RNAi-Screening auf postembryonalen Phänotypen in Identifizieren C elegans</em
Summary
Wir beschreiben eine Methode, um sensibilisiert postembryonalen Regulatoren der Protein-Expression und Lokalisation in identifizieren<em> C elegans</em> Mit Hilfe eines RNAi-basierter Genom-Bildschirm und einen integrierten Transgen, die eine funktionelle, fluoreszenzmarkierten Protein exprimiert.
Abstract
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.
Protocol
Discussion
Die RNAi-Screening hier vorgestellte Methode ermöglicht eine schnelle und sensitive Analyse von Gen-Produkten für einen normalen (oder transgene) postembryonalen Phänotyp erforderlich. Das gezeigte Beispiel ist ein Bildschirm für die Gene in der subzellulären Lokalisation eines fluoreszenzmarkierten Proteins beteiligt. Allerdings ist dieses Protokoll modifiziert werden, um Gene, die anderen postembryonalen Phänotypen von Interesse zu identifizieren.
Diese Methode nutzt eine Kandidateng…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Die Autoren danken Herrn Dr. Rick Padgett (Waksman Institut, Rutgers University, NJ) für das Geschenk des DBL-1 cDNA und Dr. Christopher Rongo (Waksman Institut, Rutgers University, NJ) für eine Injektion Marker danken. Dr. Barth Grants Labor durchgeführt, die das Gen für die Bombardierung Pistole mit geringer Kopienzahl Integration des GFP-markierten DBL-1-Konstrukt. Die René Garcia Labor leistete technische Hilfe bei der Erstellung der texIs100. Die René Garcia, Robyn Lints und Hongmin Qin Laboratorien zur Verfügung gestellt produktive Beratung. Diese Arbeit wurde vom Start-up-Mitteln aus dem TAMHSC Department of Molecular and Cellular Medicine finanziert. Die Verbindung Umfang und Spinning-Disk-konfokale wurden mit Mitteln der Abteilung und der TAMHSC College of Medicine Dekanat zur Verfügung gestellt gekauft.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| NGM Agar | Nematode growth medium | IPM Scientific, Inc | Can be prepared following NGM agar protocol25 |
| M9 Medium | 22mM KH2PO4, 42mM Na2HPO4, 86mM NaCl, 1 mM MgSO4 |
26 | |
| Agar-Agar | EMD Chemicals Inc. | 1.01614.1000 | 2% in water for NGM plates. 4% in water for microscope slide pads (autoclave initially and microwave to melt thereafter). |
| Bacto Peptone | Becton Dickinson – Difco CP | 211677 | 0.25% |
| IPTG | Research Products International Corp. | I56000-5.0 | 1 mM final concentration |
| carbenicillin | Research Products International Corp. | C46000-5.0 | 50 μg/ml working dilution |
| LB Broth Lennox | Becton Dickinson – Difco CP | 240230 | 20 g/liter |
| tetracycline | Sigma | 268054 | 12.5 μg/ml working dilution |
| sodium hypochlorite | Any brand | 5% household bleach | Use fresh bleach. |
| sodium hydroxide | Any Brand | CAS 1310-73-2 | 5 N stock |
| M9 medium | Wormlab Recipe Book | http://130.15.90.245/wormlab_recipe_book.htm#Commonlab | 26 |
| levamisol | Sigma | 31742 | 100 μM – 1 mM working dilution |
| sodium azide | Fisher Scientific | S227 | 10 mM in M9 working dilution |
| 24-well plate | Greiner Bio-One | 662160 | VWR distributor |
| microscope slides | Any brand | 75 x 25 x 1 mm | |
| microscope cover slips | Any brand | 22 x 22 mm No.1.5 | Use the thickness recommended by the microscope manufacturer. |
| compound microscope | Carl Zeiss, Inc. | A1m | Use objectives and filters to match the needs of the experiment. |
| media pump | Manostat Varistaltic pump | Kate model #72-620-000 |
Use tubing and settings appropriate for the machine |
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