We thank John Darga of the MGH DNA Core for preparation of the DNA screening library. Christopher Chigas of Perkin Elmer provided invaluable support for our Wallac 1420 luminometer. Steven Ciacco and Martin Thomae of Agilent provided support for the Bravo robot. We thank Ron Johnson and Steve Titus at the NIH for generously providing their dual-glow luciferase assay protocol.

For an experimental overview, see Figure 1.
1. Prepare Reporter Plasmids Containing the Alpha-Synuclein Promoter
Regulatory elements of the alpha-synuclein gene (Figure 2) span approximately 10 kb, from the upstream NACP (non-A beta component of amyloid peptide) dinucleotide repeat sequence1,2 through intron 2 3. We include a portion of this region in our reporter construct. We found that plasmids containing intron 2 required special procedures for growth, as outlined below. The luciferase plasmids, pGL4.10 and pGL4.75 (Figure 3), are commercially available from Promega and can be propagated using conventional molecular biology protocols.
<ol>
	<li>Amplify the 2&#160;components of the alpha-synuclein promoter in separate PCRs using the recipe in Table 1 and the primers in Table 2.</li>
	<li>Verify PCR products on an agarose gel and clean using the Qiaquick PCR Purification kit (Qiagen), eluting in 50 &#956;l of TE (Tris-EDTA). Store the eluted 0.9 kb fragment at -20 &#176;C for future use.</li>
	<li>Digest the entire volume of the 3.4 kb PCR fragment, as well as 5 &#956;g of pGL4.10, with SacI and XhoI. Treat the vector with Calf Intestine Alkaline Phosphatase (Roche) and gel purify using the PureLink Quick Gel Extraction kit (Invitrogen).</li>
	<li>Ligate the fragment and vector using T4 DNA ligase (NEB). Clean the completed reaction using the PureLink PCR Micro kit (Invitrogen), eluting in 10 &#956;l TE buffer.</li>
	<li>Transform 2 &#956;l of the cleaned, ligation reaction into 20 &#956;l of ElectroMAX Stbl4 competent cells (Invitrogen) and electroporate according to the manufacturer&#39;s instructions. Shake in a 30 &#176;C incubator at 225 rpm for 90 min. Spread 2&#160;volumes on LB plates containing 100 mg/ml ampicillin and incubate at 30 &#176;C for 2&#160;days.</li>
	<li>Using a toothpick, select 4-6 small colonies for inoculation into 2 ml of TB (Terrific Broth). Grow these miniprep cultures in a 30 &#176;C shaker O/N.</li>
	<li>Purify plasmid DNA from 1.5 ml of each miniprep culture using the PureLink HiPure Plasmid Miniprep kit (Invitrogen).</li>
	<li>Digest the minipreps with BamHI and electrophorese on an agarose gel. The correct clone should produce fragments of 2.8 kb and 4.9 kb.</li>
	<li>Restreak the remaining miniprep culture from the correct clone on a fresh LB (Luria Broth) plate and grow at 30 &#176;C for 2&#160;days.</li>
	<li>Select a small colony and inoculate a 1.5 ml TB starter culture. Shake O/N at 30 &#176;C. Do not let the starter culture grow to saturation.</li>
	<li>Dilute the entire starter culture into 150 ml of prewarmed TB and shake at 30 &#176;C for 2&#160;days. Before proceeding to the next step, use 1.5 ml of this culture for an additional miniprep and digestion to confirm that the clone did not mutate during replication.</li>
	<li>Purify plasmid DNA from the remaining culture using the PureLink HiPure Plasmid Maxiprep kit (Invitrogen). Expected yields of this intermediate plasmid are 50-150 &#956;g per 150 ml of culture.</li>
	<li>Thaw the 0.9 kb fragment frozen in Step 1.2. Repeat Steps 1.3 to 1.12 to clone the 0.9 kb fragment into the intermediate plasmid, digesting instead with KpnI and SacI. Ligate, transform, select, and grow for maxipreps as above. Digestion with BamHI should yield fragments of 5.8 kb and 2.8 kb. This is the plasmid that will be used for the screen.</li>
