexes. In order to obtain viral particles carrying different fluorescent protein markers, perform each transfection independently with the marker of choice. Remember that the retroviral transfer vector is around 7 kb in length and cannot be packaged if above 10 kb.</li>
	</ol>
	</li>
	<li>Incubate plates at 37 &#176;C. In order to increase viral production place plates at 30 &#176;C or 32 &#176;C instead. 
	NOTE: 24 hr post transfection media can be replaced with 7 ml of fresh media, and although this may improve cell health it may decrease viral load in the supernatant.</li>
	<li>48 hr post transfection collect the supernatant-containing viral particles and filter with a 0.45 &#956;M polytetrafluoroethylene filter. Use freshly collected viral supernatant for transduction. Otherwise keep supernatant in aliquots at -80 &#176;C and avoid freeze-thaw. Remember that viral titer will decrease probably up to 50% with each freezing-thawing cycle.</li>
	<li>Plate cell line of choice 24 hr prior to transduction if adherent, or immediately before transduction if non-adherent. Seed 2.0 x 105 to 3.0 x 105 adherent cells (here Huh 7.5.1 and HEK 293T) or 5.0 x 105 to 1 x 106 non-adherent cells (here SupT1) per well in a 6-well plate. 
	NOTE: Make sure to calibrate the number of cells to be seeded prior to transduction, especially so for adherent cells, so cells achieve a confluency of around 60% at the time of transduction.</li>
	<li>Add 5 &#956;g/ml Polybrene (hexadimethrene bromide) to seeded cells and then add previously collected viral supernatant to obtain around 20-40% infection (here 2 ml). 
	NOTE: The MOI or percentage of infection is mostly arbitrary, as sorting will allow to amplify any subpopulation of cells. Obviously, a higher rate of infection eases and speeds up the process of amplification.</li>
	<li>Transduce cells by centrifugation in a hanging bucket rotor at 1,500 x g for 120 min at 32 &#176;C. Resuspend non-adherent cells with fresh media prior to incubation. For adherent cells, replace with fresh media 24 hr post-transduction. 
	NOTE: It is recommended to seal the plates prior to centrifugation with parafilm to avoid spilling over. To increase genetically barcoded cell diversity, transduce cells with viral particles carrying different fluorescent proteins (obtained from step 1.4). When choosing fluorescent markers, make sure they are compatible with the instrumentation and they have distinguishable fluorescent parameters.</li>
</ol>
2. Selection and Amplification of Genetically Barcoded Cells
<ol>
	<li>Analyze transduced mammalian cells by flow cytometry at least 72 hr post transduction to quantitate the percentage rate of transduction. Use a comparable non-transduced cell line as negative control. 
	NOTE: As sorting will be used to purify individual barcoded cell populations, a high percentage rate of initial transduction, while not necessary, should speed up the process of amplification.</li>
	<li>Utilize the cell-sorter of choice to obtain the cells that express the protein of interest.
	<ol>
		<li>For that purpose, ensure the gates are properly set in the right channels.
		<ol>
			<li>To do so use the same na&#239;ve cell line as negative control and set the parameter/channel voltage so that the negative population is below the value of 103 on each fluorescent axis. Determine gating for positive fluorescence as events that appear above that of the negative control for each fluorescent channel.</li>
		</ol>
		</li>
		<li>Be aware that each instrument may vary and the correct voltage for determining gating should be adjusted accordingly, making positive and negative controls imperative in every experimental setup.
		<ol>
			<li>For fluorescence detection in this experimental setup use the FITC channel (530/30 band pass filter proceeded by a 502 long pass filter) for eGFP, the PE channel (585/42 band pass filter proceeded by a 556 long pass filter) for td Tomato, and the APC channel (660/20 band pass filter) for E2 Crimson. 
