Name: Optimierte quantitative Bewertung der Stabilität der Enhancer-RNA in embryonalen Mausstammzellen (Video) | JoVE Journal
Uploaded: 2025-11-21T14:45:00.000+00:00
Duration: 3 min 34 s
Description: Erfahren Sie, wie man die Stabilität von Enhancer-RNAs in embryonalen Mausstammzellen mit Actinomycin D, RT-qPCR und nichtlinearer Regression quantitativ bewertet. Diese Methode ermöglicht präzise Halbwertszeitbestimmungen.

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</ol>

<table><tbody><tr><td>2-Mercapptoethanol</td><td>Gibco</td><td>21985023</td><td></td></tr><tr><td>Actinomycin D</td><td>Sigma-Aldrich</td><td>A9415</td><td></td></tr><tr><td>Chloroform</td><td>Daejung-Chemikalien</td><td>2548-4105</td><td></td></tr><tr><td>DMSO</td><td>GenDEPOT</td><td>D2680</td><td></td></tr><tr><td>Fetales Rinderserum (FBS)</td><td>Sigma-Aldrich</td><td>F0392</td><td></td></tr><tr><td>Gelatine</td><td>Sigma-Aldrich</td><td>G1890-100G</td><td></td></tr><tr><td>Glasgow' s Minimum Essential Medium (GMEM)</td><td>Gibco</td><td>11710035</td><td></td></tr><tr><td>GraphPad Prism (Version 9)</td><td>GraphPad-Software</td><td></td><td></td></tr><tr><td>Isopropanol</td><td>Daejung-Chemikalien</td><td>5035-4105</td><td></td></tr><tr><td>KnockOut Serum-Ersatz (KOSR)</td><td>Gibco,&nbsp;</td><td>10828028</td><td></td></tr><tr><td>Leukämie-Inhibitorfaktor (LIF)</td><td>Merck</td><td>ESG1106</td><td></td></tr><tr><td>MEM-Lösung für nicht-essentielle Aminosäuren (MnEA)</td><td>Welgene</td><td>LS005</td><td></td></tr><tr><td>M-MLV cDNA-Synthesekit&nbsp;</td><td>Enzynomik</td><td>EZ006S</td><td></td></tr><tr><td>OPTIZEN NanoQ Plus&nbsp;</td><td>KLAB</td><td>01-QI685-M-16-C</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Welgene</td><td>LS202-02</td><td></td></tr><tr><td>Power SYBR-Green PCR Mastermix&nbsp;</td><td>Angewandte Biosysteme</td><td>4368708</td><td></td></tr><tr><td>QuanStudio1 Echtzeit-PCR-System&nbsp;</td><td>Angewandte Biosysteme</td><td>A40425</td><td></td></tr><tr><td>Natriumpyruvat</td><td>Welgene</td><td>LS013</td><td></td></tr><tr><td>TRIzol&nbsp;</td><td>Invitrogen</td><td>15596018</td><td></td></tr></tbody></table>

optimierte quantitative bewertung der stabilität der enhancer-rna in embryonalen mausstammzellen

Optimized Quantitative Assessment of Enhancer RNA Stability in Mouse Embryonic Stem Cells

Optimierte quantitative Bewertung der Stabilität der Enhancer-RNA in embryonalen Mausstammzellen

Enhancer RNAs (eRNAs) are non-coding transcripts produced from transcriptionally active enhancer regions and are increasingly recognized as key regulators ...

optimized-quantitative-assessment-enhancer-rna-stability-mouse

Research

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JoVE Journal

Biochemistry

Biochemie

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

Einzelzell-Genexpression Test wird für das Verständnis der Stammzell Heterogenitäten benötigt.

Profiling Individuelle humanen embryonalen Stammzellen durch quantitative RT-PCR

Die experimentelle Validierung der Enhancer-Aktivität erfolgt am besten durch eine Funktionsverlustanalyse. Hier wird ein effizientes Protokoll vorgestellt, das die CRISPR/Cas9-vermittelte Deletion verwendet, um die allelspezifische Regulation der Gentranskription in F1-ES-Zellen zu untersuchen, die ein Hybridgenom (Mus musculus129 x Mus castaneus) enthalten.

