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 for lesing og redigering av manuskriptet. Denne forskningen ble støttet av National Institute of Environmental Health Sciences, National Institutes of Health egenutført Research Program Z01ES102745 (til GH).

1. Oct4GiP Mouse ES Cell Maintenance Oct4GiP celler ble vennlig levert av Dr. Austin Smith. De ble utledet fra de 129/Ola mus som frakter en Oct4-GFPiresPac transgenet 4,5. De er opprettholdt i gelatin-belagte vevskulturstudier plater i ESGRO komplett pluss klonal klasse medium (Millipore), eller i M15 medium: DMEM (Invitrogen) supplert med 15% FBS, 1000 U / ml ESGRO (Millipore), 1x Ikke- essensielle aminosyrer (Invitrogen), 1x EmbryoMax Nucleasides (Millipore), og 10 mm β-mercapto...

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Den Oct4GiP reporter analysen beskriver vi ovenfor kan kvantitativt måle omfanget av selvfornyelse vs differensiering. Sammenlignet med andre tilgjengelige metoder, for eksempel morfologi-baserte 12 og spredning / levedyktighet-baserte analyser, tilbyr høyere følsomhet og gjennomstrømning, samt en mer direkte måling av ES cellen staten. Det er derfor godt egnet for storskala-skjermer og genetisk epistasis analyse. Faktisk har vi og andre med hell brukt den Oct4GiP reporter analysen for genom-wide RNAi sk...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> ESGRO komplett pluss klonal karakteren middels </td><td> Millipore </td><td> SF001-500P </td></tr><tr><td> DMEM (High glukose 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 (redusere 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 (Non-essensielle aminosyrer) </td><td> Invitrogen </td><td> 11140 </td></tr><tr><td> 100x Nukleosider 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-kvalifisert føtal 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-046256-02 </td></tr><tr><td> Sox2 siRNA </td><td> Dharmacon </td><td> M-058489-01 </td></tr></tbody></table> Kontroll siRNA: siRNA duplex målretting Firefly luciferase (5&#39;-CGTACGCGGAATACTTCGA) syntetisert av Dharamcon.

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

Pluripotency og selvfornyelse er to definerende karakteristikkene av embryonale stamceller (ES-celler). Forstå den underliggende molekylære mekanismen vil i stor grad forenkle bruken av ES celler for utviklingsbiologi studier, sykdom modellering, drug discovery, og regenerativ medisin (anmeldt i 1,2). For å påskynde identifisering og karakterisering av nye regulatorer av ES celle vedlikehold og selvfornyelse, utviklet vi en fluorescens reporter-baserte analysen til kvantitativt måle selvfornyelse status i mus ES celler ved hjelp av Oct4GiP cellene tre. De Oct4GiP celler uttrykker den grønne fluorescerende protein (GFP) under kontroll av Oct4 genet promoter-regionen 4,5. Oct4 kreves for ES celle selvfornyelse, og er høyt uttrykt i ES cellene og raskt nedregulert i løpet differensiering 6,7. Som et resultat, korrelerer GFP uttrykk og fluorescens i reporteren cellene trofastmed ES cellen identitet 5, og fluorescens-aktivert celle sortering (FACS) analyse kan brukes til å nøye overvåke selvfornyelse status av cellene på celle-nivå 3,8. Sammen med RNAi, kan Oct4GiP reporteren analysen brukes til å raskt identifisere og studere regulering av ES celle vedlikehold og selvfornyelse 3,8. Sammenlignet med andre metoder for å analysere selvfornyelse, er det mer praktisk, følsom, kvantitativ, og av lavere kostnader. Det kan utføres i 96 - eller 384-brønnen plater for store studier som high-throughput skjermer eller genetisk epistasis analyse. Til slutt, ved hjelp av andre slektslinje-spesifikke reporter ES cellelinjer, kan analysen vi beskriver her også bli modifisert for å studere skjebnen spesifikasjonen under ES celledifferensiering.

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

Vi beskriver en fluorescens reporter analyse for å raskt identifisere og karakterisere gener som regulerer mus embryonale stamceller vedlikehold og selvfornyelse.

Oct4GiP Reporter Assay for å studere gener som regulerer Mouse Embryonic Stem Cell Maintenance and 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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