			NOTE: The 488 nm laser excites eGFP and td Tomato while E2 Crimson is excited by the 633 nm laser.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Sort into 15 ml conical tubes with 1 ml of 100% FCS. 
	NOTE: The process of sorting fluorescent cell populations is not compulsory as one can directly proceed with the sort of individual clones (see below). Sorting tight populations based on chosen fluorescence ensures a backup population for future selection of individual clones. Keep in mind that individual cells are at times difficult to grow while a large population of cells can be easily frozen, and then thawed and amplified at a later stage.</li>
	<li>Spin down the sorted populations in a hanging bucket rotor centrifuge at 524 g at 32 &#176;C for 5 min to pellet cells. Re-suspend in 1 ml of fresh media, and re-plate cells so that cell confluency is below 60% to allow growth and division for at least two days.
	<ol>
		<li>For example, plate adherent cells at around 3 x 105 cells in 2 ml of media per well in a 6-well plate and 2 x 106 cells in 2 ml of media in a 6 cm plate. Use DMEM for adherent cells and RPMI for non-adherent cells (or any specified medium if required). Place plated cells in an incubator at 37 &#176;C for several days to allow amplification.</li>
	</ol>
	</li>
</ol>
3. Obtain Clonal Populations of Genetically Barcoded Cells at Different Intensities
<ol>
	<li>Obtain clonal populations from cells originally transduced (step 1.7) or from the sorted population (step 2.3). 
	NOTE: This will ensure equal or similar level of fluorescent protein expression among all the cells within the population (a tight population with a small CV in mean fluorescence intensity of up to 40%).
	<ol>
		<li>For that purpose, sort single cells into 96-well plates with an automated cell deposition unit (ACDU). Set gates for sorting according to populations of interest. Ensure gates are set at least one log-scale apart to obtain distinguishable populations when choosing cells at different fluorescent intensities. For the procedure shown here, use the same flow cytometry experimental set up as the one described in step 2.2.</li>
	</ol>
	</li>
	<li>Amplify individual clones in an incubator at 37 &#176;C for at least 2 weeks. Through the process of amplification analyze the cells under the microscope to ensure that growing populations arise from individual cells and not from more than one. 
	NOTE: Remember that the sorter will place one cell per well on average, and while some wells may not have any cell at all, others may have more than one.
	<ol>
		<li>To ensure that a population arises from one cell only, be extremely gentle with the plate and never disturb the wells by pipetting. Observe the plate, well by well, under the microscope. 
		NOTE: While individual cells may at times be difficult to identify, once they start dividing they will be easily recognizable.</li>
		<li>Be aware that often cells &#8216;clump&#8217; along the edges. Discard those wells where multiple clonal populations can be observed and keep those that have one tight population. 
		NOTE: It is advisable to transfer those into larger plates for amplification.</li>
	</ol>
	</li>
	<li>Screen the putative clonal populations by analyzing them by flow cytometry. Compare them to a negative control utilizing the same flow cytometry experimental setup.
	<ol>
		<li>Analyze only a percentage of the sample and keep the rest as backup. Choose the most distinctly separate populations based on average intensity and tightness of the population. Amplify them as needed or freeze stocks in freezing media with 20% glycerol at -80 &#176;C for short periods of time or liquid nitrogen for longer. 
		NOTE: Remember that a &#8216;true&#8217; clonal population should have a very tight fluorescence pattern.</li>
	</ol>
	</li>
</ol>
4. Ensure Multiplexing Capabilities for High Throughput Screening (HTS)
<ol>
