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.

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Wir haben nichts zu offenbaren.

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...

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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

Wir beschreiben eine Methode, um sensibilisiert postembryonalen Regulatoren der Protein-Expression und Lokalisation in identifizieren<em&gt; C elegans</em&gt; Mit Hilfe eines RNAi-basierter Genom-Bildschirm und einen integrierten Transgen, die eine funktionelle, fluoreszenzmarkierten Protein exprimiert.

RNAi-Screening auf postembryonalen Phänotypen in Identifizieren C elegans</em

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

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

16.9K 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.