C. elegans transgenes were provided by the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN). This work was supported by NIH R21 grant NS0667678-01 to KS. All authors participated in the collection, analysis, and interpretation of data. CKL, KS, and KPC participated in writing and revising the manuscript.

1. Preparation of bacterial worm food
Day 1
<ol>
<li>Add 5 ml of saturated E. coli OP50 bacterial culture to 500 ml Terrific broth supplemented with 50 &mu;g/ml streptomycin and grow in a shaking incubator (225 rpm) overnight at 37&deg;C.</li> 
</ol>
Day 2
<ol start="2">
<li>Split the overnight bacterial culture into ten 50 ml tubes and centrifuge bacteria in a refrigerated centrifuge at 2,500 rcf for 20 minutes.</li>
<li>Decant off LB broth and resuspend each bacterial pellet in 10 ml of liquid nematode growth media (NGM). Shake horizontally in a floor shaker for 15 minutes to resuspend the bacteria. To make NGM buffer, add 3 g NaCl to 1 L deionized water and autoclave. Cool to 55&deg;C and add in order the following sterile solutions: 1 ml of 1 M CaCl2, 1 ml of 1 M MgSO4, and 25 ml of 1 M potassium phosphate, pH 6.0.</li>
<li>Centrifuge the bacterial culture in a refrigerated centrifuge at 2,500 rcf for 20 minutes.</li>
<li>Decant off NGM buffer and weigh the bacterial pellet.</li>
<li>Add an equal volume of NGM buffer to resuspend the pellets, aliquot 3 ml of bacteria concentrate into 15 ml tubes, and store at -20&deg;C.</li>
</ol>
2. Large-scale C. elegans liquid culture
We use a transgenic line VP596 (dvIs19[pAF15(gst-4::GFP::NLS)];vsIs33[dop-3::RFP]) carrying two fluorescent constructs: Pgst-4::GFP14 to monitor SKN-1 activity and Pdop-3::RFP17 to serve as a standard for worm number normalization.
Day 1
<ol>
<li>Mix 150 ml NGM buffer, 1.5 ml LB broth, 150 &mu;l of 5 mg/ml cholesterol (in ethanol), and 75 &mu;l of 100 mg/ml streptomycin. Filter-sterilize the mixture and add to a 1 liter sterile flask. 
NOTE: Adding 1% LB broth to NGM buffer helps to prevent worms from sticking to glassware and plasticware, but this amount is not enough to support bacteria growth.</li>
<li>Synchronize worms with the standard hypochlorite procedure18. Up to 0.5 ml of gravid worms can be processed in a single 15 ml tube with 5 ml of hypochlorite solution (3.75 ml sterile water, 1 ml household bleach, and 250 &mu;l 10 N NaOH). Wash the released eggs with sterile water and resuspend them in 10 ml of NGM buffer.</li> 
<li>Dilute a sample of the eggs 100 fold in NGM buffer (e.g., 100 &mu;l in 10 ml) and count the number of eggs in three separate 5 &mu;l aliquots. Multiple the average number by 200,000 (100 dilution factor x 2,000) to estimate the total number of eggs.</li>
<li>Add 200,000 to 2 million eggs to the flask with NGM buffer and shake the culture at 100 rpm at 20&deg;C.</li>
</ol>
Day 2
<ol start="5">
<li>At least 16 h after adding eggs to the flask, use a sterile serological pipet to remove approximately 0.5 ml of the suspended worm culture. Pipet three 5 &mu;l drops onto the sterile lid of a Petri dish. Place the lid in a -20&deg;C freezer for 1-2 minutes to paralyze worms. Count the average number of live hatched worms per 5 &mu;l and then multiple by 30,000 (150 ml/5 &mu;l) to estimate the total number of hatched worms.</li>
<li>Thaw a tube of frozen OP50 bacterial culture (Protocol 1.6) and add 3 ml of 50% OP50 into the worm culture per 500,000 hatched worms. Shake the flask at 100 rpm at 20&deg;C. Worms will develop into L4 larvae and young adults in about 51 hours. The OD600 of bacteria should be about 2.200 at the beginning. Monitor the amount of bacteria by absorbance and add more if the OD 600 drops below 0.900.</li>
</ol>
3. Worm collection and dispensing 
Day 4
<ol>
<li>Use a sterile serological pipet to transfer approximately 0.5 ml of the suspended worm culture to a standard NGM agar plate. View the worms with a stereomicroscope to make sure that most are L4 larvae or young adults. L4 larvae will have a ventral clear spot in the middle of the body. Young adults will be slightly larger and will not have a clear spot.</li>
<li>Pour the worm culture into sterile 50 ml tubes and place the tubes in a test tube rack on the benchtop. Allow the worms to settle for about 10 minutes. Remove the supernatant by aspiration or pipetting. 
NOTE: This step can be important to remove unhatched eggs or worms that did not develop because they will settle slower than L4 larvae and young adults.</li>
