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 trabajo fue financiado por el Fondo de Investigación de Islandia y la Universidad de Islandia Fondo de Investigación.

1. Transfección El clon molecular está contenida en dos plásmidos, p8XSp5-EGFP y p67r, de 12 kb y 4,5 kb, respectivamente, 9,10. <ol><li> Corte cantidades equimolares de los dos plásmidos, es decir, 4,4 g de p8XSp5-EGFP y 1,6 g de p67r con XbaI y ligar antes de la transfección. </li><li> Para la ligadura, utilice una unidad de Weiss de la ligasa en una reacción de 50 l, y se incuba a 16 ° C durante la noche. </li><li> Cuando la ligadura se haya completado, se incuba la me...

<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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+visna+virus+in+tissue+culture.">Cultivation of visna virus in tissue culture.</a> Arch Gesamte Virusforsch. 10, 368-380 (1960).</li><li>Gudmundsdottir, H. S., Olafsdottir, K., Franzdottir, S. R., 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=Construction+and+characterization+of+an+infectious+molecular+clone+of+maedi-visna+virus+that+expresses+green+fluorescent+protein.">Construction and characterization of an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein.</a> J. Virol. Methods. 168, 98-102 (2010).</li><li>Andresson, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence+and+biological+properties+of+a+pathogenic+proviral+molecular+clone+of+neurovirulent+visna+virus.">Nucleotide sequence and biological properties of a pathogenic proviral molecular clone of neurovirulent visna virus.</a> Virology. 193, 89-105 (1993).</li><li>Skraban, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+mutations+within+39+amino+acids+in+the+envelope+glycoprotein+of+maedi-visna+virus+alter+the+neutralization+phenotype.">Naturally occurring mutations within 39 amino acids in the envelope glycoprotein of maedi-visna virus alter the neutralization phenotype.</a> J. Virol. 73, 8064-8072 (1999).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> American Journal of Hygiene. 27, 493-497 (1938).</li><li>Berkowitz, R. D., Ilves, H., Plavec, I., Veres, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+systems+derived+from+Visna+virus:+analysis+of+virus+production+and+infectivity.">Gene transfer systems derived from Visna virus: analysis of virus production and infectivity.</a> Virology. 279, 116-129 (2001).</li></ol>

La GFP expresar clon molecular de MVV que aquí se presenta debe ser útil para el análisis de las interacciones de la célula huésped y la patogenicidad de MVV tanto in vitro como in vivo. El virus obtenido después de 7-14 días de la replicación, sin duda, ha adquirido algunas mutaciones debido a la inexactitud de la transcriptasa inversa. Sin embargo, hay limitaciones en el uso de este clon como un vector de ciclo único. Una de ellas es que el uso de la clonación se limita a las células de ovejas y cabras de o...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </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 virus (MVV) es un lentivirus de ovejas, causando neumonía intersticial lentamente progresiva y encefalitis 1. Las células diana principal de MVV in vivo son considerados como una de las 2 linaje monocitos. Ciertas cepas de MVV puede replicar en otros tipos celulares, sin embargo, 3,4. La proteína verde fluorescente es un indicador comúnmente utilizado para el estudio de los lentivirus en las células vivas. Hemos insertado el gen EGFP en el gen de la dUTPase de MVV. El gen dUTPase está bien conservada en la mayoría de las cepas de lentivirus de ovejas y cabras y se ha demostrado que es importante en la replicación de CAEV 5. Sin embargo, dUTPase ha demostrado ser prescindible para la replicación del clon molecular de MVV utilizado en este estudio, tanto in vitro como in vivo 6. Replicación de MVV se limita estrictamente a las células de oveja o de cabra de origen. Nosotros usamos una línea celular primaria del plexo coroideo de oveja (células SCP) para la transfección y la propagación del virus de la 7. La MVV fluorescente es totalmente infecciosa y la expresión EGFP es estable durante al menos seis pasajes 8. Existe una buena correlación entre las mediciones de DICT 50 y EGFP. Este virus por lo tanto, debe ser útil para la detección rápida de las células infectadas en los estudios de patogenicidad y tropismo celular 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

Se describe un clon molecular del virus Maedi-visna que expresa GFP y es totalmente contagioso. La replicación de este virus puede ser detectado mediante el uso de microscopía de fluorescencia y citometría de flujo.

Propagación y detección de un clon infeccioso molecular de Maedi-Visna virus que expresa la 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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