The authors would like to thank Dr. Rick Padgett (Waksman Institute, Rutgers University, NJ) for the gift of the dbl-1 cDNA and Dr. Christopher Rongo (Waksman Institute, Rutgers University, NJ) for an injection marker. Dr. Barth Grant's lab performed the gene gun bombardment for low copy number integration of the gfp-tagged dbl-1 construct. The Ren&eacute; Garcia laboratory provided technical assistance during the creation of texIs100. The Ren&eacute; Garcia, Robyn Lints, and Hongmin Qin laboratories provided productive advice. This work was funded by start-up funds from the TAMHSC Department of Molecular and Cellular Medicine. The compound scope and spinning disk confocal were purchased with funds provided by the department and the TAMHSC College of Medicine Office of the Dean.

1. Screening strain construction
The careful design of the screening strain is critical for the success of the screen and has been described elsewhere3. For some researchers, using a strain that expresses a visible product from a transgene is needed for the experiment. Many strains harboring integrated transgenes are available from the CGC or individual researchers. If a transgenic strain is required for the screen but is not available, then it can be generated using a published method like bombardment4, UV/TMP5, or Mos transposon insertion6. In order to visualize our protein of interest, we inserted the gfp-coding sequence in frame with the mature cDNA sequence (we used dbl-1 sequence). Because this GFP fusion protein is not visible by dissecting scope, we used a coinjection marker that was visible and did not affect the viewing of our protein of interest (ttx-3p::rfp, for a review of several other standard markers, see 7). We then created an integrated transgene from the extrachromosomal array. We found that bombardment of the transgene yielded a low-copy number integrated line that neither rescued the dissecting microscope phenotype nor produced visible levels of GFP-tagged product (Beifuss and Gumienny, unpublished). UV/TMP integration of a multiple copy extrachromosomal array originally made by injection8,9 yielded visible, rescuing levels of transgene product (Figure 3A). Western blot with anti-GFP antibody confirmed that the transgene (allele name texIs100) is expressed and processed correctly (Beifuss and Gumienny, unpublished). Integrated transgenes should be outcrossed five times to remove extraneous background mutations regardless of source.
For our screen, we wanted the only protein of interest to be the transgenic, tagged form. Therefore, we removed the functional endogenous gene by introducing a loss-of-function mutant dbl-1 allele. 
Lastly, we sensitized the strain to the effects of RNAi. The RNAi from the library will, for many genes, produce a more severe reduction in gene product if animals contain a mutation that sensitizes the animals to the effects of RNAi10,11 The tissue(s) of interest should be considered when choosing an appropriate RNAi sensitizing background11. We used the canonical rrf-3(pk1426) allele to make our screening strain. RRF-3 is an RNA-directed RNA polymerase (RdRP) homolog that normally inhibits somatic RNAi10. Mutations in other RNAi hypersensitizing genes like eri-1 or eri-1 lin-15b can be used instead to increase the effectiveness of the RNAi11-13. 
The screening strain we made has the genotype rrf-3(pk1426); texIs100; dbl-1(nk3).
2. Choosing and preparing an RNAi library
Commercially available C. elegans cDNA libraries represent about 55% or 87% of the predicted genes in C. elegans individually (Vidal lab or Ahringer lab libraries, respectively). Individual clones are available for purchase (Open Biosystems, Geneservice, Ltd.). We chose the ORF-RNAi library constructed by the Vidal lab (Open Biosystems) because its clones are mostly full-length cDNAs Gateway cloned and ready for downstream applications (Invitrogen Corporation, Carlsbad, CA). The original Ahringer lab genomic cDNA library contains cDNA fragments that are not as widely useful for downstream characterization experiments14,15. Both libraries use a vector that contains two T7 promoters flanking the insert (Figure 1). The constructs are grown in bacterial strain HT115, which expresses T7 polymerase upon induction by isopropyl-&beta;-D-thiogalactopyranoside (IPTG). Bacteria containing library clones are induced with IPTG to produce dsRNA (see Step 3.4). 