<li>Collect all worms into a single 50 ml tube and wash with NGM buffer with 1% LB broth (NGM+LB) 3-4 times to remove bacteria. 
NOTE: This is best done in a swinging-bucket centrifuge by collecting worms at 500 rcf for 30 sec and replacing the supernatant with fresh NGM+LB buffer.</li>
<li>After washing, fill the tube with NGM+LB buffer to 50 ml and pour into a custom worm dispensing flask. Add a small stir bar and stir to keep the worms suspended. Count the number of worms in three 5 &mu;l drops as indicated above. Dilute with NGM+LB until you get 12-15 worms per 5 &mu;l drop (&tilde;2.5-3.0 worms /&mu;l). 
NOTE: A minimum of about 20 ml (60,000 worms) is needed to fill the dead space of the dispensing flask and dispenser cassette. Each 384 well plate requires about 35,000 worms or 11.5 ml of suspended worms.</li>
<li>Load a 10 &mu;l dispensing cassette and sterilize by priming with 70% ethanol. Rinse out the ethanol by priming with sterile water.</li>
<li>Insert the end of the dispensing cassette into the flask so that it is just above the stir bar without disrupting its movement. Make sure that the worms remain suspended throughout the whole flask. Program the dispenser to dispense at low speed with 2 pre-pulses. Prime and run at least 10 ml of suspended worms. Fill plates as needed rapidly to prevent the worms from settling in the tubing.</li>
<li>Seal the plates with breathable tape.</li>
<li>Place the plates on a shaking platform in an incubator at the appropriate temperature for your assay. Do not stack the plates.</li>
</ol>
4. Fluorescence analysis
<ol>
<li>Place the target plate into a microplate reader to measure the fluorescence intensity of each well with the appropriate emission and excitation wavelength (Filters for our assay: GFP 485/20ex 528/20em; RFP 540/25ex 590/35em).</li>
<li>Calculate the ratio of GFP/RFP and normalize with the readings of the control wells (without activating compound) to determine the exact fold difference of the fluorescence intensity derived from individual treatments.</li>
</ol>
5. Representative Results: 
Our SKN-1 assay uses a chromosomally integrated dual reporter strain (VP596). As shown in Figure 1, the number of worms is well correlated to volume dispensed. As shown in Figures 2A and 2B, total GFP and RFP fluorescence per well is highly reproducible from well to well across a 384 well plate. As shown in Figure 2C, un-induced Pgst-4::GFP fluorescence is linearly correlated to Pdop-3::RFP across 384 wells. When expressed as a ratio of GFP/RFP, fluorescence becomes highly reproducible from well to well across a 384 well plate (compare coefficient of variation from Figure 2D to 2A) demonstrating the ability of the Pdop-3::RFP reporter to reduce variability. As shown in Figure 3, the induction of GFP relative to RFP with a SKN-1 activating xenobiotic (38 &mu;M juglone) is robust and highly reproducible across a 384 well plate.
<img src="/files/ftp_upload/2745/2745fig1.jpg" alt="Figure 1"/> Figure 1. Number of worms versus volume dispensed. Worms were diluted to approximately 2/&mu;l and dispensed into a 24 well plate for manual counting with a stereomicroscope. N = 8 wells per volume.
<img src="/files/ftp_upload/2745/2745fig2.jpg" alt="Figure 2"/> Figure 2. Total fluorescence of worms dispensed into a 
384 well plate is reproducible. Worms were diluted to approximately 2.5/&mu;l and 30 &mu;l was dispensed into every well of a 384 well plate. GFP (A) and RFP (B) 
fluorescence of all wells had a coefficient of variation below 9%. (C) GFP fluorescence is highly correlated to RFP fluorescence. (D) Calculating the ratio of GFP to 
RFP reduced the coefficient of variation to below 6% (A, B, and D) Solid lines indicate the means and broken lines indicate three standard deviations above or below 
the mean.
<img src="/files/ftp_upload/2745/2745fig3.jpg" alt="Figure 3"/> Figure 3. Activation of Pgst-4::GFP is robust and consistent. Approximately 75 L4 worms were dispensed into all wells of a 384 well plate and 38 &mu;M juglone was added in every other column. GFP and RFP fluorescence was measured after 21 h of incubation. The mean relative fluorescence ratios of all control (1.0) and juglone wells (8.9) are marked with solid lines. Three standard deviations above the control mean and below the juglone mean are marked with broken lines.

