Overvågning Cell-selvstændige døgnrytmen ur Rhythms of Gene Expression Brug Luciferase bioluminescens Reporters
Summary
Døgnrytmen ure fungerer i de enkelte celler, dvs, de er celle-autonome. Her beskriver vi metoder til at generere celle-autonome ur modeller bruger ikke-invasiv, luciferase-baseret real-time bioluminescens teknologi. Reporter celler giver medgørlig, funktionelle modelsystemer til at studere døgnrytmen biologi.
Abstract
Hos pattedyr, er mange aspekter af adfærd og fysiologi, såsom søvn-vågne cyklus og lever metabolisme reguleres af endogene døgnrytmen ure (revideret 1,2). Den circadian tidtagning systemet er en hierarkisk multi-oscillator netværk med den centrale ur placeret i suprachiasmatic kerne (SCN) at synkronisere og koordinere ekstra-SCN og perifere ure andetsteds 1,2. Individuelle celler er de funktionelle enheder til generering og vedligeholdelse af cirkadiske rytmer 3,4, og disse oscillatorer i forskellige vævstyper i organismen deler en bemærkelsesværdigt ens biokemisk negativ feedback-mekanisme. Men på grund af vekselvirkninger på det neurale netværk niveau i SCN og gennem rytmiske, systemiske tidskoder på organismal niveau, er døgnrytmen på organismal niveau ikke nødvendigvis celle-autonome 5-7. Sammenlignet med traditionelle undersøgelser af lokomotorisk aktivitet in vivo og SCN eksplantater ex vivo, cell-baserede in vitro assays mulighed for opdagelsen af celle-autonome cirkadiske defekter 5,8. Strategisk celle-baserede modeller er mere eksperimentelt medgørlig for fænotypisk karakterisering og hurtig opdagelse af grundlæggende ur mekanismer 5,8-13.
Fordi døgnrytmer er dynamiske, er langsgående målinger med høj tidsopløsning nødvendig for at vurdere urfunktion. I de senere år har real-time bioluminescens optagelse ved hjælp ildflueluciferase som journalist blevet en fælles teknik til at studere døgnrytmen hos pattedyr 14,15, da det giver mulighed for undersøgelse af persistens og dynamik af molekylære rytmer. At overvåge celle-selvstændige døgnrytmer for genekspression, kan luciferase reportere indføres i celler via transient transfektion 13,16,17 eller stabil transduktion 5,10,18,19. Her beskriver vi en stabil transduktion protokollen med lentivirus-medieret genlevering. THan lentiviral vector system er overlegen i forhold til traditionelle fremgangsmåder, såsom forbigående transfektion og kimcellelinjetransmission grund af dens effektivitet og alsidighed: den tillader effektiv levering og stabil integration i værtsgenomet af både delende og ikke-delende celler 20. Når et reporter-cellelinien er etableret, kan dynamikken i urfunktion undersøges gennem bioluminescens optagelse. Vi først beskrive generation af P (Per2)-d Luc reporter linjer, og derefter præsentere data fra denne og andre cirkadiske journalister. I disse assays er 3T3-musefibroblaster og U2OS humane osteosarcomceller anvendes som cellulære modeller. Vi diskuterer også forskellige måder at bruge disse ur modeller i cirkadiske undersøgelser. Fremgangsmåder beskrevet her kan anvendes til en lang række celletyper til at studere cellulære og molekylære basis for døgnrytmen ure, og kan være nyttig i løse problemer i andre biologiske systemer.
Protocol
Discussion
1. Ændringer af nuværende protokol
1,1 Optagelse enheder og gennemløb overvejelser
På grund af sin kommercielle tilgængelighed har LumiCycle (Actimetrics) blevet den mest almindeligt anvendte automatiserede luminometer enhed for real-time optagelse 4,5,9,19,29-31. The LumiCycle anvender fotomultiplikatorrør (PMT'er), som lysdetektorer, der giver ekstremt høj følsomhed og lav støj 14, og derfor er særlig egnet til dataregistreringen …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet delvist af National Science Foundation (IOS-0.920.417) (ACL).
Materials
| Name of reagent | Company | Catalogue number | Comments |
| DMEM | HyClone | SH30243FS | For regular cell growth |
| DMEM | Invitrogen | 12100-046 | For luminometry |
| FBS | HyClone | SH3091003 | |
| Pen/Strep/Gln(100x) | HyClone | SV3008201 | |
| B-27 | Invitrogen | 17504-044 | |
| D-Luciferin | Biosynth | L-8220 | |
| Poly-L-lysine | Sigma | P4707 | |
| Polybrene | Millipore | TR-1003-G | |
| Forskolin | Sigma | F6886 | |
| All other chemicals | Sigma | ||
| Equipment | |||
| Tissue culture incubator | 5% CO2 at 37°C | ||
| Tissue culture hood | BSL-2 certified | ||
| Light & fluorescent microscope | Phase contrast optional | ||
| LumiCycle | Actimetrics |
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