Name: using rna-mediated interference feeding strategy to screen for genes involved in body size regulation in the nematode c. elegans
Uploaded: 2013-02-13T12:00:00
Description: Watch this Scientific Journal Video about Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans at JoVE.com

Vi takker James Clark til tilvejebringelse af tal for dyr på forskellige udviklingsmæssige tidspunkter. C. elegans stammer i denne undersøgelse blev indhentet fra Caenorhabditis Genetics Center, som er støttet af NIH National Center for Forskning Ressourcer (NCRR). Dette arbejde blev støttet af CIRG 1817 fra CUNY til JL og CSD, og ​​ved NIH 1R15GM073678-01 og 1R15GM097692-01 til CSD Vi takker Dr. William J. Rice, Dr. Nathalia Holtzman, og Melissa Silvestrini om kommentarer til manuskriptet. Dette arbejde blev udført i delvis opfyldelse af kravene til en ph.d.-grad fra Graduate Center of the City University of New York (S. Xiong).

1. Forberedelse RNAi Fodring Plates Carrying målgensekvens <ol><li> Hvis du bruger kommercielt tilgængelige RNAi biblioteker (fx fra Source BioScience LifeSciences), skal du fortsætte til trin 1,4. Alternativt klone målgensekvenser i vektor L4440, et almindeligt anvendt orm RNAi plasmid 8, ved standardklonings-protokol. </li><li> Omdanne det rekombinante plasmid i bakteriestamme HT115 (DE3), kultur dem på LB-agarplader med 25 ug / ml carbenicillin og 12,5 ug / ml tetracyclin, og vælge en enke...

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I denne protokol, beskriver vi vores metode til identifikation af kropsstørrelse mutanter af C. elegans by RNAi fodring. Denne metode anvendes til identifikation af TGF-β signal komponenter. Da sådanne komponenter er stærkt konserverede ved evolution 15, sådanne skærme er relevante for at belyse de molekylære mekanismer af TGF-β-signalering i alle metazoans. En vigtig overvejelse i udformningen af ​​en sådan skærm er den udgangsstamme. Vi har vist, at både ERI-1, lin-15b og <e...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> RNAi orm plader 17,7 g Worm Medium Mix 60 g Agar Tilføj H2O til 3 liter. Autoklav. Tilsættes 3 ml 1 M IPTG (sterilt), og 3 ml 25 mg / ml carbenicillin (steril), og derefter hældes i 60 mm petriskåle. Worm plader 17,7 g Worm Medium Mix 0,6 g streptomycinsulfat 60 g Agar Tilføj H2O til 3 liter. Autoklav. Hæld i 60 mm petriskåle. Worm Medium Mix 55 g Tris-CI 24 g Tris-OH 310 g Bacto-pepton 800 mg Cholesterol 200 g NaCl Bland grundigt og vær sikker på at undgå klumper, hvilket pulveriseret blanding kan opbevares i mange måneder før at bruge. 2% agarose 0,5 g Agarose Add 25 ml dH 2 O. Varme i mikrobølgeovn indtil det er opløst. 1M Isopropyl β-D-1-thiogalactopyranosid (IPTG) 2 g IPTG Opløs in10 ml dH 2 O 1 M NaN3 6,5 g NaN3 Tilføj dH 2 O til 100 ml 25 mM NaN3 2,5 ml 1 M NaN3 Tilføj dH 2 O til 100 ml Caenorhabditis elegans fra Caenorhabditis Genetics center L4440 plasmid (bærer ampicillinresistensgen) Bakterier stamme HT115 (DE3) (indeholder IPTG inducerbar T7-polymerase, deficient for RNAse III-gen (a dsRNAse), som også bærer tetracyclinresistensgenet) 16 60 mm petriskåle 100 mm petriskåle 14 ml rundbundet Falcon kultur rør objektglas dækglas 1-20 gl pipette 20-200 ul pipette 200-1000 pipipettor 1-200 gl pipettespidser 200-1000 ul pipettespidser 1,5 ml Eppendorf-rør platintråd orm pick </td></tr></tbody></table>

using rna-mediated interference feeding strategy to screen for genes involved in body size regulation in the nematode c. elegans

Dobbeltstrenget RNA-medieret interferens (RNAi) er en effektiv strategi til at vælte målgenekspression 1-3. Det er blevet anvendt til mange modelsystemer, herunder planter, invertebrater og vertebrater. Der er forskellige metoder til at opnå RNAi in vivo 4,5. For eksempel kan målgenet omdannes til en RNAi vektor, og derefter enten permanent eller transient omdannes til cellelinier og primære celler til opnåelse gen knockdown virkninger, alternativt syntetiseres dobbelt-oligonukleotider fra specifikke målgener (RNAi oligoer) kan være transient omdannet til cellelinjer eller primære celler til tavshed målrette gener, eller syntetiseres dobbelt-strengede RNA-molekyler kan mikroinjiceres i en organisme. Da nematoden C. elegans bruger bakterier som en fødekilde, fodring af dyrene med bakterier udtrykker dobbelt-strenget RNA mod målgener giver en levedygtig strategi 6. Her præsenterer vi en RNAi fodringFremgangsmåde til at score kropsstørrelse fænotype. Kropsstørrelse i C. elegans reguleres primært af det TGF-β - ligesom ligand DBL-1, så dette assay er egnet til identifikation af TGF-β signalering komponenter 7. Vi brugte forskellige stammer herunder to RNAi hypersensitive stammer at gentage de RNAi fodring eksperimenter. Vores resultater viste, at RRF-3-stammen gav os den bedste forventede RNAi fænotype. Fremgangsmåden er let at udføre, reproducerbar og let kvantificeres. Desuden er vores protokol minimerer brugen af ​​specialiseret udstyr, så det er velegnet til mindre laboratorier eller personer, overvejende bachelor institutioner.

I vores forskning har vi fokus på kropsstørrelse regulering af den DBL-1/TGF-β vej. Tabet af funktion af de DBL-1 metaboliseringsvej resulterer i lille kropsstørrelse, herunder en kortere kropslængde sammenlignet med vildtype-dyr 7,10,11. Således skærme til C. elegans kropsstørrelse mutanter er i stand til at identificere TGF-β signal komponenter og modifikatorer. DBL-1 signalering medieret af en konserveret TGF-β signaltransduktionsvej, der omfatter celleoverfladereceptorer og intracellul?...

Watch this Scientific Journal Video about Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans at JoVE.com

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

Vi viser, hvordan du bruger RNAi fodring teknik til at vælte målgener og score kropsstørrelse fænotype i<em&gt; C. elegans</em&gt;. Denne metode kunne anvendes til stor skala skærmen for at identificere potentielle genetiske komponenter af interesse, såsom de involveret i kropsstørrelse regulering ved DBL-1/TGF-β signalering.

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 ...

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

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