We thank the Italian Neuroblastoma Foundation for the full financial support to this project.

NOTE: The throughput of such assay systems depends on the available HTS lab equipment. This protocol is facilitated by a Tecan Freedom EVO 200 robot, which performs liquid handling in 96-well format. Miniaturization to 384-well format is also possible. The robotic liquid handling system is positioned under a laminar flow hood in order to maintain aseptic conditions during all experimental steps. If no liquid-handling automation is available, the protocol can be readily adapted to low-throughput format.
Primary Screening
1. Day 1: Prepare and Seed Cells
NOTE: Seed cells to yield 80% confluence at the time of assaying luciferase activity, i.e., 48 hr after seeding.
<ol>
	<li>Resuspend trypsinized CHP134-mycn3UTR neuroblastoma cells to a concentration of 2 &#215; 105 cells/ml in RPMI containing 10% FBS and 1% L-glutamine. For 27 plates prepare at least 250 ml of cell suspension taking into account liquid handling, troughs dead volumes and repeated pipetting steps. Pour the cell suspension into sterile reservoir and place it on the robot desk.</li>
	<li>Place 9 barcoded white flat-bottom 96-well plates and a box of 200 &#956;l sterile pipet tips on the robot desk. Mix the cell suspension by pipetting before each dispensation step to avoid cells settling under gravity. Dispense 75 &#956;l of cells into each well of 9 plates to get a final density of 1.5 &#215; 104 cells per well.</li>
	<li>Cover the plates with the lids and leave them outside of the incubator for 30 min to allow the cells to sediment to the bottom of the well. This will minimize edge effects.</li>
	<li>Repeat steps 1.2 and 1.3 twice in order to prepare a total number of 27 plates.</li>
	<li>Place the plates at 37 &#176;C and 5% CO2 and incubate for 24 hr.</li>
</ol>
2. Day 2: Treat the Cells with Library Compounds
NOTE: The Spectrum Collection small molecule library consists of 2,000 compounds arranged in 8 rows and 10 columns in 25 96-well plates at the concentration of 10 mM in DMSO. The wells on the first and last columns are left empty for controls. Compound screening assays are typically performed at 1&#8211;10 &#181;M compound concentration8. The screening protocol described here fixes 2 &#956;M as the working concentration and 24 hr as the assay end point.
<ol>
	<li>Thaw 9 compound library plates at room temperature.</li>
	<li>Prepare two troughs with sterile PBS and PBS-0.5% DMSO solutions and place them on the robot desk. For each library plate, prepare and label accordingly one dilution plate &#8212; a polypropylene U-bottom 96-well plate with a working volume of at least 400 &#956;l. Fill each well of columns 2 to 11 of dilution plates with 398 &#956;l of sterile PBS using the liquid handler&#8217;s fixed tips. Fill columns 1 and 12 with 50 &#956;l of sterile PBS with 0.5% DMSO in order to reproduce the vehicle concentration in the control wells (0.02%).</li>
	<li>Place two library plates, the correspondingly labeled dilution plates, two boxes of 50 &#956;l sterile pipet tips and six cells plates on the robot desk. Uncover cells plates.</li>
	<li>Pipet 2 &#956;l of the 10 mM stock solution from one of two library plates in the corresponding dilution plate and mix well. This will result in an intermediate compounds concentration of 50 &#956;M. Using the same set of tips, dispense 3 &#956;l of the 50 &#956;M dilution in 3 barcoded white 96-well plates with cells. This dilution scheme results in 2 &#956;M working concentration of each tested compound. 
	NOTE: To avoid unnecessary manipulations with the tips, use a full set of 96 tips from the beginning, even though the library plates contain only 80 compounds. This is possible since the first and the last columns of the library plates are empty.</li>
	<li>Replace the pipet tips and repeat Step 2.4 for the second library plate.</li>
	<li>Cover the cells plates and return them to the incubator for 24 hr.</li>
	<li>Repeat Steps 2.3-2.6 two more times for the remaining library plates.</li>
</ol>
3. Day 3: Perform Luciferase Assay
NOTE: Before performing the luciferase assay, it is possible to multiplex it with a cell viability assay based on reduction of resazurin in order to obtain a cell-viability index from each well9. Multiplexing luciferase and viability assay assays results in a reduction of luciferase signal that, however, can be ignored if the cells provide high enough level of luciferase activity. Luciferase activity can be detected by a variety of commercial and homemade10,11 luciferase assay reagents with stable luminescent signal.
<ol>
	<li>Equilibrate the reconstituted luciferase reagent of choice to room temperature. Make sure the volume is enough for all the assayed plates. For 27 plates 170 ml of luciferase reagent will be required taking into account liquid handling, troughs dead volume and repeated pipetting steps. Pour the luciferase reagent into a reservoir and place it on the robot desk.</li>
