Cette recherche a été soutenue par l&#39;Office of Science (BER), Département américain de l&#39;Énergie, Accord de coopération non. De-FC02-02ER63453, la National Science Foundation Programme de séquençage du génome microbien (numéros 0,626,826 et 0,731,916 prix). Nous remercions John Glass pour son expertise technique et des conseils et K. Eric Wommack pour son aide dans la collecte des échantillons environnementaux.

1. Préparation des solutions Avant d&#39;effectuer la chromatographie d&#39;hydroxyapatite, des tampons phosphates doivent être préparés et l&#39;hydroxyapatite doivent être correctement hydraté. <ol><li> Solution de phosphate 1M, pH 6,8: Dans un flacon 1L dissoudre 119.98g de phosphate de sodium monobasique dans 1L d&#39;stérile, traitée par DEPC H 2 O. Préparer un phosphate de sodium dibasique 1M solution dans un flacon de 1L en dissolvant 141.96g de phosphate de sodium dibasique dans 1L d&#39;stérile, traitée par DEPC H 2 O. Combinez les mono-et di-solutions à un ratio de 1:1. Flacon Placer sur une plaque d&#39;agitation et mélanger à l&#39;aide d&#39;un barreau magnétique. Ajuster le pH à 6,8 par addition d&#39;hydroxyde de sodium (pour augmenter le pH) ou l&#39;acide phosphorique (pour diminuer le pH). </li><li> Tampon phosphate 0,12 M: Dans un flacon de 500ml, mélanger 30 ml de solution de phosphate 1M, pH 6,8, 2,5 ml de 10% de sodium dodécyl sulfate (SDS), et 5 ml de 0,5 M EDTA. Ajouter stérile, traitée par DEPC H2O à un volume de 225ml. Ajuster le pH à 6,8. Ajouter stérile, traitée par DEPC H2O 250ml. Autoclave. </li><li> Tampon phosphate 0,18 M: Dans un flacon de 500ml, mélanger 45 ml de solution de phosphate 1M, pH 6,8, 2,5 ml de 10% de sodium dodécyl sulfate (SDS), et 5 ml de 0,5 M EDTA. Ajouter stérile, traitée par DEPC H 2 O pour un volume de 225ml. Ajuster le pH à 6,8. Ajouter stérile, traitée par DEPC H 2 O pour 250ml. Autoclave. </li><li> Tampon phosphate 0,20 M: Dans un flacon de 500ml, mélanger 50 ml de solution de phosphate 1M, pH 6,8, 2,5 ml de 10% de sodium dodécyl sulfate (SDS), et 5 ml de 0,5 M EDTA. Ajouter stérile, traitée par DEPC H 2 O pour un volume de 225ml. Ajuster le pH à 6,8. Ajouter stérile, traitée par DEPC H 2 O pour 250ml. Autoclave. </li><li> 0,40 M de tampon phosphate: dans un flacon de 500ml, 100ml de combiner de solution de phosphate 1M, pH 6,8, 2,5 ml de 10% de sodium dodécyl sulfate (SDS), et 5 ml de 0,5 M EDTA. Ajouter stérile, traitée par DEPC H 2 O pour un volume de 225ml. Ajuster le pH à 6,8. Ajouter stérile, traitée par DEPC H 2 O pour 250ml. Autoclave. </li><li> &quot;Tampon phosphate 1,00 M&quot; (concentration réelle de phosphate dans cette solution est 0,91 M): Dans un flacon de 500ml, mélanger 30 ml de solution de phosphate 1M, pH 6,8, 2,5 ml de 10% de sodium dodécyl sulfate (SDS), et 5 ml de 0,5 M d&#39;EDTA. Ajouter stérile, traitée par DEPC H2O à un volume de 225ml. Ajuster le pH à 6,8. Ajouter stérile, traitée par DEPC H 2 O pour 250ml. Autoclave. </li><li> Hydratation Hydroxyapatite: Poids à 1g d&#39;hydroxyapatite et d&#39;ajouter à un tube de 50ml. Ajouter 6ml de tampon phosphate 0,12 M à l&#39;hydroxyapatite et renverser pour mélanger. Avant d&#39;utiliser porter la température à 60 ° C et bien mélanger. Store pour les longues périodes