</ol>
2. Prepare 293T Cells, Reporter Plasmids, and Library DNA for Screening
293T cells are first seeded into 96-well plates and transfected the following day. Reporter plasmids and library DNAs are diluted into 96-well plates. We obtained plates of transfection-grade library DNA from the DNA Core at Massachusetts General Hospital (<a href="http://dnacore.mgh.harvard.edu" target="_blank">dnacore.mgh.harvard.edu</a>). This protocol transfects a single library plate into 2&#160;identical plates of 293T cells (Figure 1), thus 2&#160;plates of cells should be seeded for each library plate to be screened.
Note: Grow cells in media without phenol red or antibiotics, as these interfere with the luciferase assays and increase toxicity during transfection, respectively.
<ol>
	<li>For each library plate to be screened, resuspend trypsinized 293T cells to a concentration of 1.5 x 105 cells/ml in DMEM containing 14% FBS. Pour into a sterile reservoir (Axygen).</li>
	<li>Place the reservoir, 2&#160;clear-bottomed, 96-well plates (Corning), and a box of filter pipet tips (Agilent) on the robot deck. Dispense 100 &#956;l of cells into each well of both plates, for a density of 1.5 x 104 cells per well.</li>
	<li>Centrifuge the cell plates at 50 x&#160;g for 2 min, using low ramp speeds to ensure equal cell density throughout each well. Incubate at 37 &#176;C and 10% CO2 O/N.</li>
	<li>Dilute the firefly and Renilla reporter plasmids to 5 ng/&#956;l in serum-free media. Combine the dilutions in a ratio of 5:3 firefly to Renilla plasmid (by volume) in a 15 ml conical tube.</li>
	<li>Pipet the combined plasmids into each well of a 96-well plate, dispensing 17 &#956;l per library plate, plus 2-10 &#956;l excess, into each well.</li>
	<li>Obtain transfection-grade DNA library plates as above and dilute to 13.3 ng/&#956;l with endotoxin-free water. The minimum volume per well should be 28 &#956;l.</li>
</ol>
3. Perform Transfections
On day 2&#160;of the screen, reporter plasmids and library DNAs are transfected into 293T cells.
<ol>
	<li>For each library plate, mix 107.5 &#956;l of RT Optifect (Invitrogen) with 4.3 ml serum-free media (1:40 dilution) in a polystyrene tube. Incubate for 5 min at RT, and then dispense 44 &#956;l (per library plate) into each well of a 96-well, polystyrene V-bottomed plate (Greiner).</li>
	<li>Place the plate of diluted Optifect, reporter plasmids (Step 2.5), library DNAs (Step 2.6), and a box of pipet tips on the robot deck.</li>
	<li>Aspirate 17 &#956;l of reporter plasmids, 43 &#956;l of diluted Optifect, and 26 &#956;l of library DNA, inserting a 5 &#956;l air gap between each aspiration. The library DNA should be aspirated last to prevent cross-contamination. Dispense the contents of the tips into a new polystyrene V-bottomed plate, mix, cover, and set aside for 20 min to allow lipid/DNA complexes to form. If transfecting multiple library plates, replace the pipet tips and library plate, and repeat.</li>
	<li>Uncover the Optifect/DNA mixture plate and place it on the robot deck with 2 plates of 293T cells. Using a single box of pipet tips, aspirate 80 &#956;l of the Optifect:DNA mixture and slowly dispense 40 &#956;l onto each plate of cells. If transfecting multiple library plates, repeat for all Optifect:DNA plates. Cover cells.</li>
	<li>Centrifuge the cell plates at 1,200 x&#160;g for 30 min. Cover and return to the incubator.</li>
	<li>Incubate the cells for 4-6 hr. During this incubation, prepare fresh media by mixing 12 ml DMEM containing 10% FBS and 10 &#956;g/ml ciprofloxacin (CellGro) for each transfected library plate. Pour fresh media into a sterile reservoir.</li>
