Name: a primary neuron culture system for the study of herpes simplex virus latency and reactivation
Uploaded: 2012-04-02T13:00:00
Description: Watch this Scientific Journal Video about A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation at JoVE.com

Vi takker de anmeldere for deres gennemtænkte forslag, som har bidraget til at forbedre dette manuskript. Dette arbejde blev støttet af tilskud til MVC (NS21072, HD23315), ACW (GM61139, S10RR017970) og IM (AI073898, GM056927) fra NIH. MK blev delvist understøttet af en NIH uddannelse tilskud (5T32 AI007180).

1. Isolering og dyrkning af SCG-neuroner fra rotte fostre At give en nyttig baggrund for at forstå denne protokol, og for en omfattende diskussion af tidligere litteratur, etablerede metoder til SCG neuron kultur, herunder grundlaget for SCG in vitro-kultur, plade-belægningssubstrater, og de ​​komponenter i serum-frie medier, læseren henvises til referencer 2-4. <ol><li> Brugen af rotter som en kilde til SCG-neuroner blev gennemført i overensstemmelse med NIH re...

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Denne primære neuron kultur og infektion system giver en enkel og effektiv metode til at udforske de molekylære mekanismer, der ligger til grund for HSV-1 ventetid og reaktivering. Systemet trofast rekapitulerer de accepterede kendetegnende for ventetid er defineret i både infektioner hos mennesker og i live-dyremodeller. Når viruset er latent i SCG kulturer, kan infektiøse partikler og virale lytiske genprodukter ikke blive detekteret. Den eneste virale gen-produkt fundet i latent inficerede neuro...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalog # </td><td> Kommentarer </td></tr><tr><td> 70 um nylonfilter (cellefilter) </td><td> BD Biosciences </td><td> 352350 </td><td></td></tr><tr><td> 1x Hanks Balanced Salt Solution (HBSS - / -) </td><td> Invitrogen </td><td> 14175 </td><td> w / o CaCl2 og MgCl2 </td></tr><tr><td> 1x Minimum Essential Media (MEM) </td><td> Invitrogen </td><td> 11095-080 </td><td></td></tr><tr><td> 5-Fluor-2&#39;-deoxyuridin </td><td> Sigma </td><td> F0503 </td><td> fremstilling 20 mM stamopløsning i 1X MEM; opbevares ved -20 ° C </td></tr><tr><td> 96-brønds flade brøndbunden TC-plader </td><td> Corning </td><td> 3599 </td><td></td></tr><tr><td> Acyclovir </td><td> &lt;em&gt; Calbiochem </td><td> 114798 </td><td> forberede 31 mM stamopløsning i DMSO, opbevares ved -20 ° C </td></tr><tr><td> Aphidicolin </td><td> Calbiochem </td><td> 178273 </td><td> fremstilling 10 mM stamopløsning i DMSO, opbevares ved -20 ° C </td></tr><tr><td> B-27 Supplement </td><td> Invitrogen </td><td> 17504-44 </td><td></td></tr><tr><td> Collagenase </td><td> Sigma </td><td> C2674 </td><td> fremstille 10 mg / ml stamopløsning i HBSS - / -, opbevares ved -20 ° C </td></tr><tr><td> D-(+)-glucose </td><td> Sigma </td><td> G6152 </td><td> forberede 40% aktier i H 2 O; Filtersteriliser &amp; opbevares ved 4 ° C </td></tr><tr><td> L-glutamin </td><td> Invitrogen </td><td> 25030-081 </td><td></td></tr><tr><td> Laminin </td><td> Sigma </td><td> L2020 </td><td> forberede 1 mg / ml stamopløsning i H 2O; quick-freeze 20 pialiquats og opbevares ved -80 ° C; fortyndet til 2 mg / ml bearbejdning konc. i sterilt H2O </td></tr><tr><td> Leibovit&#39;z L-15 medier </td><td> Invitrogen </td><td> 11415 </td><td></td></tr><tr><td> Nerve Growth Factor </td><td> Harlan Laboratories </td><td> BT.5017 </td><td> fremstilling 50 ug / ml stamopløsning i HBSS - / -, opbevares ved -80 ° C </td></tr><tr><td> Neurobasal medium </td><td> Invitrogen </td><td> 12348 </td><td></td></tr><tr><td> Phosphonoeddikesyre (PAA) </td><td> Sigma </td><td> P6909 </td><td> forberede 75 mg / ml stamopløsning i H 2 O, skal opbevares ved -20 ° C </td></tr><tr><td> Poly-D-lysin-hydrobromid </td><td> Sigma </td><td> P0899 </td><td> fremstille 20 mg / ml stamopløsning i H2O; opbevares ved -20 ° C </td></tr><tr><td> Rotte-hale collagen </td><td> Millipore </td><td> 08-115 </td><td>Koncentration varierer med udbuddet parti; opbevares ved 4 ° C og fortyndet til 0,66 mg / ml arbejde conc. i sterilt H2O </td></tr><tr><td> Trichostatin A </td><td> Sigma </td><td> T8552 </td><td> fremstilling 1 mM stamopløsning i DMSO, opbevares ved -20 ° C </td></tr><tr><td> Trypsin 2,5% </td><td> Invitrogen </td><td> 15090-04 </td><td></td></tr></tbody></table>