de temps à 4 ° C. </li><li> Avant d&#39;utiliser, à équilibrer tous les tampons phosphate et d&#39;hydroxyapatite hydratés à 60 ° C. </li></ol> 2. Préparation de Econo-Colonne <ol><li> Fermer le robinet. Rincez plusieurs fois la colonne désionisée H 2 O par les reprises inversant la colonne et la décantation. </li><li> Retirer le robinet de l&#39;Econo-colonne et la colonne de l&#39;autoclave pour stériliser. </li><li> Remplacer et fermer le robinet. Ajouter 1ml de Sigmacote à l&#39;stériles Econo-colonne et manteau de toutes les surfaces vitrées. Ouvrir le robinet et décanter. </li><li> Fixez colonne pour un bain d&#39;eau circulant et régler la température à 60 ° C. Pour einsure le moins de perte de chaleur à travers la colonne de la colonne d&#39;emballage dans un agent isolants tels que du papier d&#39;aluminium ou d&#39;isolation des tuyaux en mousse est recommandé. </li><li> Rincer la colonne de Econo-deux fois avec stérile, exempt de RNase H 2 O puis deux fois avec stérile, sans RNase tampon phosphate 0,12 M. </li></ol> 3. Chromatographie Hydroxypaptite <ol><li> S&#39;assurer que le robinet est fermé, ajoutez lentement 2 ml de pré-préparés, l&#39;hydroxyapatite en suspension au fond de l&#39;Econo-colonne en utilisant une pipette stérile 2ml sérologique. </li><li> Autoriser l&#39;hydroxyapatite de s&#39;installer sous la force de gravité pendant 30 minutes. </li><li> Égoutter tampon de colonne et robinet à proximité. Combinez les acides nucléiques avec un tampon phosphate 0,12 M dans un volume final de 500 pl et incuber à 60 ° C pendant 10 minutes dans un bloc chauffant ou un bain d&#39;eau. </li><li> D&#39;appliquer rapidement les acides nucléiques préparés dans un tampon phosphate 0,12 M à l&#39;hydroxyapatite aide d&#39;une pipette sérologique de 2 ml en veillant à ne pas perturber la colonne. </li><li> Autoriser les acides nucléiques de se lier à l&#39;hydroxyapatite pendant 30 minutes. </li><li> Placer un tube 15ml sous la colonne, ouvrir le robinet et recueillir l&#39;échantillon initial contenant ADNsb. Fermer le robinet. </li><li> Ajouter 6ml de tampon phosphate 0,12 M à l&#39;hydroxyapatite en veillant à ne pas perturber la colonne, ouvrir le robinet et recueillir l&#39;ADN simple brin dans le tube de 15ml de l&#39;étape précédente. Fermer le robinet. </li><li> Ajouter 1 volume d&#39;un phénol: chloroforme: alcool isoamylique (25:25:1 v / v / v) dans le tube, mélanger vigoureusement et centrifuger à 3500 xg pendant 15 minutes. Transférer le surnageant contenant ADNss dans un nouveau tube 15ml. </li><li> Répétez 3,76 et 3,87 en utilisant 6ml de tampon phosphate 0,20 M à l&#39;eluth et ARN purifier de la colonne en veillant à utiliser un nouveau tube 15ml pour la collecte. </li><li> Répétez 3,76 et 3,87 en utilisant 6ml de tampon phosphate 0,40 m pour éluer et purifier ADNdb de la colonne en veillant à utiliser un nouveau tube 15ml pour la collecte. </li><li> Répétez 3,76 et 3,87 en utilisant 6ml de tampon phosphate 1,00 M à la bande de la colonne de toute ADNdb résiduelle en veillant à utiliser un nouveau tube 15ml pour la collecte. </li></ol> 4. Dessalage échantillons d&#39;acide nucléique <ol><li> Transfert 4-5ml d&#39;échantillon à une Amicon Ultra-4 dispositif centrifuge de filtre équipé