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

Denne studien ble finansiert av: National Research Network on Aging (NFN S93) av den østerrikske Science Foundation (FWF), EU-kommisjonen integrerte prosjekter MiMAGE og PROTEOMAGE, Nederland Genomics Initiative / nederlandske organisasjonen for Scientific Research (NGI / NWO, 05040202 og 050 - 060-810 NCHA), EU finansiert Network of Excellence Levetid (FP6 036894), og Innovasjon forskningsprogram på Genomics (SenterNovem, IGE01014 og IGE5007).

1. Plasmid Anlegg <ol><li> Bestill custom oligo-nukleotider koding for DGN (ACKNWFSSLSHFVIHL 11) og for dgnFS (HARTGSLACPTSSSICE) og ligere det inn i pEGFP-C1 vektor for å få sammensmeltingen av GFP med DGN / dgnFS (figur 1). </li><li> Forsterke kodesekvensen for GFP-DGN og GFP-dgnFS ved PCR i henhold til protokollen fra pENTR Directional TOPO Kloning Kit og fortsette med pLenti6/V5 Directional TOPO Cloning Kit (figur 6). </li></ol> 2. Virus Produksj...

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Den første publikasjonen med grønt fluorescerende protein (GFP) som reporter substrat for ubiquitin-proteasom aktivitet ble publisert i 2000 12. Siden da har GFP blitt et vanlig verktøy for å visualisere cellulære aktiviteter, spesielt ubiquitin-proteasom prosess. Å overvåke ubiquitin-proteasom aktivitet in vivo en transgen mus modell med en GFP-baserte reporter har blitt innført 13. Ytterligere in vivo undersøkelser etablert en transgen mus modell med en lignende degron-...

<table border="1"><tbody><tr><td> Navnet på reagensen </td><td> Selskapet </td><td> Katalognummer </td></tr><tr><td> pEGFP-C1 Vector </td><td> BD Biovitenskap 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 Reagens </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> Polyetylenglykol </td><td> Sigma </td><td> P2139 </td></tr><tr><td> NaCl </td><td> Merck </td><td> 1.06404.1000 </td></tr><tr><td> Dulbeccos Fosfatbufret Saline 1x (PBS) </td><td> Invitrogen </td><td> 14190 </td></tr><tr><td> hexadimethrine bromide </td><td> Sigma </td><td> 10,768-9 </td></tr><tr><td> Blasticidin </td><td> Invitrogen </td><td> R21001 </td></tr><tr><td> Krystallfiolett </td><td> Sigma </td><td> C3886 </td></tr><tr><td> FACS rør </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> Føtalt bovint serum (FBS) </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> MEM ikke-essensielle aminosyrer (NEAA) 100x </td><td> Invitrogen </td><td> 11140035 </td></tr><tr><td> MEM Sodium Pyruvat 100 mM </td><td> Invitrogen </td><td> 11360039 </td></tr><tr><td> D-(+)-glukose (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 er den viktigste intracellulære organeller involvert i proteolytisk nedbrytning av unormale, misfolded, skadet eller oksidert proteiner 1, 2. Vedlikehold av proteasome aktivitet ble innblandet i mange viktige cellulære prosesser, som celle stressrespons 3, cellesyklus regulering og cellulær differensiering 4 eller i immunsystemet respons fem. Dysfunksjon av ubiquitin-proteasom systemet har vært knyttet til utvikling av svulster og nevrodegenerative sykdommer 4, 6. I tillegg ble en reduksjon i proteasom aktivitet funnet som en funksjon av cellulær senescence og organismebiologi 7 aldring, 8, 9, 10. Her presenterer vi en metode for å måle ubiquitin-proteasom aktivitet i levende celler ved hjelp av en GFP-DGN fusion protein. Å være i stand til å overvåke ubiquitin-proteasom i levende primære celler, konstruerer komplementær DNA som koder for et grønt fluorescerende protein (GFP)-DGN fusjonsprotein (GFP-DGN og ustabil) og en variant som bærer en frameshift mutasjon (GFP-dgnFS, stabil 11) er satt inn i lentiviral ekspresjonsvektorer. Vi foretrekker denne teknikken over tradisjonelle transfeksjon teknikker fordi det garanterer en meget høy transfeksjon virkningsgrad uavhengig av celletypen eller alderen på giveren. Forskjellen mellom fluorescens som vises av GFP-dgnFS (stabil) protein og destabilisert protein (GFP-DGN) i fravær eller nærvær av proteasominhibitor kan brukes til å estimere ubiquitin-proteasom aktivitet i hvert enkelt cellestamme. Disse forskjellene kan overvåkes av epifluorescence mikroskopi eller kan måles ved strømningscytometri.

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

En metode for å overvåke ubiquitin-proteasom aktivitet i levende celler er beskrevet. En degron-destabilisert GFP-(GFP-DGN) og en stabil GFP-dgnFS fusjonsprotein genereres og transduced inn i cellen ved hjelp av en lentiviral ekspresjonsvektor. Denne teknikken gjør det mulig å generere en stabil GFP-dgn/GFP-dgnFS uttrykke cellelinje der ubiquitin-proteasom aktivitet kan lett vurdert ved hjelp epifluorescence eller flowcytometri.

Overvåking av Ubiquitin-proteasome aktivitet i levende celler ved hjelp av en Degron (DGN)-destabilisert grønt fluorescerende protein (GFP)-baserte 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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