Name: oct4gip reporter assay to study genes that regulate mouse embryonic stem cell maintenance and self-renewal
Uploaded: 2012-05-30T13:00:00
Description: Watch this Scientific Journal Video about Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal at JoVE.com

Vi takker Brad Lackford til læsning og redigering af manuskriptet. Denne forskning blev støttet af National Institute of Environmental Health Sciences, National Institutes of Health Egne Research Program Z01ES102745 (til GH).

1. Oct4GiP mus ES-celle vedligeholdelse Oct4GiP celler blev venligst tilvejebragt af Dr. Austin Smith. De er afledt fra 129/Ola mus, der bærer en Oct4-GFPiresPac transgen 4,5. De fastholdes i gelatine-coatede vævskulturplader i ESGRO fuldstændig plus klonal grad medium (Millipore) eller i M15 medium: DMEM (Invitrogen) suppleret med 15% FBS, 1000 U / ml ESGRO (Millipore), 1x Ikke essentielle aminosyrer (Invitrogen), 1x EmbryoMax Nucleasides (Millipore), og 10 uM β-mercaptoethanol...

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Den Oct4GiP reporter analysen beskriver vi ovenfor kan kvantitativt måle omfanget af selvfornyelse vs differentiering. Sammenlignet med andre tilgængelige metoder, såsom morfologi-baserede 12 og proliferation / levedygtighed-baserede assays, giver det højere følsomhed og gennemløb, såvel som en mere direkte måling af ES-celle tilstand. Det er derfor velegnet til store skærme og genetiske epistasis analyse. Faktisk har vi og andre med succes brugt Oct4GiP reporter analysen for genom-dækkende RNAi sk?...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> ESGRO komplet plus klonale kvalitet medium </td><td> Millipore </td><td> SF001-500P </td></tr><tr><td> DMEM (høj glucose 1X) </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> 0,25% trypsin-EDTA </td><td> Invitrogen </td><td> 25200 </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 1001817 </td></tr><tr><td> OPTI-MEM (reducere serum-medium) </td><td> Invitrogen </td><td> 31985 </td></tr><tr><td> ESGRO mLIF (10 7 units/1ml) </td><td> Millipore </td><td> DAM1776540 </td></tr><tr><td> MEM NEAA (ikke-essentielle aminosyrer) </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> 100x Nucleosides for ES-celle </td><td> Millipore </td><td> 10620-1 </td&gt; </tr><tr><td> 2-mercaptoethanol </td><td> Sigma </td><td> M7522-100ml </td></tr><tr><td> ES-kvalificeret føtalt bovint serum </td><td> Invitrogen </td><td> 10437 </td></tr><tr><td> Nanog siRNA </td><td> Invitrogen </td><td> MSS231181 </td></tr><tr><td> Oct4 siRNA </td><td> Dharmacon </td><td> D-046.256 til 02 </td></tr><tr><td> Sox2 siRNA </td><td> Dharmacon </td><td> M-058.489 til 01 </td></tr></tbody></table> Kontrol siRNA: siRNA duplex målretning ildflueluciferase (5&#39;-CGTACGCGGAATACTTCGA) syntetiseres ved Dharamcon.

oct4gip reporter assay to study genes that regulate mouse embryonic stem cell maintenance and self-renewal

Pluripotency og selv-fornyelse er to definerende karakteristika af embryonale stamceller (ES-celler). Forstå den underliggende molekylære mekanisme vil i høj grad lette brugen af ES-celler for Developmental Biology undersøgelser, sygdom modellering, Drug Discovery, og regenerativ medicin (revideret i 1,2). At fremskynde identifikation og karakterisering af hidtil ukendte regulatorer af ES-celle vedligeholdelse og selvfornyelse, udviklede vi en fluorescens reporter-baseret assay til kvantitativt at måle selvfornyelse status i muse ES-celler under anvendelse af Oct4GiP cellerne tre. De Oct4GiP celler udtrykker det grønt fluorescerende protein (GFP) under kontrol af Oct4 genpromotorregion 4,5. Oct4 kræves for ES-celle selvfornyelse, og er overudtrykt i ES-celler og hurtigt nedreguleres under differentiering 6,7. Som et resultat korrelerer GFP-ekspression og fluorescens i reportergen-celler nøjagtigtmed ES-celle identitet 5, og fluorescens-aktiveret celle sortering (FACS)-analyse kan bruges til nøje at overvåge selvfornyelse status af cellerne på enkelt celle niveau 3,8. Sammen med RNAi, kan Oct4GiP reporter analysen bruges til hurtigt at identificere og studere regulatorer af ES-celle vedligeholdelse og selvfornyelse 3,8. Sammenlignet med andre fremgangsmåder til assaybestemmelse selvfornyelse, er det mere bekvemt, følsom, kvantitativ, og lavere omkostninger. Det kan udføres på 96 - eller 384-brønds plader for omfattende undersøgelser, såsom høj-throughput skærme eller genetisk epistasis analyse. Endelig, ved hjælp af andre slægt-specifikke reporter ES-cellelinier, kan assayet er beskrevet her også modificeres til at studere skæbne specifikation i ES-celle-differentiering.

Watch this Scientific Journal Video about Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal at JoVE.com

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Beskriver vi en fluorescens Reporter Assay til hurtigt at identificere og karakterisere gener, der regulerer muse embryonale stamceller vedligeholdelse og selvfornyelse.

Oct4GiP Reporter Assay til at studere gener, der regulerer mus embryonale stamceller Vedligeholdelse og Self-fornyelse

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

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