d&#39;un poids moléculaire de 30 000 coupé la membrane Ultracel. </li><li> Concentré échantillon à &lt;500μl par centrifugation à 6000 xg, 30 ° C, à 5 minutes (ou jusqu&#39;à ce que le volume concentré est atteint) dans un rotor à angle fixe. Jeter les traverser. </li><li> Ajouter le reste de l&#39;échantillon (s) pour Amicon Ultra-4 dispositif de filtre centrifuge et porter le volume jusqu&#39;à 4 à 5 ml avec du tampon 1X RNAse TE. </li><li> Concentré échantillon à &lt;500μl par centrifugation à 6000 xg, 30 ° C, à 5 minutes (ou jusqu&#39;à ce que le volume concentré est atteint) dans un rotor à angle fixe. Jeter les traverser. </li><li> Ajouter 4 à 5 ml de RNAse tampon 1X TE Amicon Ultra-4 dispositif de filtre centrifuge. Concentré échantillon à &lt;500μl par centrifugation à 6000 xg, 30 ° C, à 5 minutes (ou jusqu&#39;à ce que le volume concentré est atteint) dans un rotor à angle fixe. Jeter les traverser. </li><li> Répétez l&#39;étape 4.5 un supplément de 5 fois pour dessaler complètement échantillon d&#39;acide nucléique (s). </li><li> Transférer l&#39;échantillon restant (s) dans un tube stérile 1.7ml Eppendorf. Ajouter 1/10ème du volume d&#39;acétate de sodium 3M, pH 7,0, deux volumes d&#39;éthanol 100%, et 1 microlitre d&#39;Glycoblue. Mélangez bien au vortex. </li><li> Centrifuger à 28000 xg, 4 ° C, 60 minutes. Décanter veillant à ne pas perturber le culot. Ajouter 300μl d&#39;éthanol à 70%. Centrifuger à 28000 xg température ambiante, pendant 10 minutes. Décanter, sec et remettre chaque fraction dans un volume approprié de RNase tampon TE. </li></ol> 5. Les résultats représentatifs: La figure 1 récapitule les méthodes présentées pour l&#39;utilisation de la chromatographie d&#39;hydroxyapatite de fractionner ADNsb, ADNdb et des acides nucléiques ARN à partir d&#39;un assemblage mixte virale. Cette méthode exploite l&#39;interaction entre la charge de squelette phosphate chargé négativement des acides nucléiques et les chargés positivement ions Ca 2 + présent dans l&#39;hydroxyapatite et permet le fractionnement efficace de sous-types d&#39;acides nucléiques (ADN simple brin, ADN double brin et ARN) avec des concentrations croissantes de tampon phosphate 12. Le fractionnement d&#39;hydroxyapatite in vitro &quot;communauté&quot; des ADNss connue (M13mp18), ADNdb (lambda) et l&#39;ARN (MS2 et phi6) génomes viraux est illustrée dans la Figure 2. Les acides nucléiques ont été combinées à des concentrations égales, appliquées à la colonne d&#39;hydroxyapatite et ont été élues en utilisant une concentration croissante de tampon phosphate. Chaque sous-type acide nucléique élue indépendante des autres avec moins de 9% le report d&#39;une fraction à la suivante. L&#39;application de cette technique pour les acides nucléiques isolés à partir d&#39;une communauté virale recueillies auprès de la baie de Chesapeake est montré dans la figure 3. Le rendement total d&#39;acide nucléique isolé à partir de virus purifié a été appliquée à la colonne d&#39;hydroxyapatite. Matériel génomique composé d&#39;ADN simple brin, l&#39;ARN et ADN double brin a été éluée avec 0,12 M de manière indépendante, et les concentrations de 0,20 M de tampon phosphate 0.40M/1.00M respectivement. ADN double-brin est élue à la concentration de