Duplicate the entire library upon receipt and use the duplicate for all experiments. The original and duplicate libraries should be stored in different -80&deg;C freezers that are connected to separate electrical lines.
3. Preparation of bacteria with library clones
<ol>
 <li>(Within two months of the experiment) Streak bacteria from selected feeding library clones, as well as positive and negative controls, on labeled LB- carbenicillin/ tetracycline plates (we grow 8 cultures per 100 mm plate) and incubate at 37&deg;C overnight (&gt;16 hours) (Figure 1). For each 24-well plate, we use two controls. The positive control, bli-4 (K04F10.4), produces a dose-dependent post-embryonic pheontype16,17. The negative control, C06C3.5, is a predicted pseudogene (<a href="http://WormBase.org">WormBase.org</a>) that causes no observed phenotypes (Beifuss and Gumienny, unpublished). </li>
 <li> (Within two months of the experiment, preferably sooner) Prepare 24-well Nematode Growth Medium (NGM) plates containing 25 &#x2013; 50 &mu;g/ml carbenicillin and 1mM IPTG. Store at 4&deg;C. </li>
 <li>(Day 1) For each clone, inoculate 2 ml LB media containing 50 &mu;g/ml carbenicillin with a single colony from the streaked plate(s) (in Step 3.1) using standard sterile technique. Incubate at 37&deg;C overnight (&gt;16 hours) in a shaker set to 220 &#x2013; 240 rpm (Figure 1). The solution should be cloudy after 16 hours. </li>
 <li>(Day 2) The next morning, induce double stranded RNA (dsRNA) production by adding IPTG to the cultures to a final concentration of 1mM. Incubate cultures at 37&deg;C, 220 &#x2013; 240 rpm, for an additional 4 &#x2013; 5 hours. This step ensures a more consistent and robust RNAi-induced knockdown of gene product than relying solely on the IPTG in the agar of the NGM plate (in Step 3.5). </li>
 <li>Spot 30 &mu;l of each induced bacterial culture into separate wells of a 24-well NGM RNAi plate containing 25 &#x2013; 50 &mu;g/ml carbenicillin and 1mM IPTG (Figure 1, see Figure 4 for template used to track RNAi experiments). Put plates, uncovered, in a sterile flow hood for 20 minutes or until the bacterial spots are dry. Do not overdry. Overdrying causes the edges of the agar to pull away from the plate and animals will be irretrievable if they crawl into the resulting crevices. </li>
</ol>
4. Preparation of nematodes
Start with a staged population of nematodes. Staging animals decreases the chance that differences observed between the control and the experimental RNAi trials are simply due to differences in developmental stage of the animals (however, if the RNAi causes a developmental delay, this should be noted by the screener (Figure 2)). Furthermore, starting the RNAi experiment with L1 larvae obviates potential confounding effects of embryonic lethality by RNAi of genes that may also play postembryonic roles of interest. 
Staging is accomplished by first bleaching a mixed-stage population of the screening strain, which only eggshell-protected embryos survive. This stages animals to within about 12 hours at 20&deg;C or about 18 hours at 16&deg;C18. To more tightly stage animals, arrest animals in the first larval stage (L1) by letting embryos hatch in M9 medium without food. Starved L1 animals placed on food resume growth from the same starting age19. 
<ol>
 <li>(Day 1) Use three 100 mm plates of fed, clean screening strain animals that contain gravid adults and embryos. Wash animals from plates using sterile M9 by pipeting liquid across the plate surface gently to loosen animals and embryos. Transfer the wash into a sterile 15 ml tube with screw cap. </li>
 <li> If the wash is greater than 3.5 ml, spin down the animals (~3000 rpm for 30 seconds) and remove liquid to 3.5 ml. If the wash is less than 3.5 ml, add H20 or M9 to the tube to total 3.5 ml. </li>
 <li>Wear appropriate protective gear (lab coat, gloves, and goggles) while handling chemicals. Mix 0.5 ml 5 N NaOH with 1 ml fresh bleach (5% sodium hypochlorite, less than 1 year old). Add this mixture to the 3.5 ml nematode wash. Start a timer counting up. This process should not take more than 10 minutes. If it does, then embryos will be endangered and yield may be reduced, or the bleach is old and should be replaced. </li>
 <li>Vortex the tube for about 10 seconds. Repeat the vortexing every two minutes for 4 to 10 minutes. After each vortexing, observe the solution under a dissecting scope for animal carcasses. </li>
 <li>When embryos are released and adults are fragmented (6 &#x2013; 8 minutes), pellet the embryos by spinning the tube in a table top centrifuge (~3000 rpm, 30 seconds). </li>