	<li>Place 2&#160;boxes of robot tips, a single plate of cells, the reservoir containing fresh media, and a waste reservoir onto the robot deck. Aspirate 100 &#956;l of media from the cells and dispense into the waste reservoir partially filled with 70% ethanol. Using fresh tips, aspirate 100 &#956;l of fresh media and slowly dispense onto the cells. Repeat for all plates of cells.</li>
	<li>Return plates to the incubator for 48 hr.</li>
</ol>
4. Perform Dual-Luciferase Assays
On day 4&#160;of the screen, the firefly and Renilla luciferase assays are performed.
Note: The dual-luciferase assay requires the sequential addition of 2&#160;assay buffers, 3X firefly assay buffer and 3X Renilla assay buffer, as described in Table 3. Firefly and Renilla luciferases are not secreted, thus cells must be lysed prior to measuring luciferase activity. Firefly assay buffer lyses cells and provides substrate for the firefly luciferase; Renilla assay buffer quenches the firefly signal and provides substrate for the Renilla luciferase.
<ol>
	<li>For each library plate, prepare 8 ml of 3X firefly assay buffer from stock solutions as described in Table 3, and dispense 82 &#956;l into each well of a 96-well V-bottomed plate.</li>
	<li>For each library plate, also prepare 12 ml of 3X Renilla assay buffer and dispense 122 &#956;l into each well of a 96-well V-bottomed plate. Set aside both the firefly and Renilla assay buffers.</li>
	<li>Place a box of pipet tips, a waste reservoir, and 2&#160;plates of cells (transfected from the same library plate) on the robot deck.</li>
	<li>Aspirate 60 &#956;l of media from each plate and discard in the waste reservoir partially filled with 70% ethanol. Cover plates and set aside. If transfecting multiple library plates, repeat for all sets of plates.</li>
	<li>Place 2&#160;boxes of pipet tips, the plate of 3X firefly assay buffer, and a set of cell plates on the robot deck.</li>
	<li>Aspirate 40 &#956;l of 3X firefly assay buffer and add it to the first plate of cells, mixing thoroughly. Switching to new pipet tips, repeat for the second cell plate. Place cells at RT for 10 min to complete lysis. If transfecting multiple library plates, replace the boxes of tips and cell plates, and repeat.</li>
	<li>Record luminescence from each well of each cell plate on a luminometer, recording for 1 sec per well. Return plates to the robot deck.</li>
	<li>Place 2&#160;boxes of pipet tips, the plate of 3X Renilla assay buffer, and a set of cell plates on the robot deck.</li>
	<li>Aspirate 60 &#956;l of 3X Renilla assay buffer, and add it to the first plate of cells, mixing thoroughly. Switching to a new box of pipet tips, repeat for the second cell plate. If transfecting multiple library plates, repeat for all sets of cell plates.</li>
	<li>Record luminescence from all plates on a luminometer, recording each well for 1 sec as above. Discard plates.</li>
</ol>
5. Data Analysis
<ol>
	<li>Calculate the firefly:Renilla luminescence ratio (&#34;F:R ratio&#34;) for each well by dividing the counts of firefly signal by the counts of Renilla signal.</li>
	<li>Calculate the induction value by dividing the F:R ratio of each well by the average F:R ratio of the plate, excluding the highest and lowest 25% of wells.</li>
	<li>Average the induction values across replicates for a single transfected library plate. Genes inducing or repressing alpha-synuclein more than three-folds are considered &#34;hits&#34; in this screen and are subject to further validation and secondary screens.</li>
</ol>