Generieren von CRISPR / Cas9 Mediated monoallelic Löschungen Enhancer-Funktion in embryonalen Stammzellen der Maus zu studieren

Dieses Protokoll stellt eine Methode quantitative, single-cell in situ Analyse der Proteinexpression durchzuführen lineage in mouse Spezifikation Präimplantationsembryonen zu studieren. Die Verfahren, die für die Sammlung von Blastozysten, Vollmontage Immunfluoreszenz-Nachweis von Proteinen, Imaging von Proben auf einem konfokalen Mikroskop und Kern Segmentierung und Bildanalyse beschrieben.

Quantitative Analyse der Proteinexpression zu studieren Lineage Spezifikation in Maus Präimplantationsembryonen

In dieser Arbeit stellen wir einen experimentellen Workflow zur Verfügung, wie aktive Enhancer identifiziert und experimentell validiert werden können.

Vorhersage und Validierung von Gen-regulatorischen Elementen Activated Während Retinsäure Induced Embryonic Stem Zelldifferenzierung

Proteine ​​enthalten oft mehrere Domänen, die verschiedene zelluläre Funktionen ausüben kann. Gen-Knock-outs (KO) nicht über diese funktionelle Vielfalt berücksichtigen. Hier berichten wir über eine Rekombination vermittelten Kassettenaustausch (RMCE) -basierte Struktur-Funktions-Ansatz in KO embryonalen Stammzellen, die für die molekulare Präparation von verschiedenen funktionellen Domänen oder Varianten eines Proteins ermöglicht.

Struktur-Funktions-Studien in embryonalen Maus-Stammzellen Rekombinase-vermittelten Kassettenaustausch

Dieses Protokoll beschreibt die erforderlichen Schritte zum Entwerfen und ausführen gemultiplexten Ausrichtung der Enhancer mit deaktivieren Fusionsproteins SID4X-dCas9-KRABBE, auch bekannt als Enhancer Störungen (Enhancer-i). Dieses Protokoll ermöglicht die Identifizierung von Enhancer, die Genexpression regulieren und erleichtert das Sezieren von Beziehungen zwischen Enhancer Regulierung ein gemeinsames Zielgen.

Dissektion der Enhancer-Funktion mit Multiplex CRISPR-basierte Enhancer Einmischung in Zell-Linien

Wir berichten über die Anwendung der quantitativen Chromosomenkonformationsaufnahme, gefolgt von einer Hochdurchsatzsequenzierung in embryonalen Körpern, die aus embryonalen Stammzellen erzeugt werden. Diese Technik ermöglicht es, die Kontakte zwischen vermeintlichen Enhancern und Promotorregionen eines bestimmten Gens während der embryonalen Stammzelldifferenzierung zu identifizieren und zu quantitieren.

Identifizierung von Enhancer-Promoter-Kontakten in embryoiden Körpern durch quantitative Chromosomenkonformationserfassung (4C)

Dieses Protokoll beschreibt einen neuartigen rAAV-basierten transienten Enhancer-Reporter-Assay. Dieser Assay kann verwendet werden, um eine enhancergesteuerte Expression in vivo im Gehirn der Maus zu induzieren.

AAV-Einsatz von Enhancer-basierten Expressionskonstrukten in vivo im Gehirn von Mäusen

Dieses Protokoll bietet eine optimierte rechnerische Pipeline zur Quantifizierung von naszierenden Enhancer-Transkripten. Durch die Integration von Chromatinzugänglichkeit, Chromatinmerkmal und Transkriptionsdaten ermöglicht es einen genauen Nachweis und eine strangspezifische Analyse der Enhanceraktivität in komplexen intragenen Regionen, während es für Forscher ohne umfangreiche bioinformatische Ausbildung zugänglich bleibt.

Eine computergestützte Pipeline für die Quantifizierung intergener/intragener Enhancer-RNA in embryonalen Stammzellen der Maus

Erfahren Sie, wie man die Stabilität von Enhancer-RNAs in embryonalen Mausstammzellen mit Actinomycin D, RT-qPCR und nichtlinearer Regression quantitativ bewertet. Diese Methode ermöglicht präzise Halbwertszeitbestimmungen.

Optimierte quantitative Bewertung der Stabilität der Enhancer-RNA in embryonalen Mausstammzellen

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