 <li>Aspirate as much of the supernatant as possible without disturbing the pellet. Be careful, not greedy. </li>
 <li>Add sterile M9 to ~ 10ml. Vortex briefly or shake well to resuspend the pellet. </li>
 <li> Spin and remove supernatant again. If the smell of bleach can be detected in the tube, repeat rinse as necessary. </li>
 <li>Resuspend embryos in 10 &#x2013; 15 ml sterile M9 and transfer to a small (25 ml) Erlenmeyer flask (for aeration). Shake at the proper temperature for the strain and experiment (15 &#x2013; 25&deg;C) overnight (&gt; 16 hours at 20&deg;C). In this time, the animals will all hatch and arrest in the L1 diapause. </li>
 <li>(Day 2) Spin down L1 stage animals in a 15 ml tube and resuspend in 0.5 &#x2013; 1 ml M9 (~3000 rpm, 30 sec.). Place 5 &mu;L solution on a separate plate and count the number of animals. Make adjustments to the volume, if needed, to yield 30 worms/ 3 &#x2013; 10 &mu;L solution. </li>
 <li>Spot approximately 30 hypochlorite synchronized L1 stage animals onto the bacteria in each well of the prepared 24-well plate. Too many more than 30 animals per well may result in the population starving before reaching maturity. Let the plate dry with the lids askew. Replace the lid and secure it with a rubber band. Invert the plate and place it at the appropriate temperature for the time needed to score at the desired stage. Because rrf-3(pk1426) is a temperature sensitive allele, our screening strain animals were grown at 15 &#x2013; 17&deg;C for three to four days to observe L4 to adult stage animals (Figure 1). </li>
</ol>
5. Observation of nematodes
(Day 5) Once the nematodes have grown to the desired stage (Figure 2A), confirm that the positive and negative controls produce the expected phenotypes by using a dissecting microscope. We use bli-4 as a control for RNAi efficacy, because it produces dose-dependent post-embryonic defects that range from blistered adults (mild RNAi phenotype, not shown) to developmentally arrested, tiny larvae (strong, expected RNAi phenotype) (Figure 2B). Then observe the animals in each experimental well using a dissecting microscope and note obvious abnormal phenotypes induced by RNAi (Figure 2C). After the controls are confirmed and gross phenotypes noted, screen the RNAi experiments for phenotypes of interest. We use a compound microscope equipped with fluorescence and a 63x objective to observe at least five animals from each RNAi experiment, beginning with controls (Figure 3). 
<ol>
 <li>Prepare a slide (standard 75 x 25 mm) by making a 4% agar pad (4% agar in H2O). To ensure a uniform thickness of agar pad, place a clean glass slide between two spacers (each spacer is made of a microscope slide with two layers of lab tape on it). Pipette about 100 &mu;L (two to three drops from a glass Pasteur pipet) of molten 4% agar onto the center of the clean glass slide. Form an agar pad by covering the molten agar using an additional slide placed quickly but gently on top of the spacers and the molten agar. Expose the agar pad by gently sliding the glass slides apart, leaving the pad adhering to one slide.</li>
 <li> Place a ~5 &mu;L drop of anesthetic (levamisole or sodium azide, for example) on the agar pad. Mount 8 &#x2013; 12 animals in the anesthetic. Cover with a 22 x 22 mm #1.5 coverslip (or the thickness of coverslip best suited for the compound microscope) and observe animals using a compound microscope. </li>
 <li>Record gene, number of animals observed, and phenotype results for each RNAi experiment. For a screen, make and use a standard template (see example, Figure 5). </li>
 <li>Verify the RNAi-produced phenotype by another, secondary phenotype, if possible, and by repeating the experiment. (For example, the example screen shown used alteration of GFP-tagged DBL-1 localization as a primary screen and body size as a secondary screen20.) Bacteria can be used from the same streak (stored at 4&deg;C) for up to two months. Older streaks may result in poor growth in liquid overnight cultures and should be restreaked first. </li>
 <li>Sequence at least one end of the cDNA insert to confirm that the insert matches the library label (the Ahringer library has had a reliability analysis performed; see <a href="http://biocompute.bmi.ac.cn/CelRNAi/">http://biocompute.bmi.ac.cn/CelRNAi/</a>)21. Recommended primers (for inserts from either library) anneal on sites in the L4440-derived vector and flank the insertion site: 5'-AGCGAGTCAGTGAGCGAG-3' and 5'-GTAAAACGACGGCCAGT-3' (M13f20 primer). </li>
