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 vil gerne takke Dr. Rick Padgett (Waksman Institute, Rutgers University, NJ) for gaven af DBL-1 cDNA og Dr. Christopher Rongo (Waksman Institute, Rutgers University, NJ) for en injektion markør. Dr. Barth Grant laboratorium udførte genkanonen bombardement for lavt kopital integration af GFP-mærkede DBL-1-konstruktionen. Den René Garcia Laboratoriet ydet faglig bistand under oprettelsen af texIs100. Den René Garcia, Robyn Lints, og Hongmin Qin laboratorier forudsat produktiv rådgivning. Dette arbejde blev finansieret af start-up midler fra TAMHSC Institut for Molekylær og cellemedicin. Den forbindelse omfang og spinning disk konfokal blev købt med midler fra afdelingen og TAMHSC College of Medicine kontor dekanen.

1. Screening stammen byggeri Den omhyggelig udformning af screeningen stammen er kritisk for succesen af skærmen, og er blevet beskrevet andetsteds 3. For nogle forskere ved anvendelse af en stamme, der udtrykker et synligt produkt fra en transgen nødvendig for eksperimentet. Mange stammer, der indeholdt integrerede transgener er tilgængelige fra CGC eller individuelle forskere. Hvis en transgen stamme er nødvendig for skærmen, men ikke er tilgængelig, kan det dannes ved hjæl...

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RNAi screeningsmetode præsenteret her muliggør en følsom og hurtig analyse af genprodukter, der kræves for en normal (eller transgene) postembryonic fænotype. Det viste eksempel er en screening for gener involveret i den subcellulære lokalisering af en fluorescens-mærket protein. Imidlertid kan denne protokol kan modificeres til at identificere gener, der påvirker andre postembryonic fænotyper af interesse. Denne fremgangsmåde drager fordel af en kandidat-gen fremgangsmåde ved anv...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> NGM Agar </td><td> Nematode vækstmedium </td><td> IPM Videnskabelige, Inc </td><td> Kan blive udarbejdet NGM agar-protokollen 25 </td></tr><tr><td> M9-medium </td><td> 22mm KH 2 PO 4, 42mm Na 2 HPO 4, 86mm NaCl, 1 mM MgSO4 </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 vand til NGM plader. 4% i vand til mikroskop slidklodser (autoklaveres i første omgang og mikrobølgeovn til at smelte derefter). </td></tr><tr><td> Bacto-pepton </td><td> Becton Dickinson - Difco CP </td><td> 211677 </td><td> 0,25% </td&gt; </tr><tr><td> IPTG </td><td> Research Products International Corp </td><td> I56000-5.0 </td><td> 1 mM slutkoncentration </td></tr><tr><td> carbenicillin </td><td> Research Products International Corp </td><td> C46000-5.0 </td><td> 50 ug / ml fortynding </td></tr><tr><td> LB-medium 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 ug / ml fortynding </td></tr><tr><td> natriumhypochlorit </td><td> Enhver brand </td><td> 5% husholdningsblegemiddel </td><td> Brug frisk blegemiddel. </td></tr><tr><td> natriumhydroxid </td><td> Enhver Brand </td><td> CAS 1310-73-2 </td><td> 5 N lager </td></tr><tr><td> M9-medium </td><td> Wormlab Opskriftsbog </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 pM - 1 mm fortynding </td></tr><tr><td> natriumazid </td><td> Fisher Scientific </td><td> S227 </td><td> 10 mM i M9 fortynding </td></tr><tr><td> 24-brønds plade </td><td> Greiner Bio-One </td><td> 662160 </td><td> VWR distributør </td></tr><tr><td> mikroskopobjektglas </td><td> Enhver brand </td><td> 75 x 25 x 1 mm </td><td></td></tr><tr><td> mikroskop dækglas </td><td> Enhver brand </td><td> 22 x 22 mm No.1.5 </td><td> Anvende tykkelsen anbefalet af mikroskopet fabrikanten. </td></tr><tr><td> Forbindelsen mikroskop </td><td> Carl Zeiss, Inc. </td><td> A1m </td><td> Anvende mål og filtre til de behov for eksperimentet. </td></tr><tr><td> medier pumpe</td><td> Manostat Varistaltic pumpe </td><td> Kate model # 72-620-000 </td><td> Brug slanger og indstillinger, der passer til maskinen </td></tr></tbody></table>

rnai screening to identify postembryonic phenotypes in c. elegans

C. elegans har vist sig at være en værdifuld modelsystem til opdagelse og funktionelle karakterisering af mange gener og genprodukter veje 1. Mere avancerede værktøjer og ressourcer til studier i dette system er lettere fortsat opdagelsen af ​​gener med mere subtile fænotyper eller roller. Her præsenterer vi en generel protokol, vi er tilpasset til at identificere C. elegans gener med postembryonic fænotyper af interesse ved hjælp af RNAi 2. Denne fremgangsmåde kan let modificeres til bestemmelse af fænotype valg, hvad enten de lette eller fluorescens optik på et dissektionsmikroskop eller forbindelse mikroskop. Denne screening protokol udnytter de fysiske aktiver i organismen og molekylære værktøjer til C. elegans forskning fællesskab har frembragt. Som et eksempel viser vi anvendelse af en integreret transgen, som udtrykker et fluorescerende produkt i en RNAi skærmen for at identificere gener, der kræves for normal lokalisering af detteprodukt i sent larver og voksne. Først brugte vi en kommercielt tilgængelig genomisk RNAi bibliotek med fuld-længde cDNA skær. Dette bibliotek letter den hurtige identifikation af flere kandidater ved RNAi reduktion af kandidat-genproduktet. For det andet har vi skabt en integreret transgen som udtrykker vores fluorecently taggede protein af interesse i et RNAi-følsom baggrund. Det tredje ved at udsætte skraverede dyr RNAi, tillader dette skærmbillede identifikation af genprodukter, der har en afgørende embryonisk rolle, som ellers ville maskere en post-embryonale rolle i reguleringen af ​​proteinet af interesse. Endelig Denne screening anvender en forbindelse mikroskop udstyret til enkelt celle opløsning.

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

Beskriver vi en sensibiliseret metode til at identificere postembryonic regulatorer af proteinekspression og lokalisering i<em&gt; C. elegans</em&gt; Ved hjælp af en RNAi-baseret genomisk skærm og en integreret transgen som udtrykker et funktionelt, fluorescens mærket protein.

RNAi screening for at identificere Postembryonic fænotyper 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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