Name: large-scale gene knockdown in c. elegans using dsrna feeding libraries to generate robust loss-of-function phenotypes
Uploaded: 2013-09-25T19:00:00
Description: Watch this Scientific Journal Video about Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes at JoVE.com

This work was supported by funds from the National Institutes of Health MH097163. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40-OD010440).

Note: This protocol can be used to identify genes required for a variety of biological processes in a variety of tissue types. As a necessary quality control during the screen, animals are fed both positive and negative control dsRNA-expressing bacteria to demonstrate RNAi effects in the targeted tissue.
Other published feeding protocols grow dsRNA-expressing bacteria in the presence of either 50 &mu;g/ml ampicillin or 25 &mu;g/ml carbenicillin 8-10. We have found that bact...

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We have described a protocol that uses a commercially available dsRNA feeding library to perform large-scale screens in C. elegans. We provide all instructions to duplicate the library and to use it effectively. We demonstrate that this approach is able to identify genes involved in different biological processes in different tissues of the animal. While the phenotypes we describe here are easily observable (Unc, Egl, and Dpy), this protocol can be adapted to search for genes required for nearly any biological p...

Historically, genetic screens in C. elegans have been performed by treating animals with a chemical mutagen such as ethyl methanesulfonate to create random mutations within DNA. Progeny or grandprogeny of mutagenized animals are then isolated that exhibit a desired abnormal phenotype. The mutation responsible for causing the phenotype is then identified through a labor-intensive mapping procedure or by sequencing the mutant worm's entire genome. There are many advantages to performing such chemical mutagenesis screens as they create permanent lesions within the worm genome that can result in both gain-of-function and loss-of-function phenotypes, but the mapping procedure is slow and expensive.
Double-stranded RNA interference (dsRNAi) provides an alternative means to identify genes involved in important biological processes. In this method, dsRNA matching the coding sequence of an endogenous gene is introduced into the worm. The dsRNA is processed in vivo to generate small (21-22 nucleotide) RNAs that serve as guides to find and bind the matching endogenous mRNAs, targeting them for destruction 1. This procedure is relatively quick, but because dsRNA targets mRNA rather than DNA, only reduction-of-function phenotypes are observed using this approach.
Introduction of dsRNAs into an animal can be achieved by direct injection of dsRNA 2, by soaking animals in dsRNA 3, or by feeding animals bacteria that express dsRNA 4. Each of these delivery methods cause systemic knockdown effects as most cells of the animal express SID-1, a transmembrane channel capable of importing dsRNA 5. Originally used as a reverse genetics approach to knockdown the expression of individual genes one at a time, the development of two feeding libraries now permits large-scale genetic screens. The commercially-available dsRNA libraries contain bacterial clones representing either 55% or 87% of all C. elegans genes 6, 7.
Unfortunately, large scale dsRNAi feeding screens often result in variable knockdown efficiencies 8-10. While the absolute level of knockdown by RNAi is not easily measured, the reduced knockdown efficiency observed in large scale screens must be caused by residual gene-specific mRNAs that escape degradation by RNAi. This, in turn, could be a direct result of dsRNA plasmid loss from bacteria fed to animals in these screens. Supporting this idea, animals fed mixtures of bacteria, in which only 50% of the bacteria express dsRNAs against a targeted gene, show dramatically reduced (if any) knockdown effects when compared to control fed animals 11. The extent of gene knockdown becomes a critical concern when performing dsRNAi screens as most genes in C. elegans are haplosufficient and thus gene expression must be reduced by at least 50% to cause loss-of-function phenotypes 12.
We have used previously published feeding protocols to perform large-scale screens and found the same variable knockdown effects reported by others 8-10. In a search for possible causes, we found that nearly 60% of the bacteria fed to animals in the screen had lost the dsRNA plasmid. We have developed a library duplication and screening protocol that dramatically reduces or eliminates plasmid loss and greatly improves knockdown efficiency. This protocol is suitable for performing large scale screens in C. elegans and can be used to screen either one of the commercially available dsRNA libraries. Using this protocol, we find that essentially 100% of the bacteria fed to animals during the screen retain the dsRNA-encoding plasmid and cause robust and highly penetrant loss of function phenotypes in all tissues examined.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>96-well Falcon flat bottom plate </td>
 <td>Fisher Scientific</td>
 <td>353072</td>
 <td>sterile</td>
 </tr>
 <tr>
 <td>adhesive foil</td>
 <td>USA Scientific</td>
 <td>2923-0100</td>
 <td></td>
 </tr>
 <tr>
 <td>Nunc omniplate</td>
 <td>Fisher Scientific</td>
 <td>267060</td>
 <td></td>
 </tr>
 <tr>
 <td>2.0 ml deep 96-well polypropylene Masterblock</td>
 <td>USA Scientific</td>
 <td>5678-0285</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>Lids for deep well plates</td>
 <td>USA Scientific</td>
 <td>5665-6101</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml combitips</td>
 <td>USA Scientific</td>
 <td>4796-5000</td>
 <td>individually wrapped</td>
 </tr>
 <tr>
 <td>Reservoir</td>
 <td>USA Scientific</td>
 <td>2977-8500</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>12-well plates</td>
 <td>USA Scientific</td>
 <td>CC7682-7512</td>
 <td>individually wrapped</td>
 </tr>
 <tr>
 <td>dsRNA feeding library</td>
 <td>OpenBiosystems</td>
 <td>RCE1181</td>
 <td>store -80 &deg;C</td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Fisher Scientific</td>
 <td>BP1760-5</td>
 <td></td>
 </tr>
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 <td>Tetracycline</td>
 <td>Fisher Scientific</td>
 <td>BP912-100</td>
 <td></td>
 </tr>
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 <td>Carbenicillin</td>
 <td></td>
 <td></td>
 <td>allow 2-4 weeks for delivery <a href="http://www.biochemicaldirect.com" target="_blank">www.biochemicaldirect.com</a></td>
 </tr>
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 <td>Bacteriolgical Agar Ultrapure grade A </td>
 <td>Affymetrix</td>
 <td>10906</td>
 <td>for worm plates</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Brayer roller</td>
 <td>USA Scientific</td>
 <td>9127-2940</td>
 <td></td>
 </tr>
 <tr>
 <td>Boekel 96-pin replicator</td>
 <td>Fisher Scientific</td>
 <td>05-450-9</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>microshaker</td>
 <td>Thomas Scientific</td>
 <td>1231A93</td>
 <td>3 mm orbit</td>
 </tr>
 <tr>
 <td>12 channel multichannel pipet (0.5-10 &mu;l)</td>
 <td>USA Scientific</td>
 <td>7112-0510</td>
 <td></td>
 </tr>
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 <td>12 channel multichannel pipet (30-300 &mu;l)</td>
 <td>USA Scientific</td>
 <td>7112-3300</td>
 <td></td>
 </tr>
 <tr>
 <td>Repeater Plus manual pipettor</td>
 <td>USA Scientific</td>
 <td>4026-0201</td>
 <td></td>
 </tr>
 </tbody>
 </table>

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.

P0 knockdown phenotypes will begin to appear after 2-3 days of feeding animals dsRNA expressing bacteria. Some early phenotypes include larval arrest and lethality and should be observed in 2-3% of all feeding wells. These same phenotypes will also be observed in the F1 generation animals. The presence of F1 eggs that fail to hatch is another common F1 phenotype, and together, these three phenotypes will be observed in nearly 10% of all wells in F1 screens.
We used the protocol described here ...

Watch this Scientific Journal Video about Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes at JoVE.com

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.

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 ...

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