</ol>
Using this method, a single person can reasonably stage and observe two to four sets of experiments every week. For instance, animals staged on Monday and Tuesday can be observed Thursday and Friday, respectively, and can again be staged on Friday and Saturday for Monday and Tuesday observation. Depending on the phenotype(s) screened each day, a set may comprise one 24-well plate by compound microscopy or more if the phenotype is quickly identified. Thus, at least 88 different RNAi experiments can be observed in one week, taking into account positive and negative controls and rate of screening. A dissecting scope screen could be performed much faster, without the need for mounting animals on slides for viewing. Multiple people could also increase a lab's throughput by staggering schedules and/or using multiple microscopes. An alternative method for growing animals on a thin film of agar and transferring a slice of the agar containing animals directly to a slide for viewing may save time. This variation was successfully used for screening male tail abnormalities at 400x magnification22.
6. Representative Results
Examples of normal and altered localization of GFP-tagged DBL-1 are shown in Figure 3. Normal expression of GFP-tagged DBL-1 includes ventral nerve cord cell bodies and a row of punctae (Figure 3A). DBL-1 is severely attenuated when animals are fed RNA that prevents dbl-1 mRNA translation (Figure 3B). dbl-1(RNAi) also produces small animals, the secondary screen in this example (data not shown). Genes that affect DBL-1 localization are readily identified by RNAi using a strain designed for this screen (Figure 3C). 
<img src="/files/ftp_upload/3442/3442fig1.jpg" alt="Figure 1" /> 
Figure 1. Screen scheme to identify extracellular regulators of DBL-1 signaling. Bacteria from the feeding library are grown overnight, induced with IPTG, and incubated at 37&deg;C for an additional 4 hours to allow the bacteria to produce double stranded RNA (dsRNA). 30 &mu;l of the induced bacterial culture is spotted per well onto a 24-well NGM (nematode growth medium) plate containing 25 &mu;g/ml carbenicillin and 1mM IPTG and allowed to dry in a sterile flow hood. We stage first larval stage (L1) larvae by letting hypochlorite-treated embryos hatch in media without food, which induces an L1 diapause (arrested development). Approximately 30 synchronized L1 stage animals are plated onto each well containing bacteria from the RNAi library, which allows staged animals to resume growth and to consume the dsRNA created by the bacteria23. After 72 hr at 15&deg;C, the young adult worms are screened for a visible phenotype. At this age, body size defects are evident and fluorescence is bright from texIs100 transgene expression. 
<img src="/files/ftp_upload/3442/3442fig2.jpg" alt="Figure 2" /> 
Figure 2. Examples of phenotypes to identify before screening. All images of animals in a plate well were taken at the same magnification using a dissecting microscope. Animals were all imaged about 72 hours after plating as starved L1 larvae. All images were treated identically. A) RNAi of a gene that gives no gross morphological defects. Animals appear wild type. B) RNAi of bli-4. Animals display arrested development, and are tiny compared to the animals in panel A. C) RNAi of dpy-13. Animals are at the same stage of development as animals in panel A, but display a "dumpy" body morphology. 
<img src="/files/ftp_upload/3442/3442fig3.jpg" alt="Figure 3" /> 
Figure 3. Examples of fluorescently tagged protein and how RNAi of specific genes alters localization pattern. All images were taken at 630x magnification with spinning disk confocal microscopy, 5 sec. exposure. Scale bar = 10 &mu;m. All images were treated identically. Open arrowheads indicate cell bodies. Arrows mark line of punctae. Filled arrowheads indicate some aberrantly localized GFP-tagged DBL-1. A) RNAi-fed pseudogene C06C3.5 ("wild-type"). B) dbl-1(RNAi) control. C) RNAi of a gene required for normal localization of GFP-tagged DBL-1.
<img src="/files/ftp_upload/3442/3442fig4.jpg" alt="Figure 4" /> 
Figure 4. Template example for tracking RNAi experiments (library clones) in 24-well plates. This template can be used to create a permanent record of the experiments, unlike directly labeling plates.
<img src="/files/ftp_upload/3442/3442fig5.jpg" alt="Figure 5" /> 
Figure 5. Template example for recording RNAi phenotypes. Expand as needed.

