Name: propagating and detecting an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein
Uploaded: 2011-10-09T14:00:00
Description: Watch this Scientific Journal Video about Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein at JoVE.com

Questo lavoro è stato finanziato dal Fondo di ricerca islandese e l&#39;Università di Fondo Islanda ricerca.

1. Trasfezione Il clone molecolare è contenuto in due plasmidi, p8XSp5-EGFP e p67r, di 12 kb e 4,5 kb rispettivamente. 9,10 <ol><li> Tagliare quantità equimolare di due plasmidi, ovvero 4,4 mg di p8XSp5-EGFP e 1,6 mg di p67r con XbaI e legare prima di trasfezione. </li><li> Per la legatura, usare 1 unità Weiss di ligasi in una reazione 50 microlitri e incubare a 16 ° C durante la notte. </li><li> Quando la legatura è completata, incubare la miscela legatura a 65 ° C per 15...

<ol>
	<li>Sigurdsson, B., Palsson, P. A., Grimsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visna,+a+demyelinating+transmissable+disease+of+sheep.">Visna, a demyelinating transmissable disease of sheep.</a> J. Neuropathol. Exp. Neurol. 14, 389-403 (1957).</li><li>Gorrell, M. D., Brandon, M. R., Sheffer, D., Adams, R. J., Narayan, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovine+lentivirus+is+macrophagetropic+and+does+not+replicate+productively+in+T+lymphocytes.">Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes.</a> J. Virol. 66, 2679-2688 (1992).</li><li>Andresdottir, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+and+genetic+differences+between+lung-+and+brain-derived+isolates+of+maedi-visna+virus.">Biological and genetic differences between lung- and brain-derived isolates of maedi-visna virus.</a> Virus Genes. 16, 281-293 (1998).</li><li>Georgsson, G., Houwers, D. J., Palsson, P. A., Petursson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+viral+antigens+in+the+central+nervous+system+of+visna-infected+sheep:+an+immunohistochemical+study+on+experimental+visna+induced+by+virus+strains+of+increased+neurovirulence.">Expression of viral antigens in the central nervous system of visna-infected sheep: an immunohistochemical study on experimental visna induced by virus strains of increased neurovirulence.</a> Acta Neuropathol. 77, 299-306 (1989).</li><li>Turelli, P., Guiguen, F., Mornex, J. F., Vigne, R., Querat, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dUTPase-minus+caprine+arthritis-encephalitis+virus+is+attenuated+for+pathogenesis+and+accumulates+G-to-A+substitutions.">dUTPase-minus caprine arthritis-encephalitis virus is attenuated for pathogenesis and accumulates G-to-A substitutions.</a> J. Virol. 71, 4522-4530 (1997).</li><li>Petursson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visna+virus+dUTPase+is+dispensable+for+neuropathogenicity.">Visna virus dUTPase is dispensable for neuropathogenicity.</a> J. Virol. 72, 1657-1661 (1998).</li><li>Sigurdsson, B., Thormar, H., Palsson, P. 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La GFP che esprimono clone molecolare di MVV qui presentato dovrebbe essere utile per analizzare le interazioni della cellula ospite e la patogenicità dei MVV sia in vitro che in vivo. Il virus ottiene dopo 7-14 giorni di replica ha indubbiamente acquisito alcune mutazioni a causa della inesattezza della trascrittasi inversa. Tuttavia, ci sono limitazioni per l&#39;utilizzo di questo clone come vettore singolo ciclo. Una è che l&#39;uso del clone è limitata alle cellule di pecora e capra origine. L&#39;efficienza di ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 10938025 </td></tr><tr><td> Opti-MEM </td><td> Gibco </td><td> 51985018 </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668019 </td></tr></tbody></table>

propagating and detecting an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein

Maedi-Visna virus (MVV) è un lentivirus di pecore, lentamente progressiva che causa la polmonite interstiziale e l&#39;encefalite 1. Le cellule bersaglio primario della MVV in vivo sono considerate dei 2 lignaggio monociti. Alcuni ceppi di MVV può replicare in altri tipi cellulari, ma 3,4. La proteina verde fluorescente è un marker comunemente usato per lo studio lentivirus nelle cellule viventi. Abbiamo inserito il gene EGFP nel gene per dUTPase di MVV. Il gene dUTPase è ben conservato in ceppi più lentivirus di pecore e capre e ha dimostrato di essere importante nella replicazione del CAEV 5. Tuttavia, dUTPase ha dimostrato di essere indispensabili per la replica del clone molecolare di MVV usato in questo studio sia in vitro che in vivo 6. Replica MVV è strettamente limitata alle cellule di pecora o capra origine. Noi usiamo una linea principale di cellule dal plesso coroideo di pecore (cellule SCP) per la trasfezione e la propagazione del virus 7. La MVV fluorescente è completamente infettive e di espressione EGFP è stabile per almeno 6 passaggi 8. C&#39;è una buona correlazione tra le misurazioni di TCID 50 e EGFP. Questo virus dovrebbe quindi essere utile per il rilevamento rapido delle cellule infette negli studi di tropismo cellulare e patogenicità in vitro e in vivo 8.

Watch this Scientific Journal Video about Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein at JoVE.com

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Descriviamo un clone molecolare di Maedi-Visna virus che esprime GFP ed è completamente infettive. Replicazione di questo virus possono essere rilevati tramite microscopio a fluorescenza e citometria a flusso.

Di moltiplicazione e di Rilevamento di un clone infettive molecolare di Maedi-Visna Virus che esprime la proteina fluorescente verde

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

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