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This work was supported by royalties obtained by licensing BacMamreagents (US patent 5,731,182).

Alpha-synuclein has been implicated in Parkinson&#39;s disease (PD) as a component of Lewy bodies4, intracellular inclusions considered pathognomonic for the disease. Numerous genome-wide association studies have linked single nucleotide polymorphisms in alpha-synuclein with increased risk for sporadic PD5,6,7. Although less common than sporadic PD, familial PD may be also caused by mutations in alpha-synuclein8, as well as duplication and triplication of the alpha-synuclein locus9,1...

The ability to identify key genetic regulatory elements and the factors that act on them is fundamental for the exploration of numerous biological processes. However, identifying factors that regulate expression of genes in rare cell types, such as specific neuronal populations, can be challenging. Here, we present a protocol for identifying novel transcriptional regulators of alpha-synuclein (SNCA), a gene associated with Parkinson&#39;s disease and expressed in dopaminergic neurons in the substantianigra pars compacta region of the midbrain. We accomplish this using a high-throughput, dual-luciferase, in vitro reporter screen to deconstruct alpha-synuclein expression in 293T cells. The alpha-synuclein promoter is first cloned into a reporter plasmid containing the firefly luciferase gene. A commercially available plasmid containing the Renilla luciferase under the control of a constitutively active promoter serves as an internal control. These reporter constructs are co-transfected into 293T cells in microplates with plasmids from a DNA expression library, such that each well is transfected with a single library plasmid. After 48 hr, luciferase activity for each reporter is measured sequentially using a dual-glow assay. The relative expression of alpha-synuclein in response to each library gene is inferred by the ratio of firefly:Renilla luciferase activity in each well (F:R ratio) after plate-based normalization.
This protocol provides the ability to screen a large number of genes (~2,500 per week) for their ability to transactivate a reporter gene using minimal manpower (1-2 people) and minimal cost (about $3 reagent cost per microplate assay). Transcriptional regulators of neuronal genes (e.g., alpha-synuclein), which are difficult to study in cultured neurons refractory to genetic manipulation, can be compared side-by-side in this direct functional assay. We include in our protocol a detailed method for cloning and growth of plasmids containing the alpha-synuclein promoter, since we found that plasmids containing this region are unstable when grown with conventional procedures (see Discussion). We also include an inexpensive alternative to commercially available dual-luciferase reagents for high-throughput assays. This protocol can be easily adapted to target other regulatory elements of interest, or to any process requiring high-throughput transient transfections.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Material&lt;/strong&gt;</td></tr><tr><td>QIAQuick PCR Purification Kit</td><td>Qiagen</td><td>28106</td><td></td></tr><tr><td>PureLink Quick Gel Extraction Kit</td><td>Invitrogen</td><td>K2100-12</td><td></td></tr><tr><td>PureLink PCR Micro Kit</td><td>Invitrogen</td><td>K310250</td><td></td></tr><tr><td>PureLinkHiPure Plasmid Miniprep Kit</td><td>Invitrogen</td><td>K2100-03</td><td></td></tr><tr><td>PureLinkHiPure Plasmid Maxiprep Kit</td><td>Invitrogen</td><td>K2100-07</td><td></td></tr><tr><td>Bravo Robot</td><td>Agilent</td><td>-</td><td></td></tr><tr><td>Robot Pipet Tips with Filter</td><td>Agilent</td><td>19477-022</td><td></td></tr><tr><td>Robot Pipet Tips without Filter</td><td>Agilent</td><td>19477-002</td><td></td></tr><tr><td>Clear-bottomed 96-well Plates</td><td>Corning</td><td>3610</td><td></td></tr><tr><td>Reservoirs</td><td>Axygen</td><td>RES-SW96-HP-SI</td><td></td></tr><tr><td>Polystyrene V-bottomed 96-well Plate</td><td>Greiner</td><td>651101</td><td></td></tr><tr><td>Polypropylene V-bottomed 96-well Plate</td><td>Greiner</td><td>651201</td><td></td></tr><tr><td>Adhesive Plate Covers</td><td>CryoStuff</td><td>#FS100</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Phusion DNA Polymerase</td><td>New England Biolabs</td><td>M0530L</td><td></td></tr><tr><td>BAC 2002-D6</td><td>Invitrogen</td><td>2002-D6</td><td></td></tr><tr><td>Calf Intestine Alkaline Phosphatase</td><td>Roche</td><td>10713023001</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202L</td><td></td></tr><tr><td>pGL4.10</td><td>Promega</td><td>E6651</td><td></td></tr><tr><td>pGL4.74</td><td>Promega</td><td>E6931</td><td></td></tr><tr><td>ElectroMAX Stbl4 Competent Cells</td><td>Invitrogen</td><td>11635-018</td><td></td></tr><tr><td>Subcloning Efficiency DH5&amp;alpha; Competent Cells</td><td>Invitrogen</td><td>18265-017</td><td></td></tr><tr><td>DMEM without Phenol Red</td><td>Invitrogen</td><td>31053-028</td><td></td></tr><tr><td>Optifect</td><td>Invitrogen</td><td>12579017</td><td></td></tr><tr><td>Ciprofloxacin</td><td>CellGro</td><td>61-277-RF</td><td></td></tr><tr><td>Tris-Hydrochloride Powder</td><td>Sigma</td><td>93287</td><td></td></tr><tr><td>Tris-Base Powder </td><td>Sigma</td><td>93286</td><td></td></tr><tr><td>Triton X-100 Pure Liquid</td><td>Fisher</td><td>BP151-100</td><td></td></tr><tr><td>DTT</td><td>Invitrogen</td><td>15508-013</td><td></td></tr><tr><td>C&amp;#8211;nzyme A</td><td>Nanolight</td><td>309</td><td></td></tr><tr><td>ATP</td><td>Sigma</td><td>A6419</td><td></td></tr><tr><td>Luciferin</td><td>Invitrogen</td><td>L2912</td><td></td></tr><tr><td>h-CTZ</td><td>Nanolight</td><td>301</td><td></td></tr><tr><td>PTC124</td><td>SelleckBiochemicals</td><td>S6003</td><td></td></tr></tbody></table>

