Name: large-scale gene knockdown in c. elegans using dsrna feeding libraries to generate robust loss-of-function phenotypes
Uploaded: 2013-09-25T19:00:00
Description: Watch this Scientific Journal Video about Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes at JoVE.com

Dette arbejde blev støttet af midler fra National Institutes of Health MH097163. Nogle stammer blev leveret af CGC, som er finansieret af NIH Office of Research Infrastructure Programmer (P40-OD010440).

Bemærk: Denne protokol kan anvendes til at identificere gener, der er nødvendige for en række biologiske processer i en række forskellige vævstyper. Som et nødvendigt kvalitetskontrol under skærmen, dyrene fodres både positiv og negativ kontrol dsRNA udtrykker bakterier at demonstrere RNAi effekter i målrettede væv. Andre offentliggjorte fodring protokoller vokse dsRNA-udtrykkende bakterier i nærvær af enten 50 ug / ml ampicillin eller 25 pg / ml carbenicillin 8-10....

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Vi har beskrevet en protokol, der anvender en kommercielt tilgængelig dsRNA fodring bibliotek til at udføre store skærme i C. elegans. Vi leverer alle instruktioner til at duplikere biblioteket og at bruge det effektivt. Vi viser, at denne fremgangsmåde er i stand til at identificere gener, der er involveret i forskellige biologiske processer i forskellige væv i dyret. Mens fænotyper vi beskriver her, er let observerbare (Unc EGL, og Dpy), kan denne protokol tilpasses for at søge efter gener, der er nød...

