Name: rnai screening to identify postembryonic phenotypes in c. elegans
Uploaded: 2012-02-13T13:00:00
Description: Watch this Scientific Journal Video about RNAi Screening to Identify Postembryonic Phenotypes in C. elegans at JoVE.com

Forfatterne ønsker å takke Dr. Rick Padgett (Waksman Institute, Rutgers University, NJ) for gave dbl-1 cDNA og Dr. Christopher rongo (Waksman Institute, Rutgers University, NJ) for en injeksjon markør. Dr. Barth Grants lab utført genet pistolen bombardementet for lavt kopi nummer integrering av GFP-merket dbl-1 konstruksjon. Den René Garcia laboratoriet gitt teknisk assistanse under opprettelsen av texIs100. Den René Garcia, Robyn Lints, og Hongmin Qin laboratorier gitt produktiv råd. Dette arbeidet ble finansiert av oppstart midler fra TAMHSC Institutt for molekylær-og Cellular Medicine. Den sammensatte omfang og spinning disk confocal ble kjøpt med midler gitt av instituttet og TAMHSC College of Medicine kontor Dean.

1. Screening belastning konstruksjon Den forsiktige utformingen av screening belastning er kritisk for suksess på skjermen og har blitt beskrevet tidligere tre. For noen forskere, bruker en påkjenning som uttrykker en synlig produkt fra en transgenet nødvendig for forsøket. Mange stammer huser integrerte transgener er tilgjengelige fra CGC eller individuelle forskere. Hvis en transgen belastningen er nødvendig for skjermen, men er ikke tilgjengelig, så det kan genereres ved hj...

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Den RNAi screening metoden presentert her gir en sensitiv og rask analyse av genprodukter som kreves for en normal (eller transgen) postembryonic fenotype. Eksempelet som vises er en skjerm for gener som er involvert i subcellulære lokalisering av en fluorescently tagget protein. Men dette kan protokollen endres for å identifisere gener som påvirker andre postembryonic fenotyper av interesse. Denne metoden utnytter en kandidat genet tilnærming ved hjelp av en RNAi bibliotek. Videresend g...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> NGM Agar </td><td> Nematode vekstmedium </td><td> IPM Scientific, Inc </td><td> Kan være forberedt etter NGM agar protokoll 25 </td></tr><tr><td> M9 Medium </td><td> 22mm KH 2 PO 4, 42mm Na 2 HPO 4, 86mm NaCl, 1 mm MgSO 4 </td><td></td><td> 26 </td></tr><tr><td> Agar-agar </td><td> EMD Chemicals Inc. </td><td> 1.01614.1000 </td><td> 2% i vann for NGM plater. 4% i vann for mikroskop glideplater (autoklaver først og mikrobølgeovn til å smelte deretter). </td></tr><tr><td> Bacto Peptone </td><td> Becton Dickinson - Difco CP </td><td> 211677 </td><td> 0,25% </td&gt; </tr><tr><td> IPTG </td><td> Forskning Products International Corp </td><td> I56000-5.0 </td><td> 1 mm endelig konsentrasjon </td></tr><tr><td> carbenicillin </td><td> Forskning Products International Corp </td><td> C46000-5.0 </td><td> 50 mikrogram / ml arbeider fortynning </td></tr><tr><td> LB Broth Lennox </td><td> Becton Dickinson - Difco CP </td><td> 240230 </td><td> 20 g / liter </td></tr><tr><td> tetracyclin </td><td> Sigma </td><td> 268054 </td><td> 12,5 mikrogram / ml arbeider fortynning </td></tr><tr><td> natriumhypokloritt </td><td> Enhver merke </td><td> 5% klorin </td><td> Bruk fersk blekemiddel. </td></tr><tr><td> natriumhydroksid </td><td> Enhver Brand </td><td> CAS 1310-73-2 </td><td> 5 N lager </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://1</a>30.15.90.245/wormlab_recipe_book.htm # Commonlab </td><td> 26 </td></tr><tr><td> levamisol </td><td> Sigma </td><td> 31742 </td><td> 100 mM - 1 mm jobbe fortynning </td></tr><tr><td> natriumazid </td><td> Fisher Scientific </td><td> S227 </td><td> 10 mm i M9 arbeidsmiljø utvanning </td></tr><tr><td> 24-brønn plate </td><td> Greiner Bio-One </td><td> 662160 </td><td> VWR distributør </td></tr><tr><td> objektglass </td><td> Enhver merke </td><td> 75 x 25 x 1 mm </td><td></td></tr><tr><td> mikroskop cover slips </td><td> Enhver merke </td><td> 22 x 22 mm No.1.5 </td><td> Bruk tykkelse anbefales av mikroskop produsenten. </td></tr><tr><td> sammensatte mikroskop </td><td> Carl Zeiss, Inc. </td><td> A1M </td><td> Bruk mål og filtre for å dekke behovene til forsøket. </td></tr><tr><td> media pumpe</td><td> Manostat Varistaltic pumpe </td><td> Kate Modellen # 72-620-000 </td><td> Bruk slangen og innstillinger som passer for maskinen </td></tr></tbody></table>

rnai screening to identify postembryonic phenotypes in c. elegans

C. elegans har vist seg å være en verdifull modell system for oppdagelsen og funksjonell karakterisering av mange gener og gen stier 1. Mer sofistikerte verktøy og ressurser for studier i dette systemet legger til rette for fortsatt funn av gener med mer subtile fenotyper eller roller. Her presenterer vi en generalisert protokoll vi tilrettelagt for identifisering C. elegans gener med postembryonic fenotyper av interesse å bruke RNAi to. Denne prosedyren er enkelt endres til analysen fenotypen av valget, enten ved lys eller fluorescens optikk på en dissekere eller sammensatt mikroskop. Dette screening protokollen kapitaliserer på de fysiske eiendeler i organismen og molekylære verktøy C. elegans forskningsmiljøet har produsert. Som et eksempel, demonstrerer vi bruk av en integrert transgenet som uttrykker en fluorescerende produkt i en RNAi skjermen for å identifisere gener som kreves for normal lokalisering av denneprodukt i sent stadium larver og voksne. Først brukte vi en kommersielt tilgjengelig genomisk RNAi bibliotek med full lengde cDNA innsatser. Dette biblioteket muliggjør rask identifisering av flere kandidater ved RNAi reduksjon av kandidaten genproduktet. Second, genererte vi en integrert transgenet som uttrykker vår fluorecently tagget protein av interesse i en RNAi-sensitive bakgrunn. Tredje, ved å utsette klekket dyr til RNAi, tillater dette skjermbildet identifisering av genprodukter som har en viktig embryonale rolle som ellers ville maskere en post-embryonisk rolle i regulering av protein av interesse. Til slutt bruker denne skjermen et sammensatt mikroskop utstyrt for enkelt celle oppløsning.

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

Vi beskriver en sensitivisert metode for å identifisere postembryonic regulatorer av protein uttrykk og lokalisering i<em&gt; C. elegans</em&gt; Med en RNAi-baserte genomisk skjerm og en integrert transgenet som uttrykker en funksjonell, fluorescently tagget protein.

RNAi Screening for å identifisere Postembryonic fenotyper i 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 ...

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Biology

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

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