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

Ringraziamo Brad Lackford per la lettura e la modifica del manoscritto. Questa ricerca è stata finanziata dal National Institute of Environmental Health Sciences, National Institutes of Health Research Programma intramurale Z01ES102745 (a GH).

1. Oct4GiP mouse cellule ES di manutenzione Cellule Oct4GiP sono stati gentilmente forniti dal Dr. Austin Smith. Essi sono stati ricavati dai topi portatori di una 129/Ola Oct4-GFPiresPac transgene 4,5. Essi sono mantenuti in piastre rivestite con tessuti gelatina coltura nella più completa clonale ESGRO grado medio (Millipore), o nel mezzo M15: DMEM (Invitrogen) supplementato con 15% FBS, 1000 U / ml ESGRO (Millipore), 1x non aminoacidi essenziali (Invitrogen), 1x Nucleasides Embr...

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Il saggio giornalista Oct4GiP abbiamo descritto sopra può misurare quantitativamente il grado di auto-rinnovamento vs differenziazione. Rispetto ad altri metodi disponibili, come la morfologia a base 12 e la proliferazione / vitalità saggi basati, offre maggiore sensibilità e velocità, nonché una misura più diretta dello stato della cellula ES. E &#39;quindi adatto per i grandi schermi e analisi epistasi genetica. In effetti, noi e altri hanno utilizzato con successo il saggio giornalista Oct4GiP per la...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> ESGRO medio grado completa e clonale </td><td> Millipore </td><td> SF001-500P </td></tr><tr><td> DMEM (High glucosio 1X) </td><td> Invitrogen </td><td> 11965 </td></tr><tr><td> 0,25% tripsina-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 (riduzione media del siero) </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 (aminoacidi non essenziali) </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> Nucleosidi 100x di cellule ES </td><td> Millipore </td><td> 10620-1 </td&gt; </tr><tr><td> 2-mercaptoetanolo </td><td> Sigma </td><td> M7522-100ml </td></tr><tr><td> ES-qualificato siero fetale bovino </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> SiRNA di controllo: duplex siRNA mira lucciola luciferasi (5&#39;-CGTACGCGGAATACTTCGA) sintetizzato Dharamcon.

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

Pluripotenza e di auto-rinnovamento sono due caratteristiche che definiscono le cellule staminali embrionali (cellule ES). Comprendere il meccanismo molecolare agevolerà notevolmente l&#39;uso di cellule staminali embrionali per gli studi di biologia dello sviluppo, modelli di malattia, scoperta di farmaci, e la medicina rigenerativa (recensione in 1,2). Per velocizzare l&#39;identificazione e la caratterizzazione di nuovi regolatori di manutenzione delle cellule ES e di auto-rinnovamento, abbiamo sviluppato una fluorescenza giornalista-based test per misurare quantitativamente il self-renewal stato in cellule staminali embrionali di topo usando le cellule Oct4GiP 3. Le cellule Oct4GiP esprimono la proteina fluorescente verde (GFP) sotto il controllo della regione del promotore del gene Oct4 4,5. Oct4 è necessaria per cellule ES auto-rinnovamento, ed è altamente espressa nelle cellule ES e rapidamente down-regolato durante la differenziazione 6,7. Come risultato, l&#39;espressione della GFP e fluorescenza nelle cellule del reporter correla fedelmentecon l&#39;identità della cellula ES 5, e fluorescenza selezione delle cellule attivate (FACS) analisi può essere utilizzato per monitorare attentamente la auto-rinnovamento stato delle celle a livello di singola cellula 3,8. Accoppiato con RNAi, il saggio giornalista Oct4GiP può essere utilizzato per identificare rapidamente e studiare regolatori di manutenzione cellule ES e auto-rinnovamento 3,8. Rispetto ad altri metodi per saggiare auto-rinnovamento, è più conveniente, sensibile, quantitativa, e di costo inferiore. Essa può essere effettuata in 96 - o 384 pozzetti per studi su larga scala, quali ad alto rendimento schermi o analisi epistasi genetica. Infine, utilizzando altri lineage-specifici reporter di linee cellulari ES, il saggio qui descritte possono anche essere modificato per studiare il destino specifica durante la differenziazione delle cellule ES.

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

Descriviamo un saggio di fluorescenza giornalista per identificare rapidamente e caratterizzare i geni che regolano il mouse sulle cellule staminali embrionali di manutenzione e di auto-rinnovamento.

Oct4GiP Reporter Assay per studiare i geni che regolano il mouse sulle cellule staminali embrionali di manutenzione e di auto-rinnovamento

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

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