Historisk, genetiske skærme i C. elegans er blevet udført ved behandling af dyr med et kemisk mutagen, såsom ethylmethansulfonat at skabe tilfældige mutationer i DNA. Afkom eller grandprogeny af mutageniserede dyr isoleres derefter som udviser en ønsket unormal fænotype. Mutationen ansvarlig for at forårsage fænotypen identificeres derefter gennem en arbejdskrævende kortlægning procedure eller ved sekventering mutant ormens hele genomet. Der er mange fordele ved at udføre sådanne kemisk mutagenese skærme da de skaber permanente læsioner inden ormen genomet, der kan resultere i både gain-of-funktion og tab af funktion fænotyper, men kortlægning procedure er langsomt og dyrt. Dobbelt-strenget RNA-interferens (dsRNAi) giver en alternativ måde at identificere gener involveret i vigtige biologiske processer. I denne fremgangsmåde dsRNA matcher den kodende sekvens af et endogent gen indført i ormen.DsRNA&#39;et behandles in vivo til at generere små (21-22 nukleotider) RNA, der tjener som guider til at finde og binde de matchende endogene mRNA&#39;eme, rettet dem til destruktion 1. Denne procedure er forholdsvis hurtig, men fordi dsRNA målrettet mRNA snarere end DNA, der kun reduktion-af-funktion fænotyper observeret ved anvendelse af denne fremgangsmåde. Indførelse af dsRNAs i et dyr kan opnås ved direkte injektion af dsRNA 2 ved neddypning dyr i dsRNA 3 eller ved at fodre dyr bakterier, der udtrykker dsRNA 4. Hver af disse metoder til levering forårsage systemiske knockdown virkninger som de fleste celler af dyret udtrykker SID-1, et transmembrant kanal stand til at importere dsRNA 5. Oprindeligt brugt som en omvendt genetik tilgang til Knockdown udtryk for enkelte gener én ad gangen, udvikling af to fodring biblioteker tillader nu genetiske skærme store. De kommercielt tilgængelige dsRNA biblioteker indeholder bacterial kloner repræsenterer enten 55% eller 87% af al C. elegans gener 6, 7. Desværre storstilede dsRNAi fodring skærme ofte resultere i variable knockdown effektivitetsgevinster 8-10. Mens det absolutte niveau af knockdown af RNAi ikke let måles, skal den reducerede knockdown effektivitet observeret i store skærme være forårsaget af tilbageværende gen-specifikke mRNA&#39;er, der undslipper nedbrydning af RNAi. Dette, til gengæld kan være et direkte resultat af dsRNA plasmid tab fra bakterier fodret til dyrene i disse skærme. Støtte denne idé, fodres dyrene blandinger af bakterier, hvor kun 50% af bakterierne udtrykke dsRNAs mod en målrettet gen, udviser dramatisk reduceret (hvis nogen) knockdown virkninger sammenlignet med kontrolpatienter fodrede dyr 11. Omfanget af gen knockdown bliver en kritisk bekymring, når du udfører dsRNAi skærme som de fleste gener i C. elegans er haplosufficient og dermed genekspression skal reduceres med mindst 50% to forårsage tab af funktion Fænotypers 12. Vi har brugt tidligere offentliggjorte fodring protokoller til at udføre store skærme og fandt den samme variabel knockdown effekter rapporteret af andre 8-10. I en søgning efter mulige årsager, fandt vi, at næsten 60% af de bakterier, der gives til dyrene i skærmen havde mistet dsRNA plasmid. Vi har udviklet et bibliotek dobbeltarbejde og screening protokol, der drastisk reducerer eller eliminerer plasmid tab og i høj grad forbedrer knockdown effektivitet. Denne protokol er egnet til at udføre store skærme i C. elegans og kan anvendes til at screene for en af de kommercielt tilgængelige dsRNA biblioteker. Ved hjælp af denne protokol, finder vi, at væsentlige 100% af de bakterier, der gives til dyr under skærmen bevarer dsRNA&#39;et-kodning plasmid og forårsage robust og stærkt penetrant tab af funktion fænotyper i alle undersøgte væv.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>96-well Falcon flat bottom plate </td>
 <td>Fisher Scientific</td>
 <td>353072</td>
 <td>sterile</td>
 </tr>
 <tr>
 <td>adhesive foil</td>
 <td>USA Scientific</td>
 <td>2923-0100</td>
 <td></td>
 </tr>
 <tr>
 <td>Nunc omniplate</td>
 <td>Fisher Scientific</td>
 <td>267060</td>
 <td></td>
 </tr>
 <tr>
 <td>2.0 ml deep 96-well polypropylene Masterblock</td>
 <td>USA Scientific</td>
 <td>5678-0285</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>Lids for deep well plates</td>
 <td>USA Scientific</td>
 <td>5665-6101</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml combitips</td>
 <td>USA Scientific</td>
 <td>4796-5000</td>
 <td>individually wrapped</td>
 </tr>
 <tr>
 <td>Reservoir</td>
 <td>USA Scientific</td>
 <td>2977-8500</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>12-well plates</td>
 <td>USA Scientific</td>
 <td>CC7682-7512</td>
 <td>individually wrapped</td>
 </tr>
 <tr>
 <td>dsRNA feeding library</td>
 <td>OpenBiosystems</td>
 <td>RCE1181</td>
 <td>store -80 &deg;C</td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Fisher Scientific</td>
 <td>BP1760-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Tetracycline</td>
 <td>Fisher Scientific</td>
 <td>BP912-100</td>
 <td></td>
 </tr>
 <tr>
 <td>Carbenicillin</td>
 <td></td>
 <td></td>
 <td>allow 2-4 weeks for delivery <a href="http://www.biochemicaldirect.com" target="_blank">www.biochemicaldirect.com</a></td>
 </tr>
 <tr>
 <td>Bacteriolgical Agar Ultrapure grade A </td>
 <td>Affymetrix</td>
 <td>10906</td>
 <td>for worm plates</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Brayer roller</td>
 <td>USA Scientific</td>
 <td>9127-2940</td>
 <td></td>
 </tr>
 <tr>
 <td>Boekel 96-pin replicator</td>
 <td>Fisher Scientific</td>
 <td>05-450-9</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>microshaker</td>
 <td>Thomas Scientific</td>
 <td>1231A93</td>
 <td>3 mm orbit</td>
 </tr>
 <tr>
 <td>12 channel multichannel pipet (0.5-10 &mu;l)</td>
 <td>USA Scientific</td>
 <td>7112-0510</td>
 <td></td>
 </tr>
 <tr>
 <td>12 channel multichannel pipet (30-300 &mu;l)</td>
 <td>USA Scientific</td>
 <td>7112-3300</td>
 <td></td>
 </tr>
 <tr>
 <td>Repeater Plus manual pipettor</td>
 <td>USA Scientific</td>
 <td>4026-0201</td>
 <td></td>
 </tr>
 </tbody>
 </table>

