Name: monitoring of ubiquitin-proteasome activity in living cells using a degron (dgn)-destabilized green fluorescent protein (gfp)-based reporter protein
Uploaded: 2012-11-10T14:00:00
Description: Watch this Scientific Journal Video about Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein at JoVE.com

Diese Studie wurde gefördert durch: National Research Network on Aging (NFN S93) von der österreichischen Science Foundation (FWF), Europäische Kommission Integrierte Projekte MiMAGE und PROTEOMAGE, Niederlande Genomics Initiative / Niederländische Organisation für wissenschaftliche Forschung (NGI / NWO, 05040202 und 050 - 060-810 NHG), das EU Network of Excellence Lebensdauer (FP6 036.894) finanziert und Innovation Oriented Research Program am Genomics (SenterNovem; IGE01014 und IGE5007).

Ein. Plasmidkonstruktion <ol><li> Um individuelle Oligonucleotide Codierung für dgn (ACKNWFSSLSHFVIHL 11) und zum dgnFS (HARTGSLACPTSSSICE) und ligieren es in den pEGFP-C1-Vektor, um die Fusion des GFP mit dgn ​​/ dgnFS (Abbildung 1) zu erhalten. </li><li> Amplify die kodierende Sequenz für GFP-DGN-und GFP-dgnFS mittels PCR nach dem Protokoll des pENTR Directional TOPO Cloning Kit und fahren mit dem pLenti6/V5 Directional TOPO Cloning Kit (Abbildung 6). </li></ol><p c...

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Die erste Veröffentlichung mit grün fluoreszierendem Protein (GFP) als Reporter Substrat für Ubiquitin-Proteasom-Aktivität wurde in 2000 12 veröffentlicht. Seitdem hat GFP zu einem gemeinsamen Instrument zur zellulären Aktivitäten, insbesondere das Ubiquitin-Proteasom-Prozess zu visualisieren. Um Ubiquitin-Proteasom-Aktivität in vivo zu überwachen ein transgenes Mausmodell mit einem GFP-basierte Reporter wurde 13 eingeführt. Zusätzliche in vivo Forschung etabliert ander...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> pEGFP-C1 Vektor </td><td> BD Bioscience Clontech </td><td> 6084-1 </td></tr><tr><td> pENTR Directional TOPO Cloning Kit </td><td> Invitrogen </td><td> K2400-20 </td></tr><tr><td> pLenti6/V5 Directional TOPO Cloning Kit </td><td> Invitrogen </td><td> V496-10 </td></tr><tr><td> Lipofectamine 2000 Reagent </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> DMEM </td><td> Sigma </td><td> D5546 </td></tr><tr><td> PVDF-Filter (Rotilabo-Spritzenfilter) </td><td> Roth </td><td> P667.1 </td></tr><tr><td> Polyethylenglykol </td><td> Sigma </td><td> P2139 </td></tr><tr><td> NaCl </td><td> Merck </td><td> 1.06404.1000 </td></tr><tr><td> Dulbeccos Phosphat-gepufferte Saline 1x (PBS) </td><td> Invitrogen </td><td> 14190 </td></tr><tr><td> Hexadimethrinbromid </td><td> Sigma </td><td> 10,768-9 </td></tr><tr><td> Blasticidin </td><td> Invitrogen </td><td> R21001 </td></tr><tr><td> Kristallviolett </td><td> Sigma </td><td> C3886 </td></tr><tr><td> FACS-Röhrchen </td><td> BD Biosciences </td><td></td></tr><tr><td> Penicillin Streptomycin (Pen-Strep) </td><td> Invitrogen </td><td> 15140130 </td></tr><tr><td> L-Glutamin 200 mM </td><td> Invitrogen </td><td> 25030024 </td></tr><tr><td> Fötales Rinderserum (FBS) </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> MEM Non-Essential Amino Acids (NEAA) 100x </td><td> Invitrogen </td><td> 11140035 </td></tr><tr><td> MEM Natriumpyruvat 100 mM </td><td> Invitrogen </td><td> 11360039 </td></tr><tr><td> D-(+)-Glucose (45%) </td><td> Sigma </td><td> G8769 </td></tr><tr><td> Geneticin </td><td> Invitrogen </td><td> 11811023 </td></tr><tr><td> CaCl2 </td><td> Merck </td><td> C5080 </td></tr><tr><td> Hepes </td><td> Sigma </td><td> H3375 </td></tr><tr><td> Trypsin-EDTA (0,05%) </td><td> Invitrogen </td><td> 25300054 </td></tr></tbody></table>

monitoring of ubiquitin-proteasome activity in living cells using a degron (dgn)-destabilized green fluorescent protein (gfp)-based reporter protein

Proteasome ist der wichtigste intrazelluläre Organellen in den proteolytischen Abbau von abnormal, fehlgefalteten, beschädigte oder oxidierte Proteine ​​1, 2 beteiligt. Wartung der Proteasom-Aktivität wurde in vielen wichtigen zellulären Prozessen beteiligt, wie Zelle Stressantwort 3, Regulation des Zellzyklus und Zelldifferenzierung 4 oder Reaktion des Immunsystems 5. Die Dysfunktion des Ubiquitin-Proteasom-System hat zu der Entwicklung von Tumoren und neurodegenerativen Krankheiten 4, 6 in Verbindung gebracht. Zusätzlich wurde eine Abnahme der Proteasom-Aktivität als Funktion der zellulären Seneszenz und organismal Alterung 7, 8, 9, 10 gefunden. Hier präsentieren wir eine Methode, um Ubiquitin-Proteasom-Aktivität in lebenden Zellen mit einem GFP-dgn Fusionsprotein zu messen. Um Ubiquitin-Proteasom-Aktivität in lebenden primären Zellen zu überwachen, konstruiert komplementäre DNA, die für ein grün fluoreszierendes Protein (GFP)-dgn Fusionsprotein (GFP-dgn, instabil) und eine Variante Tragen einer Frameshift-Mutation (GFP-dgnFS, stabil 11) werden in lentiviralen Expressionsvektoren insertiert. Wir bevorzugen diese Technik gegenüber herkömmlichen Transfektionstechniken weil es eine sehr hohe Transfektionseffizienz garantiert unabhängig von dem Zelltyp oder dem Alter des Spenders. Der Unterschied zwischen der Fluoreszenz, die von GFP-dgnFS (stabilen) Protein und dem destabilisierten Protein (GFP-dgn) in Abwesenheit oder Gegenwart von Proteasom-Inhibitor angezeigt kann zum Ubiquitin-Proteasom-Aktivität in jeder bestimmten Zellstamm abzuschätzen. Diese Unterschiede können durch Epifluoreszenzmikroskopie überwacht werden können oder mittels Durchflusszytometrie gemessen werden.

Watch this Scientific Journal Video about Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein at JoVE.com

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

Eine Methode, um Ubiquitin-Proteasom-Aktivität in lebenden Zellen zu überwachen beschrieben. A Degron-destabilisierten GFP-(GFP-DGN) und eine stabile GFP-dgnFS Fusionsprotein erzeugt und transduziert in die Zelle mit einem lentiviralen Expressionsvektor. Diese Technik ermöglicht eine stabile GFP-dgn/GFP-dgnFS exprimierenden Zelllinie, in dem Ubiquitin-Proteasom-Aktivität leicht beurteilt werden kann, mit Epifluoreszenz oder Durchflusszytometrie generieren.

Überwachung des Ubiquitin-Proteasom-Aktivität in lebenden Zellen unter Verwendung eines Degron (DGN)-destabilisiert Green Fluorescent Protein (GFP)-basierte Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

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Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

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