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

Wij danken Brad Lackford voor het lezen en bewerken van het manuscript. Dit onderzoek werd ondersteund door het National Institute of Environmental Health Sciences, National Institutes of Health Intramurale Research Program Z01ES102745 (naar GH).

1. Oct4GiP muis ES Cell Onderhoud Oct4GiP cellen werden vriendelijk verschaft door Dr Austin Smith. Ze werden afgeleid van de 129/Ola muizen die een Oct4-GFPiresPac transgen 4,5. Zij worden gehandhaafd gelatine beklede weefselkweek-platen op de ESGRO volledige plus klonale gehalte medium (Millipore) of in de M15 medium: DMEM (Invitrogen) aangevuld met 15% FBS, 1000 U / ml ESGRO (Millipore) 1x Niet essentiële aminozuren (Invitrogen), 1x EmbryoMax Nucleasides (Millipore) en 10 uM β-...

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De Oct4GiP reporter test beschrijven we hierboven kan kwantitatief meten van de mate van zelf-vernieuwing versus differentiatie. Vergeleken met andere beschikbare methoden, zoals morfologie gebaseerde 12 en proliferatie / levensvatbaarheid gebaseerde assays, biedt een hogere gevoeligheid en doorvoersnelheid, alsmede een directe meting van de embryonale stamcellen staat. Het is daarom zeer geschikt voor grootschalige schermen en genetische epistase analyse. Inderdaad, hebben wij en anderen met succes gebruikt ...

<table border="1"><tbody><tr><td> Naam van het serum </td><td> Vennootschap </td><td> Catalogusnummer </td></tr><tr><td> ESGRO compleet plus kloon graad Medium </td><td> Millipore </td><td> SF001-500P </td></tr><tr><td> DMEM (High glucose 1X) </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> 0,25% trypsine-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 (te verminderen 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 (Niet-essentiële aminozuren) </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> 100x Nucleosiden voor ES-cel </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-gekwalificeerde foetaal runderserum </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-046256-02 </td></tr><tr><td> Sox2 siRNA </td><td> Dharmacon </td><td> M-058489-01 </td></tr></tbody></table> Controle siRNA: siRNA duplex gericht vuurvlieg luciferase (5&#39;-CGTACGCGGAATACTTCGA) gesynthetiseerd door Dharamcon.

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

Pluripotentie en zelfvernieuwing zijn twee definities van de kenmerken van embryonale stamcellen (ES cellen). Het begrijpen van de onderliggende moleculaire mechanisme zal aanzienlijk vergemakkelijken het gebruik van ES-cellen voor de ontwikkelingsbiologie studies, ziekte modelleren, drug discovery, en regeneratieve geneeskunde (overzicht in 1,2). Om de identificatie en karakterisering van nieuwe regulatoren van ES-cel onderhoud en zelfvernieuwing te bespoedigen, hebben we een fluorescentie-reporter-gebaseerde test om kwantitatief te meten van de zelfvernieuwing status in de muis ES-cellen met behulp van de Oct4GiP cellen 3. De Oct4GiP cellen brengen green fluorescent protein (GFP) onder controle van de Oct4 gen promotergebied 4,5. Oct4 is vereist voor ES-cel zelf-vernieuwing, en is hoog tot expressie in ES-cellen en snel down-gereguleerd tijdens de differentiatie van 6,7. Hierdoor GFP expressie en fluorescentie in de cellen reporter correleert trouwmet de ES-cel identiteit 5 en fluorescentie-geactiveerde cel sortering (FACS) analyse kan worden gebruikt om nauwlettend toezien op de zelfvernieuwing status van de cellen op de enkele cel niveau van 3,8. In combinatie met RNAi, kan de Oct4GiP reporter test gebruikt worden om snel te identificeren en te bestuderen regulatoren van ES-cel onderhoud en zelfvernieuwing 3,8. In vergelijking met andere methoden voor het bepalen zelfvernieuwing is het handig, gevoelig kwantitatieve en lagere kosten. Het kan worden uitgevoerd in 96 - of 384-platen voor grootschalige studies zoals hoge-doorvoer schermen of genetische epistase analyse. Tenslotte door andere lijn-specifieke reporterconstructieproducten ES cellijnen kan de test hier beschreven ook worden gemodificeerd lot specificatie tijdens ES celdifferentiatie bestuderen.

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

We beschrijven een fluorescentie reporter test om snel te identificeren en karakteriseren genen die muis embryonale stamcellen onderhoud en zelfvernieuwing te reguleren.

Oct4GiP Reporter Assay van genen die muis embryonale stamcellen Onderhoud en Zelfvernieuwing Regel Studie

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

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