a primary neuron culture system for the study of herpes simplex virus latency and reactivation

Herpes simplex virus type-1 (HSV-1) indfører en livslang latent infektion i perifere neuroner. Denne latente reservoir er kilden til tilbagevendende genaktiveringssignaler begivenheder at sikre transmission og bidrage til klinisk sygdom. Aktuelle antivirale ikke påvirke latente reservoiret og der er ingen vacciner. Medens de molekylære detaljer lytisk replikation er velkarakteriserede, mekanismer, der styrer latens i neuroner forbliver vanskeligt. Vores nuværende forståelse af latens er afledt fra in vivo-undersøgelser med små dyremodeller, der er nødvendige for at definere virusgenprodukter krav og den rolle i immunresponser. Men det er umuligt at skelne specifikke virkninger på virus-neuron forhold fra mere generelle virkninger af infektion medieret af immune eller ikke-neuronal bæreceller i levende dyr. Hertil kommer, er dyreforsøg dyrt, tidskrævende, og begrænset i form af tilgængelige muligheder for at manipulere værtprocesser. For at overvinde disse begrænsninger er en neuron-blot-systemet desperat behov, der gengiver in vivo egenskaber latens og reaktivering men giver fordelene ved vævsdyrkning med hensyn til ensartethed og tilgængelighed. Her præsentere et in vitro-model under anvendelse af dyrkede primære sympatiske neuroner fra rotte superior cervikal ganglie (SCG) (figur 1) for at studere HSV-1 latens og reaktivering, der passer bedst, hvis ikke alle de ønskede kriterier. Efter at eliminere ikke-neuronale celler, er nær-homogene TrkA + Neuron kulturer inficeret med HSV-1 i nærvær af acyclovir (ACV) for at undertrykke lytisk replikation. Efter ACV fjernelse af ikke-produktive HSV-1-infektioner, som trofast udviser accepterede kendetegnende for ventetid er effektivt etableret. Især lytiske mRNA&#39;er, proteiner og infektiøs virus bliver påvises, selv i fravær af selektion, men latens-associeret transkript (LAT) udtrykkerion fortsætter i neuronal kerner. Virusgenomer holdes ved et gennemsnitligt antal kopier af 25 per neuron og kan induceres til produktivt replikere ved at interferere med PI3-kinase / Akt signalering eller simple tilbagetrækning af nervevækstfaktor 1. Et rekombinant HSV-1 koder for EGFP fusioneret til det virale lytiske proteinet Us11 tilvejebringer et funktionelt realtid markør for replikation skyldes reaktivering, der let kvantificeres. Ud over kemiske behandlinger kan genetiske metoder, såsom RNA-interferens eller genlevering via lentivirale vektorer anvendt med succes til systemet tillader mekanistiske undersøgelser, der er meget vanskelige, om ikke umuligt, i dyr. Sammenfattende giver SCG-baserede HSV-1 latency / reaktivering systemet en kraftfuld, nødvendigt redskab til at opklare de molekylære mekanismer, der styrer HSV1 ventetid og reaktivering i neuroner, et langvarigt puslespil i virologi hvis løsning kan tilbyde friske indsigt i udvikling af nye behandlingsformer, der Målet tHan latent herpesvirus reservoir.

Watch this Scientific Journal Video about A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation at JoVE.com

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Protokollen beskriver en effektiv og reproducerbar model til undersøgelse herpes simplex-virus type 1 (HSV-1) latens og reaktivering. Assayet anvender homogene sympatiske neuron kulturer og tillader den molekylære dissektion af virus-neuron interaktioner med en række værktøjer, herunder RNA-interferens og ekspression af rekombinante proteiner.

En primær Neuron Kultur System for Studiet af Herpes Simplex Virus Latency og genaktivering

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

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siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

siRNA elektroporation at Modutere Autophagy i herpes simplex virus type 1-inficerede Monocyte-afledte dendritiske celler

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...