	<li>Remove 9 plates with cells from the incubator, place them on the robot desk, uncover and wait until equilibrated to room temperature. Add 50&#160;&#956;l of the luciferase reagent per well plate by plate. Between the plates, introduce a delay equal to the luminescence reading time of one plate. This will assure that all the plates are measured within equal time interval from the reagent addition.</li>
	<li>After a 30 min incubation, place the first plate in the luminometer and measure luminescence after a brief shaking step. Repeat for all plates. Record the barcode of each plate and associate it to the corresponding data file to avoid mistakes arising from a large number of handled plates.</li>
	<li>Repeat Steps 3.2-3.3 twice for the remaining 18 plates with cells.</li>
	<li>Collect the remaining luciferase reagent and store it at -20 &#176;C.</li>
</ol>
4. Day 4: Data Analysis
<ol>
	<li>Process experimental data using normalization of all samples to the average of on-plate vehicle-treated controls. For each library compound, calculate the mean value x&#773; and SD over triplicates.</li>
	<li>Select up-regulating and down-regulating hits by setting a threshold of x&#773; &#177; k &#215; SD, where the mean value x&#773; and SD are computed over all assay processed values, and k is a defined constant.</li>
	<li>Select an arbitrary threshold cut-off &#60; 20% of vehicle-treated controls when a reduction of luciferase signal is most probably associated with compound toxicity. Discarding the hits below this threshold will significantly reduce a number of false-positive hits in the counter-screening. 
	NOTE: Instead of control-based normalization, experimental data can be processed applying Z score or its robust analog B score12. In addition, alternative statistical approaches for hit selection are available12.</li>
</ol>
5. Counter-screening
NOTE: Compounds identified in the primary screen (labeled &#8216;hits&#8217;) are confirmed and evaluated for the specificity by a counter-screening.
<ol>
	<li>Cherry pick the selected hits from the aliquots stored in single 2D barcoded tubes and create new &#8220;hit plates&#8221;, saving the corresponding layout. Do not forget to leave free positions for controls. 
	NOTE: In the absence of an efficient cherry-picking system, one might test both the control and 3&#8217; UTR-bearing cells in parallel in the primary screening. In this case, specific selection of compounds with effects dependent only on the introduced 3&#8217; UTR will be possible after the primary screening eliminating the need of counter-screening. However, the parallel screening in two cell lines will double the assay costs.</li>
	<li>Following the protocol for the primary screening (section &#8220;Day 1: Prepare and Seed Cells&#8221;), for each &#8220;hit plate&#8221; prepare three 96-well plates of each CHP134-mycn3UTR and CHP134-CTRL stable cells.</li>
	<li>Following the protocol for the primary screening (section &#8220;Day 2: Treat the Cells with Library Compounds&#8221;), use each hit plate to treat three CHP134-mycn3UTR and three CHP134-CTRL plates at 2 &#956;M working concentration. 
	NOTE: While the counter-screen described here is performed in triplicates at the single dose of 2 &#956;M, it might be beneficial to substitute the standard replicates and test the hits in 3 concentrations as 10-fold dilutions ranging from 2 &#956;M to 20 nM. Applying this approach would not only allow to confirm the data of the primary screen, but also to obtain preliminary dose-response data on the primary hits, minimizing false-positive rates. In this case, a different analysis pipeline should be applied to process the experimental data and confirm the hits.</li>
	<li>Following the protocol for the primary screening (section &#8220;Day 3: Perform Luciferase Assay&#8221;), measure luciferase activity in all CHP134-mycn3UTR and CHP134-CTRL plates.</li>
	<li>Following the protocol for the primary screening (section &#8220;Day 4: Data Analysis&#8221;), normalize experimental data from treated wells to the average of on-plate vehicle-treated controls and calculate the mean and SD over replicates. For each compound tested, compute the fold change of luciferase activity in CHP134-mycn3UTR vs. CHP134-CTRL cells. Select arbitrary fold change cut-offs of &#62;1.5 and significance p-values of &#60;0.01. 
	NOTE: In the described assay settings the determinant parameter for hit specificity is a significant fold change of luciferase activity in CHP134-mycn3UTR vs. CHP134-CTRL cells. If needed, one might also assess compound toxicity by performing concurrently a cell viability assay on the selected compounds. This might be especially beneficial if the primary hits are counter-screened in a dose response analysis. For a single dose, our experience indicates a strong correlation of reduced luciferase activity with decreased cell viability6. Therefore, decreased luciferase activity in target cells as well as control cells can be considered as an indicator of compound toxicity at the tested concentration.</li>
</ol>

