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

Denna studie har finansierats av: National Research Network on Aging (NFN S93) av den österrikiska Science Foundation (FWF), Europeiska kommissionen integrerade projekt MiMAGE och PROTEOMAGE, Nederländerna Genomics Initiative / Nederländerna organisationen för vetenskaplig forskning (NGI / NWO, 05.040.202 och 050 - 060-810 NCHA), det EU-finansierade Network of Excellence Lifespan (FP6 036.894) och innovationsinriktade forskningsprogram om Genomics (SenterNovem, IGE01014 och IGE5007).

1. Plasmidkonstruktion <ol><li> Beställ anpassade oligo-nukleotider som kodar för DGN (ACKNWFSSLSHFVIHL 11) och för dgnFS (HARTGSLACPTSSSICE) och ligera det in i pEGFP-C1 vektorn att erhålla fusion av GFP med DGN-/ dgnFS (figur 1). </li><li> Förstärk den kodande sekvensen för GFP-DGN och GFP-dgnFS med PCR enligt protokollet av pENTR Directional TOPO Kloning Kit och fortsätta med pLenti6/V5 Directional TOPO Cloning Kit (Figur 6). </li></ol> 2. V...

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Den första publikationen med grönt fluorescerande protein (GFP) som reporter substrat för ubiquitin-proteasom aktivitet publicerades 2000 12. Sedan dess har GFP blivit ett vanligt verktyg för att visualisera cellulära aktiviteter, speciellt ubiquitin-proteasom process. För att övervaka ubikitin-proteasom aktivitet in vivo en transgen musmodell med en GFP-baserad reporter har införts 13. Ytterligare in vivo-forskning, inrättat en transgen musmodell med en liknande degron-d...

<table border="1"><tbody><tr><td> Namnet av reagenset </td><td> Företag </td><td> Katalognummer </td></tr><tr><td> pEGFP-C1 vektor </td><td> BD Bioscience Clontech </td><td> 6084-1 </td></tr><tr><td> pENTR Directional TOPO kloningskit </td><td> Invitrogen </td><td> K2400-20 </td></tr><tr><td> pLenti6/V5 Riktat TOPO kloningskit </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 fosfatbuffrade Saline 1x (PBS) </td><td> Invitrogen </td><td> 14.190 </td></tr><tr><td> hexadimetrinbromid </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-rör </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> Fetalt bovint serum (FBS) </td><td> Biochrom AG </td><td> S0115 </td></tr><tr><td> MEM icke-essentiella aminosyror (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-(+)-glukos (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

Proteasom är den viktigaste intracellulära organell involverade i den proteolytiska nedbrytningen av abnorma, felveckade, skadade eller oxiderade proteiner 1, 2. Underhåll av proteasom aktivitet inblandad i många viktiga cellulära processer, såsom cellens stressrespons 3, cellcykelreglering och cellulär differentiering 4 eller immunsystemet 5. Dysfunktion i den ubikitin-proteasom-systemet har kopplats till utvecklandet av tumörer och neurodegenerativa sjukdomar 4, 6. Dessutom observerades en minskning proteasom aktivitet hittades som en funktion av cellulärt åldrande och organismbiologi åldrande 7, 8, 9, 10. Här presenterar vi en metod för att mäta ubiquitin-proteasom aktivitet i levande celler med hjälp av en GFP-DGN fusionsprotein. För att kunna övervaka ubiquitin-proteasom aktivitet i levande primära celler, konstruerar komplementär DNA som kodar för ett grönt fluorescerande protein (GFP)-DGN fusionsprotein (GFP-DGN, unstable) och en variant som bär en ramskiftesmutation (GFP-dgnFS, stabil 11) sätts in i Lentiviral expressionsvektorer. Vi föredrar denna teknik jämfört med traditionella transfektionstekniker eftersom det garanterar en mycket hög transfektionseffektivitet oberoende av celltyp eller ålder hos givaren. Skillnaden mellan fluorescens visas av GFP-dgnFS (stabil) protein och den destabiliserade proteinet (GFP-DGN) i frånvaro eller närvaro av proteasomhämmare kan användas för att uppskatta ubiquitin-proteasom aktivitet i varje enskilt cellstam. Dessa skillnader kan övervakas genom epifluorescensmikroskopi eller kan mätas genom flödescytometri.

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 metod för att övervaka ubiquitin-proteasom aktivitet i levande celler beskrivs. En degron-destabiliserade GFP-(GFP-DGN) och en stabil GFP-dgnFS fusionsprotein genereras och transduceras in i cellen med användning av en Lentiviral expressionsvektor. Denna teknik gör det möjligt att generera en stabil GFP-dgn/GFP-dgnFS uttryckande cellinje i vilken ubiquitin-proteasom aktivitet kan enkelt bedömas med epifluorescens eller flödescytometri.

Övervakning av ubiquitin-proteasom aktivitet i levande celler Använda en Degron (DGN)-destabiliseras grönt fluorescerande protein (GFP)-baserad 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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