phosphate de 0.40m et 1.00m et dans ce cas, domine cette communauté virale par rapport à génotypes ADNsb et l&#39;ARN. Cette observation est cohérente avec la composition de la communauté virale attendue des milieux marins et estuariens où la majorité des acides nucléiques sont récupérables ADNdb 13. <img src="/files/ftp_upload/3146/3146fig1.jpg" alt="Figure 1"/> Schéma Figure 1. De la méthode de chromatographie d&#39;hydroxyapatite pour le fractionnement des acides nucléiques. Un mélange de ADNsb, ADNdb et l&#39;ARN préparés dans un tampon phosphate 0,12 M est chauffé à 60 ° C et appliqués à une colonne d&#39;hydroxyapatite maintenu à une constante de 60 ° C en utilisant un bain d&#39;eau circulant. Les acides nucléiques (ADN simple brin, l&#39;ARN et ADN double brin) sont élues de la hydroxyapatite avec des concentrations croissantes d&#39;un tampon phosphate contenant. Ce chiffre a été reproduite avec la permission de la Société américaine de microbiologie et a été initialement présenté dans Andrews-Pfannkoch et al. 2010 12. <img src="/files/ftp_upload/3146/3146fig2.jpg" alt="Figure 2"/> Figure 2. Séparation des acides nucléiques viraux connus en utilisant la chromatographie d&#39;hydroxyapatite. Des concentrations égales de M13mp18 ADNsb, MS2 ssRNA, phi6 ARNdb et lambda ADNdb (voies 2-5, respectivement) ont été combinés (piste 6) et appliqués à une colonne d&#39;hydroxyapatite. Single-brin d&#39;ADN (M13mp18), l&#39;ARN (MS2/phi6) et ADNdb (lambda) étaient indépendamment éluée de la colonne d&#39;hydroxyapatite en utilisant 0,12 M (piste 8), 0,18 M (voie 9) et 0.40M/1.00M (voie 10) . Une échelle 1kb (voies 1, 7) a été utilisée pour confirmer tailles de génome. Ce chiffre a été reproduite avec la permission de la Société américaine de microbiologie et a été initialement présenté dans Andrews-Pfannkoch et al. 2010 12. <img src="/files/ftp_upload/3146/3146fig3.jpg" alt="Figure 3"/> Figure 3. Séparation des acides nucléiques viraux isolés à partir d&#39;une communauté au sein de la baie de Chesapeake. Les acides nucléiques ont été isolés et appliqués à une colonne d&#39;hydroxyapatite. ADNss (voie 0,12 M), l&#39;ARN (voie 0,20 M) et ADNdb (voies 0.40M/1.0M) ont été élues en utilisant de manière indépendante des concentrations de tampon phosphate de 0,12 M, 0,20 M et 0.40M/1.00M, respectivement. Échelle Salut l&#39;ADN de masse (voie L1) et Ladder 1kb (piste L2) ont été utilisés pour confirmer visuellement approximative poids moléculaire et la masse des acides nucléiques. Ce chiffre a été reproduite avec la permission de la Société américaine de microbiologie et a été initialement présenté dans Andrews-Pfannkoch et al. 2010 12.

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Aucun conflit d&#39;intérêt déclaré.

La méthodologie présentée ici chromatographie d&#39;hydroxyapatite est un outil très efficace et robuste pour le fractionnement des acides nucléiques à partir des assemblages mixtes virales, lorsque l&#39;objectif est d&#39;étudier la composition des acides nucléiques totaux de la communauté. Généralement, ADNsb, ARN et ADN double brin sera élue de la colonne de concentration pho tampon phosphate supérieure à environ 0,40 m ~ 0et respectivement. Cependant, chaque préparation d&#39;hydroxyapatite ...