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F., Parker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+properties+of+eukaryotic+mRNPs.">Principles and properties of eukaryotic mRNPs.</a> Mol. Cell. 54 (4), 547-558 (2014).</li><li>Fu, Y., Dominissini, D., Rechavi, G., He, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+regulation+mediated+through+reversible+m6A+RNA+methylation.">Gene expression regulation mediated through reversible m6A RNA methylation.</a> Nat. Rev. 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The authors declare that they have no competing financial interests.

This protocol describes a cell-based reporter-gene assay aiming at the identification of modulators that target 3&#8217; UTR-dependent post-transcriptional processes. It encompasses the primary screening and the counter-screen and, if needed, can be accompanied by a cytotoxicity assay. The outcome of the primary screening is a number of valuable candidate compounds, whose reproducibility and specificity is further validated in the counter-screening.
The identification of primary hits is based ...

For a long period transcriptional regulation of gene expression was thought to play a major if not exclusive role in controlling protein production. Accumulating evidence, however, indicates that post-transcriptional regulation contributes as much as, if not more than, transcriptional regulation to determine cellular protein abundance1,2. Post-transcriptional control of gene expression is much more complex and elaborate than was at first thought. In fact, all the various stages of post-transcriptional control have emerged to be regulated, including mRNA processing, localization, turnover, translation3 as well as the newly described reversible RNA methylation4. From a number of post-transcriptional regulation processes potentially affected, the assay described below focuses on those involving the mRNA 3&#39; untranslated region (3&#8217; UTR). 3&#8217; UTR broadly affects mRNA fate mainly via specific interaction of RNA-binding proteins and non-coding RNAs to its regulatory sequences and/or secondary structures5. Therefore, small molecules that alter these interactions or interfere with upstream signal transduction pathways will shift the balance that regulates protein abundance. This therapeutic approach is of particular importance for human pathologies for which evidence exists showing that disease-relevant genes are subjected to post-transcriptional regulation, which thus represents a potential target for pharmacological intervention. Therefore, systems allowing for screening of mRNA levels&#8217; modulators through interference with post-transcriptional control mechanisms in a high-throughput format can become valuable tools in the identification of potential novel treatments.
The described cell-based reporter gene assay allows for the identification of compounds able to modulate mRNA fate via mechanisms dependent on its 3&#8217; UTR. It consists of several principal components (Figure 1) and requires few preliminary steps to validate the feasibility of the assay. First of all, the reporter construct is designed to include the 3&#8217; UTR from a gene of interest. The 3&#8217; UTR is fused to the end of a reporter gene such as firefly luciferase (Figure 1). Any changes in post-transcriptional control mechanisms exerted through the inserted 3&#8217; UTR will alter the stability and/or translation efficiency of this chimeric transcript, resulting in varied luciferase levels. Therefore, changes in luciferase activity serve as indirect measure of affected post-transcriptional regulation of the gene of interest. The second component of the system is a control reporter construct, an identical expression plasmid that expresses the same reporter gene but does not contain any 3&#8217; UTR. The two reporter plasmids can be designed in the lab using conventional molecular biology protocols or purchased from commercial sources.
The selection of an appropriate cell line is critical for the assay construction. Which cell line is the optimal model depends on numerous factors ranging from clinical requirements like maximal resemblance to pathology to just practical issues like availability, transfectability, and growth characteristics. Importantly, prior to proceed one should make sure that fusion of 3&#8217; UTR to the reporter gene has an expected effect on the level of the chimeric transcript. The absence of significant difference in luciferase signal from the 3&#8217; UTR-bearing and control reporter constructs transiently transfected in the selected cells would indicate that the 3&#8217; UTR is not functional in this cellular model, prompting for search of alternative ones. For the high-throughput format, the 3&#8217; UTR-bearing and control luciferase reporter constructs should be stably integrated in the selected cell line. Using a stably integrated over transiently transfected cell line is preferable for several reasons. This will broaden the choice of a cellular model including hard-to-transfect cell lines, decrease the variability in the data arising from fluctuations in transfection efficiency, subside the costs. Moreover, upon transient transfection cells are very often overloaded with a DNA plasmid leading to the saturation of the system. In these settings a further increase in the reporter expression might be challenging, resulting in decreased sensitivity towards potential upregulating compounds.