high-throughput functional screening using a homemade dual-glow luciferase assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

Typical luciferase values, F:R ratios and induction values for a single half-plate are shown in Figure 4. Note the hit in well H2, a 32-fold inducer. Genes causing excessive toxicity (for example, well E3), or wells that were poorly transfected, will produce low values for both firefly and Renilla luciferases, but an average induction value. Genes causing non-specific induction of luciferase activity, perhaps via interaction with the pGL4 backbone, will induce firefly and Renilla</e...

Watch this Scientific Journal Video about High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay at JoVE.com

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

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Research

JoVE Journal

Biology

23.5K Views.  Massachusetts General Hospital. We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Video: High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Cancer Research

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicine

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Immunology and Infection

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Cellular Bioluminescence Based Assay: A High Throughput Method for Combinatorial Drug Screening

Coat 96-well optical bottom plates with an extracellular matrix mixture such as Matrigel and incubate them for 1 hour at 37 degrees Celsius. Remove the excess mixture and gently rinse the plates once with PBS.
Then, seed XG387-Luc cells at a density of 1,000 cells in 100 microliters of culture medium into each well of the coated plates and culture them overnight. On the following day, observe the cells under a microscope to confirm their attachment to the plate.
Next, prepare a 200-micromolar temozolomide solution and 2-micromolar solutions of the targeted agents in culture medium. For combination drug screening, remove the blank medium and add the medium containing either 200-micromolar temozolomide or 2-micromolar targeted agent, or a combination of both, into each well for 3 technical replicates per treatment.
Incubate the plate at 37 degrees Celsius and 5% carbon dioxide for 3 days. Take images of the cellular bioluminescence in the plate using the IVIS spectrum imaging system. Using the built-in software, create multiple circular areas of the region of interest and quantify the cellular bioluminescence.