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 metho...

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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

16.9K Görüntüleme.  Texas A&M University System Health Science Center. Biz protein ekspresyonu ve lokalizasyonu postembryonic düzenleyiciler tanımlamak için duyarlı bir yöntem tarif C. elegans Bir RNAi tabanlı ekran genomik ve işlevsel, floresan etiketli protein ifade entegre bir transgen kullanarak.