	<li>Combine as many distinct clonal populations as needed that can be further utilized in a multiplexed format. Choose clones with distinguishable absorption/emission spectra and/or different intensities. If a 96-well plate format is used, which is suitable for HTS, seed 50,000 cells in 200 &#956;l per well. 
	NOTE: Keep in mind that the number of cells is only an estimate and may change based on cell-type and whether it is adherent or not.</li>
	<li>Analyze the sample using flow cytometry to ensure that each of the independent established fluorescent cell lines can be distinguished from each other. Prior to the analysis prepare negative and single color controls to set parameters and compensation values. 
	NOTE: Setting the parameters to clearly distinguish the mixed populations will enable them to be further utilized in a multiplexed format.</li>
</ol>
5. Adapt Genetically Barcoded Cell Lines to the Biological Application of Choice
<ol>
	<li>Transduce established genetically barcoded cell lines with viral particles that contain the assay elements of choice, as described here in Stolp et al., 201359. Remember that the assay of choice should occupy an additional and distinguishable fluorescent channel and thus it is to be transferred to the appropriate barcoded cell line. 
	NOTE: Each barcoded cell line can bear a different assay.</li>
	<li>Sort genetically fluorescent barcoded cells for those that contain assay elements. 
	NOTE: Assay elements can be engineered coupled to a tag or fluorescent protein (as a fusion or through an internal ribosome entry site) for straightforward detection through western blotting, flow cytometry or microscopy. The system utilized in this protocol relies on HA epitope surface expression alone, or with additional FLAG epitope expression.
	<ol>
		<li>Stain the clonal populations containing the assay with HA and APC-fluorescently coupled antibodies. Then analyze the clonal populations with anti-FLAG and FITC-conjugated antibody staining.</li>
		<li>To track back the assay, analyze or &#8216;decode&#8217; the populations in the appropriate channel where the distinct genetically barcoded cell lines can be distinguished. Here, decode the nature of the substrate by analyzing in the PE channel for td Tomato detection.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>10 ml syringes</td><td>BD</td><td>309604</td><td>used for filtering the virus</td></tr><tr><td>0.45 &amp;micro;m ploytetrafluoroethylene filter</td><td>Pall Corporation</td><td>4219</td><td>used for filtering the virus</td></tr><tr><td>DMEM (Dulbecco&#039;s Modified Eagle Medium)</td><td>Corning</td><td>45000-304</td><td>cell growth media for HEK 293T cells</td></tr><tr><td>PEI (Polyethylenimine)</td><td>poly sciences</td><td>23966-2</td><td>2 mg/ml concentration used</td></tr><tr><td>Hanging bucket centrifuge (refrigerated)</td><td>Eppendorf</td><td>5805 000.017</td><td>used for spin infection</td></tr><tr><td>PBS (phosphate buffered saline)</td><td>Corning</td><td>21-040-CV</td><td>used for washing of cells</td></tr><tr><td>Polybrene (hexadimethreen bromide)</td><td>Sigma-Aldrich</td><td>107689</td><td>Used to increase viral infection efficiency. Used at a 5 &amp;micro;g/ml concentration.</td></tr><tr><td>FACSAria</td><td>BD Biosciences</td><td></td><td>instrument used for sorting cell populations</td></tr><tr><td>FACSCanto</td><td>BD Biosciences</td><td></td><td>instrument used for cell analysis</td></tr><tr><td>Phoenix-GP</td><td>Gift from Gary Nolan</td><td></td><td>cell line used to produced retroviral particles</td></tr><tr><td>Fetal calf serum</td><td>Mediatech</td><td>MT35015CV</td><td>used for cell growth and sorting</td></tr><tr><td>SupT1 cells</td><td>ATCC</td><td>CRL-1942</td><td>Human T lymphoblasts</td></tr><tr><td>HEK 293T cells</td><td>ATCC</td><td>CRL-11268</td><td>Human Embryonic Kidney cells that also contain the SV40 large T-antigen</td></tr><tr><td>RPMI 1640</td><td>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 ...