Finally, when all the assay components are set up, it is critical before moving to the high-throughput format to determine the feasibility of the assay, i.e., if luciferase activity can be indeed up- and down-regulated specifically via the inserted 3&#8217; UTR. The best controls would be small molecules reported to modulate the stability or translatability of mRNA through the 3&#8217; UTR of interest. If having these is not possible, surrogate controls imitating the desired effect are accepted. These could be miRNAs or RNA-binding proteins known to exert their effects via binding to the 3&#8217; UTR of interest. Evaluation of such controls before the primary screening not only indicates the functionality of the developed assay, but also allows for estimation of its dynamic range, sensitivity and performance in the high-throughput format.
Using the described protocol we screened a 2,000-compound library6. Neuroblastoma, the most common extracranial solid tumor of infancy, was used as a biological model and the 3&#8217; UTR of the MYCN oncogene, whose amplification strongly predicts adverse outcome of neuroblastoma, as a target gene7. Figure 2 outlines the experimental layout. The screening was divided in three runs with the batch size of 27 plates screened in one run. The batch of 27 plates included Spectrum Collection library plates n.1-n.8+n.25 (first run), n.9-n.16+n.25 (second run) and n.16-n.24+n.25 (third run), each plate screened in triplicate. Thus, one library plate (n.25) was assayed within all three runs making possible inter-run comparison. The protocol below describes a single run of 9 library plates tested in triplicate.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Name of Material/ Equipment</td>
			<td style="width:239px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Culture plate 96-well White</td>
			<td style="width:239px;">PerkinElmer</td>
			<td align="right" style="width:119px;">6005688</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">StorPlate 96-well U bottom (dilution plate)</td>
			<td style="width:239px;">PerkinElmer</td>
			<td align="right" style="width:119px;">6008190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">100 ml disposable trough for reagents</td>
			<td style="width:239px;">Tecan</td>
			<td>10 613 048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">300 ml disposable robotic reservoir</td>
			<td style="width:239px;">VWR</td>
			<td style="width:119px;">PB12001301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Robot tips DITI 50&#956;L Sterile</td>
			<td style="width:239px;">Tecan</td>
			<td align="right" style="width:119px;">30038607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Robot tips DITI 200&#956;L Sterile</td>
			<td style="width:239px;">Tecan</td>
			<td align="right" style="width:119px;">30038617</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrix 1.4 ml 2D Barcoded w, Flat Bottom Tubes</td>
			<td style="width:239px;">Thermo Scientific</td>
			<td align="right">3711</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">ONE-Glo Luciferase Assay System&#160;</td>
			<td style="width:239px;">Promega</td>
			<td style="width:119px;">E6120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">RPMI</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">BE12-918F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">PBS-1X, w/o Ca++, Mg++</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">BE17-516F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">L-Glutamine 200mM</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">BE17-605E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">FBS</td>
			<td style="width:239px;">Lonza</td>
			<td style="width:119px;">DE14-801F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">DMSO</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">The Spectrum collection (compound library)</td>
			<td style="width:239px;">MicroSource Discovery Systems</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Tecan Freedom EVO200 robot&#160;</td>
			<td style="width:239px;">Tecan</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Tecan F200 multiplate reader&#160;</td>
			<td style="width:239px;">Tecan</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput screening for chemical modulators of post-transcriptionally regulated genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Using the described approach, we screened a 2,000-compound library for potential modulators of post-transcriptional control mechanisms exerted through the 3&#8217; UTR of the MYCN gene. Figure 3 depicts the results of the primary screening exemplified by a single library plate. Luciferase signal displayed as percentage of vehicle-treated controls was obtained by measuring luciferase activity in triplicate plates of CHP134-mycn3UTR cells treated with compounds of a single library plate. As expect...

Watch this Scientific Journal Video about High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes at JoVE.com

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

high-throughput-screening-for-chemical-modulators-post

Research

JoVE Journal

Biology

9.3K Aufrufe.  University of Trento. Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3’ UTR.

Serie: High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

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.

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 ...

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 ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.