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We have nothing to disclose.

The RNAi screening method presented here enables a sensitive and rapid analysis of gene products required for a normal (or transgenic) postembryonic phenotype. The example shown is a screen for genes involved in the subcellular localization of a fluorescently tagged protein. However, this protocol can be modified to identify genes affecting other postembryonic phenotypes of interest.
This method takes advantage of a candidate gene approach by using an RNAi library. Forward genetic screens usin...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>NGM Agar</td>
 <td>Nematode growth medium</td>
 <td>IPM Scientific, Inc</td>
 <td>Can be prepared following NGM agar protocol25</td>
 </tr>
 <tr>
 <td>M9 Medium </td>
 <td>22mM KH2PO4, 
 42mM Na2HPO4, 
 86mM NaCl, 
 1 mM MgSO4</td>
 <td>&nbsp;</td>
 <td>26 </td>
 </tr>
 <tr>
 <td>Agar-Agar</td>
 <td>EMD Chemicals Inc.</td>
 <td>1.01614.1000 </td>
 <td>2% in water for NGM plates. 4% in water for microscope slide pads (autoclave initially and microwave to melt thereafter).</td>
 </tr>
 <tr>
 <td>Bacto Peptone</td>
 <td>Becton Dickinson &ndash; Difco CP </td>
 <td>211677</td>
 <td>0.25%</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Research Products International Corp.</td>
 <td>I56000-5.0 </td>
 <td>1 mM final concentration</td>
 </tr>
 <tr>
 <td>carbenicillin</td>
 <td>Research Products International Corp.</td>
 <td>C46000-5.0</td>
 <td>50 &mu;g/ml working dilution</td>
 </tr>
 <tr>
 <td>LB Broth Lennox</td>
 <td>Becton Dickinson &ndash; Difco CP </td>
 <td>240230</td>
 <td>20 g/liter</td>
 </tr>
 <tr>
 <td>tetracycline</td>
 <td>Sigma</td>
 <td>268054</td>
 <td>12.5 &mu;g/ml working dilution</td>
 </tr>
 <tr>
 <td>sodium hypochlorite</td>
 <td>Any brand</td>
 <td>5% household bleach </td>
 <td>Use fresh bleach.</td>
 </tr>
 <tr>
 <td>sodium hydroxide</td>
 <td>Any Brand</td>
 <td>CAS 1310-73-2</td>
 <td>5 N stock</td>
 </tr>
 <tr>
 <td>M9 medium</td>
 <td>Wormlab Recipe Book</td>
 <td><a href="http://130.15.90.245/wormlab_recipe_book.htm#Commonlab">http://130.15.90.245/wormlab_recipe_book.htm#Commonlab</a></td>
 <td>26 </td>
 </tr>
 <tr>
 <td>levamisol</td>
 <td>Sigma</td>
 <td>31742</td>
 <td>100 &mu;M &ndash; 1 mM working dilution</td>
 </tr>
 <tr>
 <td>sodium azide </td>
 <td>Fisher Scientific</td>
 <td>S227 </td>
 <td>10 mM in M9 working dilution</td>
 </tr>
 <tr>
 <td>24-well plate</td>
 <td>Greiner Bio-One</td>
 <td>662160</td>
 <td>VWR distributor</td>
 </tr>
 <tr>
 <td>microscope slides</td>
 <td>Any brand</td>
 <td>75 x 25 x 1 mm</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>microscope cover slips</td>
 <td>Any brand</td>
 <td>22 x 22 mm No.1.5</td>
 <td>Use the thickness recommended by the microscope manufacturer.</td>
 </tr>
 <tr>
 <td>compound microscope</td>
 <td>Carl Zeiss, Inc.</td>
 <td>A1m</td>
 <td>Use objectives and filters to match the needs of the experiment.</td>
 </tr>
 <tr>
 <td>media pump</td>
 <td>Manostat Varistaltic pump</td>
 <td>Kate 
 model #72-620-000</td>
 <td>Use tubing and settings appropriate for the machine</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about RNAi Screening to Identify Postembryonic Phenotypes in C. elegans at JoVE.com

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.

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 ...

rnai-screening-to-identify-postembryonic-phenotypes-in-c-elegans

Research

JoVE Journal

Biology

17.0K Aufrufe.  Texas A&M University System Health Science Center. Wir beschreiben eine Methode, um sensibilisiert postembryonalen Regulatoren der Protein-Expression und Lokalisation in identifizieren C elegans Mit Hilfe eines RNAi-basierter Genom-Bildschirm und einen integrierten Transgen, die eine funktionelle, fluoreszenzmarkierten Protein exprimiert.

Serie: RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

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 ...

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. ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

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 ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...