genetic-barcoding-with-fluorescent-proteins-for-multiplexed

Research

JoVE Journal

Biology

9.2K Views.  San Diego State University. 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. 

Video: Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Immunology and Infection

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

Biochemistry

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

Genetics

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

Cancer Research

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Developmental Biology

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Rapid Development of Cell State Identification Circuits with Poly-Transfection

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit components remains challenging and labor-intensive. To accelerate this process, our lab developed poly-transfection, a high-throughput extension of traditional mammalian transfection. In poly-transfection, each cell in the transfected population essentially performs a different experiment, testing the behavior of the circuit at different DNA copy numbers and allowing users to analyze a large number of stoichiometries in a single-pot reaction. So far, poly-transfections that optimize ratios of three-component circuits in a single well of cells have been demonstrated; in principle, the same method can be used for the development of even larger circuits. Poly-transfection results can be easily applied to find optimal ratios of DNA to co-transfect for transient circuits or to choose expression levels for circuit components for the generation of stable cell lines.
Here, we demonstrate the use of poly-transfection to optimize a three-component circuit. The protocol begins with experimental design principles and explains how poly-transfection builds upon&#160;traditional co-transfection methods. Next, poly-transfection of cells is carried out and followed by flow cytometry a few days later. Finally, the data is analyzed by examining slices of the single-cell flow cytometry data that correspond to subsets of cells with certain component ratios. In the lab, poly-transfection has been used to optimize cell classifiers, feedback and feedforward controllers, bistable motifs, and many more. This simple but powerful method speeds up design cycles for complex genetic circuits in mammalian cells.

Complex genetic circuits are time-consuming to design, test, and optimize. To facilitate this process, mammalian cells are transfected in a way that allows the testing of multiple stoichiometries of circuit components in a single well. This protocol outlines the steps for experimental planning, transfection, and data analysis.

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit ...

Bioengineering

BEST: Barcode Enabled Sequencing of Tetrads

Tetrad analysis is a valuable tool for yeast genetics, but the laborious manual nature of the process has hindered its application on large scales. Barcode Enabled Sequencing of Tetrads (BEST)1 replaces the manual processes of isolating, disrupting and spacing tetrads. BEST isolates tetrads by virtue of a sporulation-specific GFP fusion protein that permits fluorescence-activated cell sorting of tetrads directly onto agar plates, where the ascus is enzymatically digested and the spores are disrupted and randomly arrayed by glass bead plating. The haploid colonies are then assigned sister spore relationships, i.e. information about which spores originated from the same tetrad, using molecular barcodes read during genotyping. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. Here we present a detailed description of the experimental procedures required to perform BEST in the yeast Saccharomyces cerevisiae, starting with a heterozygous diploid strain through the isolation of colonies derived from the haploid meiotic progeny.

Barcode Enabled Sequencing of Tetrads (BEST) replaces the manual processes of isolating, disrupting and spacing tetrads. BEST isolates tetrads by fluorescence-activated cell sorting onto agar plates, separates the spores by agitation with glass beads, and determines which randomly arrayed colonies were derived from the same original tetrad using molecular barcodes.

Tetrad analysis is a valuable tool for yeast genetics, but the laborious manual nature of the process has hindered its application on large scales. ...

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ within a given tissue remain limited. The EASI-FISH protocol described here has been adapted to detect the expression of dozens of genes in the intact adult Drosophila central nervous system (CNS) using commercially available reagents. This protocol includes a new gel formulation that enhances gel robustness, enabling multiple rounds of hybridization and allowing the embedding of multiple brains per gel. This improvement increases throughput, facilitates optimal comparison of experimental conditions, and reduces reagent costs. Additionally, by employing the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in specific neuronal or glial cell types. Notably, the high resolution achieved through expansion microscopy, combined with the sensitivity of the method, allows for the detection of single RNA transcripts. This approach effectively integrates high image quality with high throughput, making it a powerful tool for studying gene expression throughout the intact fly brain.

Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a robust technique that combines expansion microscopy with fluorescence in situ hybridization (FISH) to detect the expression of multiple genes in thick tissue. This protocol outlines recent advancements in the method and its adaptation for labeling the intact Drosophila central nervous system..

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ ...

Neuroscience

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Samenvatting

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