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H., Blackwell, T. 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L., Pepper, J. S., Koelle, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+extrasynaptic+dopamine+signaling+in+Caenorhabditis+elegans.">Mechanism of extrasynaptic dopamine signaling in Caenorhabditis elegans.</a> Nature Neuroscience. 7, 1096-1103 (2004).</li><li>Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans, a Practical Guide. , (2005).</li><li>Pulak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+analysis,+sorting,+and+dispensing+of+C.+elegans+on+the+COPAS+flow-sorting+system.">Techniques for analysis, sorting, and dispensing of C. elegans on the COPAS flow-sorting system.</a> Methods Mol Biol. 351, 275-286 (2006).</li></ol>

No conflicts of interest declared.

We present a method for culturing and dispensing large numbers of transgenic nematodes. The equipment used for culturing worms is standard for laboratories performing molecular cloning and the liquid handling and fluorescence equipment is standard for laboratories processing large numbers of microplates. Other methods of dispensing large numbers of live C. elegans require expensive particle sorting equipment19. The Pgst-4::GFP assay can be used to screen for small molecule modulators of ca...

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>LB broth</td>
 <td>Research Products International Corp.</td>
 <td>L24066</td>
 <td>&nbsp;</td>
 </tr>

		<tr>
 	<td>Terrific broth</td>
 <td>Research Products International Corp.</td>
 <td>T15100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Synergy&trade; HT Multi-mode Microplate Reader</td>
 <td>BioTek</td>
 <td>&nbsp;</td>
 <td>Filters: GFP 485/20ex 528/20em; RFP 540/25ex 590/35em</td>
 </tr>
 <tr>
 	<td>MicroFlo Select Dispenser</td>
 <td>BioTek</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Worm dispensing flask</td>
 <td>Southern Scientific Inc., Micanopy, FL</td>
 <td>&nbsp;</td>
 <td>Custom assembled
(352) 284-2531
</td>
 </tr>
 <tr>
 	<td>384 microplates</td>
 <td>Greiner Bio-One</td>
 <td>5678-1209</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Breathable sealing tape</td>
 <td>Nunc</td>
 <td>241205</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>(5-hydroxy-p-naphthoqinone)
Juglone
</td>
 <td>ACROS</td>
 <td>121640010</td>
 <td>Juglone is dissolved in DMSO </td>
 </tr>
 <tr>
 	<td>DMSO</td>
 <td>Sigma-Aldrich</td>
 <td>D-5879</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>C. elegans transgenic strain</td>
 <td>Author&#x2019;s laboratory</td>
 <td>VP596</td>
 <td>Pgst-4::GFP and Pdop-3::RFP</td>
 </tr>
 
 
 </tbody>
	
</table>

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.

Watch this Scientific Journal Video about High-throughput Screening and Biosensing with Fluorescent C. elegans Strains at JoVE.com

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 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 ...

high-throughput-screening-biosensing-with-fluorescent-c-elegans

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

17.9K Views.  University of Florida. 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.

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

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

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 ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

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, ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

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 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.

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 and Biosensing with Fluorescent C. elegans Strains

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Chapters in this video

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

High-throughput Purification of Affinity-tagged Recombinant Proteins

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

C. elegans Chemotaxis Assay

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

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

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