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

Este trabalho foi financiado pelo Fundo de Pesquisa da Islândia e da Universidade da Islândia Fundo de Investigação.

1. Transfecção O clone molecular está contida em dois plasmídeos, p8XSp5-EGFP e p67r, de 12 kb e 4,5 kb, respectivamente. 9,10 <ol><li> Corte quantidades equimolares dos dois plasmídeos, ou seja, 4,4 mg de p8XSp5-EGFP e 1,6 mcg de p67r com XbaI e ligar antes de transfecção. </li><li> Para a ligadura, use uma unidade de Weiss de ligase em uma reação de 50 ul e incubar a 16 ° C durante a noite. </li><li> Quando a ligadura é completada, incubar a mistura de ligadura a 65...

<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. 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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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A GFP expressar clone molecular de MVV aqui apresentados deve ser útil para analisar as interações da célula hospedeira e patogenicidade do MVV tanto in vitro como in vivo. O vírus obtidos após 7-14 dias de replicação, sem dúvida, adquiriu algumas mutações devido à imprecisão da transcriptase reversa. No entanto, existem limitações para o uso deste clone como um vetor único ciclo. Uma delas é que o uso do clone é restrita a células de ovelhas e cabras de origem. A eficiência de transfecção destas c...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </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 (MVV) é um lentivírus de ovelhas, fazendo com que lentamente progressiva pneumonia intersticial e encefalite 1. As células-alvo primário do MVV in vivo são consideradas das duas linhagem de monócitos. Determinadas estirpes de MVV pode replicar em outros tipos de células, no entanto 3,4. A proteína verde fluorescente é um marcador comumente usado para estudar lentivírus em células vivas. Temos inserido o gene EGFP no gene para dUTPase de MVV. O gene dUTPase está bem conservado em cepas mais lentivírus de ovinos e caprinos e tem se mostrado importante na replicação do CAEV 5. No entanto, dUTPase foi mostrado para ser dispensáveis ​​para a replicação do clone molecular de MVV utilizados neste estudo in vitro e in vivo 6. Replicação MVV é estritamente confinada às células de ovelhas ou cabras origem. Nós usamos uma linhagem de células primárias do plexo coróide de ovelhas (células SCP) para transfecção e propagação do vírus 7. O MVV fluorescentes é totalmente infecciosas e expressão EGFP é estável durante pelo menos seis passagens 8. Há boa correlação entre as medidas de TCID 50 e EGFP. Este vírus deve ser útil para a detecção rápida de células infectadas em estudos de tropismo celular e patogenicidade 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

Nós descrevemos um clone molecular da Maedi-visna vírus que expressa a GFP e é totalmente infecciosas. Replicação deste vírus pode ser detectado por meio de microscopia de fluorescência e citometria de fluxo.

Propagação e detecção de um Clone Infecciosas Molecular da Maedi-visna vírus que expressa a proteína 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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