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

Wir bedanken uns bei Brad Lackford zum Lesen und Editieren des Manuskripts. Diese Arbeit wurde vom National Institute of Environmental Health Sciences, National Institutes of Health Research Program Intramural Z01ES102745 (GH) unterstützt.

1. Oct4GiP Maus-ES-Zell-Wartung Oct4GiP Zellen wurden freundlicherweise von Dr. Austin Smith zur Verfügung gestellt. Sie wurden von den Mäusen, die eine 129/Ola Oct4-Transgen GFPiresPac 4,5 abgeleitet. Sie werden in mit Gelatine beschichteten Gewebekulturplatten in der Esgro komplette plus klonale Grad Medium (Millipore), oder in der M15-Medium: DMEM (Invitrogen) mit 15% FBS, 1000 U / ml Esgro (Millipore), 1x Nicht-ergänzt essentiellen Aminosäuren (Invitrogen), 1x EmbryoMax Nucl...

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Die Oct4GiP Reporter-Assay beschreiben wir oben kann quantitativ messen das Ausmaß der Selbst-Erneuerung vs Differenzierung. Im Vergleich zu anderen verfügbaren Verfahren, wie die Morphologie-basierten 12 und Proliferation / Lebensfähigkeit-basierten Assays, bietet eine höhere Empfindlichkeit und Durchsatz sowie eine direkte Messung der ES-Zelle Zustand. Es wird daher auch für große Bildschirme und genetische Analyse Epistasie geeignet. In der Tat haben wir und andere erfolgreich das Oct4GiP Reporter-As...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Esgro komplette plus klonale Klasse 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% 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 (Serum-Medium zu reduzieren) </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 (Nicht-essentielle Aminosäuren) </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> 100x Nucleosides für ES-Zell- </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-qualifizierte Rinderfötenserum </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-046.256-02 </td></tr><tr><td> Sox2 siRNA </td><td> Dharmacon </td><td> M-058.489-01 </td></tr></tbody></table> Kontroll-siRNA: siRNA-Duplex Targeting Glühwürmchen-Luciferase (5&#39;-CGTACGCGGAATACTTCGA) durch Dharamcon synthetisiert.

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

Pluripotenz und Selbsterneuerung sind zwei definierenden Eigenschaften von embryonalen Stammzellen (ES-Zellen). Das Verständnis der zugrunde liegenden molekularen Mechanismus wird dadurch wesentlich erleichtert den Einsatz von ES-Zellen für Entwicklungsbiologie Studien, Krankheit Modellierung, der Wirkstoffforschung und in der regenerativen Medizin (Übersicht in 1,2). Um die Identifizierung und Charakterisierung neuer Regulatoren der ES-Zell-Wartungs-und Selbst-Erneuerung beschleunigen, entwickelten wir eine Fluoreszenz-Reporter-basierter Assay zur quantitativen Messung der Selbst-Erneuerung Status in Maus-ES-Zellen unter Verwendung des Oct4GiP Zellen 3. Die Oct4GiP Zellen exprimieren das grün fluoreszierende Protein (GFP) unter der Kontrolle des Oct4-Gen Promotor-Region 4,5. Oct4 ist für ES-Zell-Selbsterneuerung benötigt, und ist stark in ES-Zellen exprimiert und schnell herunter reguliert während der Differenzierung 6,7. Als ein Ergebnis korreliert GFP-Expression und Fluoreszenz in den Reporter-Zellen treumit dem ES-Zell-Identität 5 und Fluoreszenz-aktivierten Zellsortierung (FACS)-Analyse kann verwendet werden, die sorgfältige Überwachung der Selbst-Erneuerung Status der Zellen auf der Ebene der Einzelzelle 3,8 sein. Gekoppelt mit RNAi, kann die Oct4GiP Reporter Assay verwendet, um schnell zu identifizieren und zu untersuchen Regulatoren der ES-Zell-Wartungs-und Selbst-Erneuerung 3,8 sein. Im Vergleich zu anderen Methoden zur Bestimmung von Selbst-Erneuerung, ist es bequemer, einfühlsam, quantitative und von geringeren Kosten. Es kann in 96 durchgeführt werden - oder 384-Well-Platten für eine groß angelegte Studien wie High-Throughput-Screens oder genetische Analyse Epistasie. Schließlich wird unter Verwendung anderen Linie-spezifischen Reporters ES-Zelllinien, kann der Assay hier beschriebenen auch modifiziert werden, Determination während der ES-Zelldifferenzierung zu untersuchen.

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

Wir beschreiben einen Fluoreszenz-Reporter-Assay schnell zu identifizieren und zu charakterisieren, Gene, die Maus embryonalen Stammzellen Wartungs-und Selbst-Erneuerung zu regulieren.

Oct4GiP Reporter Assay auf Gene, die Maus embryonalen Stammzellen Wartungs-und Self-Study Erneuerung Regulate

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

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