large-scale gene knockdown in c. elegans using dsrna feeding libraries to generate robust loss-of-function phenotypes

RNA-interferens ved at fodre orme bakterier udtrykker dsRNAs har været et nyttigt redskab til at vurdere gen funktion i C. elegans. Mens denne strategi fungerer godt, når et lille antal gener er målrettet til knockdown, storstilet fodring skærme viser variable knockdown effektivitetsgevinster, hvilket begrænser deres anvendelighed. Vi har deconstructed tidligere offentliggjorte RNAi knockdown protokoller og fandt, at den primære kilde til den reducerede knockdown kan tilskrives tab af dsRNA-kodende plasmider fra bakterier fodret til dyrene. Baseret på disse observationer, har vi udviklet et dsRNA&#39;et fodring protokol, der i høj grad reducerer eller eliminerer plasmid tab at opnå en effektiv, high throughput knockdown. Vi viser, at denne protokol vil producere robuste, reproducerbar knock down C. elegans gener i flere vævstyper, herunder neuroner, og vil muliggøre en effektiv knockdown i store skærme. Denne protokol bruger en kommercielt tilgængelig dsRNA fodringbibliotek og beskriver alle de nødvendige skridt for at duplikere biblioteket og udføre dsRNA skærme. Protokollen kræver ikke brug af enhver avanceret udstyr, og derfor kan udføres af enhver C. elegans lab.

P0 knockdown fænotyper vil begynde at dukke op efter 2-3 dage med at fodre dyr dsRNA udtrykke bakterier. Nogle tidlige fænotyper omfatter larvernes anholdelse og dødelighed og bør overholdes i 2-3% af alle fodringsforsøg brønde. Disse samme fænotyper vil også blive observeret i F1-generationen dyr. Tilstedeværelsen af ​​F1 æg, der undlader at klække er en anden almindelig F1 fænotype, og sammen vil disse tre fænotyper observeres i næsten 10% af alle brønde i F1 skærme. Vi...

Watch this Scientific Journal Video about Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes at JoVE.com

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

Mens dsRNA fodring i C. elegans er et kraftfuldt værktøj til at vurdere gen funktion, nuværende protokoller for storstilede fodring skærme resulterer i variable knockdown effektivitetsgevinster. Vi beskriver en forbedret protokol til at udføre storstilet RNAi fodring skærme, der resulterer i meget effektive og reproducerbar knockdown af genekspression.

Storstilet Gene Knockdown i<em&gt; C. elegans</em&gt; Brug dsRNA Fodring Biblioteker at skabe en robust tab af funktion fænotyper

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

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Biology

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

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Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Brug af RNA-medieret Interferens fodringsstrategien at screene for gener involveret i Body Size forordning i Nematode<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

C. elegans Chemotaxis Assay

C. elegans Chemotaxis Assay

C. elegans Kemotaksi Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

Antibody Staining in C. Elegans Using "Freeze-Cracking"

Antistoffarvning i<em&gt; C. Elegans</em&gt; Brug af &quot;Freeze-Cracking&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Metoder til at vurdere subcellulære afsnit af muskler i<em&gt; C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...