<table border="1"><tbody><tr><th> Nom du produit </th><th> Société </th><th> Numéro de catalogue </th><th> Numéro de catalogue </th></tr><tr><td> Econo-Colonne </td><td> Bio-Rad </td><td> 737-0717 </td><td> Package ID 0.7cm / 2 </td></tr><tr><td> Hydroxyapatite </td><td> Bio-Rad </td><td> 130-0520 </td><td> L&#39;ADN année de Bio-Gel HTP Gel, 100g/td&gt; </tr><tr><td> Phosphate de sodium, monobasique </td><td> VWR </td><td> VW1497-01 </td><td> Monohydrate, cristal 500g </td></tr><tr><td> Phosphate de sodium dibasique </td><td> VWR </td><td> VW1496-01 </td><td> Anhydre, poudre de 500g </td></tr><tr><td> Traitée par DEPC eau </td><td> Invitrogen </td><td> AM9922 </td><td> 1L </td></tr><tr><td> 10% Solution SDS </td><td> Invitrogen </td><td> 24730-020 </td><td> UltraPure, 1L </td></tr><tr><td> EDTA 0,5 M </td><td> Invitrogen </td><td> AM9262 </td><td> pH 8,0, 1L </td></tr><tr><td> Sigmacote </td><td> Sigma-Aldrich </td><td> SL2-25ML </td><td></td></tr><tr><td> 2ml sérologique pippette </td><td> VWR </td><td> 89130-884 </td><td> Polystyrène, stérile </td></tr><tr><td> Tubes à centrifuger BD Falcon </td><td> VWR </td><td> 21008-936 </td><td> 15ml, stérile </td></tr><tr><td> Phénol: chloroforme: alcool isoamylique (25:24:1 v / v / v) </td><td> Invitrogen </td><td> 15593-031 </td><td> UltraPure, 100ml </td></tr><tr><td> Amicon Ultra-4 dispositif centrifuge </td><td> Millipore </td><td> UFC803024 </td><td> Ultracel-30 à membrane </td></tr><tr><td> 20X tampon TE, RNAse </td><td> Invitrogen </td><td> T11493 </td><td> 100ml </td></tr><tr><td> Glycoblue </td><td> Invitrogen </td><td> AM9516 </td><td> 15mg/ml </td></tr></tbody></table>

separation of single-stranded dna, double-stranded dna and rna from an environmental viral community using hydroxyapatite chromatography

Les virus, en particulier les bactériophages (phages), sont les entités biologiques les plus nombreux sur 1,2 Terre. Les virus de moduler l&#39;abondance et la diversité de la cellule hôte, contribuent à le cycle des nutriments, de modifier l&#39;hôte phénotype cellulaire, et l&#39;influence de l&#39;évolution de la cellule hôte et communautés virales à travers le transfert latéral des gènes 3. De nombreuses études ont mis en évidence la diversité stupéfiante génétique des virus et leur potentiel fonctionnel dans une variété de milieux naturels. Techniques de métagénomique ont été utilisés pour étudier la diversité taxonomique et fonctionnelle des complexes potentiels assemblages viraux dont les membres contiennent l&#39;ADN simple brin (ADNsb), l&#39;ADN double brin (ADNdb) et les génotypes ARN 4-9. Les protocoles actuels de construction de bibliothèques utilisées pour étudier l&#39;ADN ou d&#39;ARN contenant l&#39;environnement contenant des virus nécessitent un traitement initial nucléase afin d&#39;éliminer les modèles non ciblées 10. Cependant, une compréhension globale de l&#39;effectif du gène collective de la communauté du virus et la diversité du virus nécessite la connaissance de tous les membres indépendamment de la composition du génome. Le fractionnement des sous-types d&#39;acides nucléiques purifiés fournit un mécanisme efficace par lequel l&#39;étude des assemblages virale sans sacrifier une partie de la signature génétique de la communauté. Hydroxyapatite, une forme cristalline du phosphate de calcium, a été employé dans la séparation des acides nucléiques, ainsi que des protéines et des microbes, depuis les années 1960 11. En exploitant l&#39;interaction entre les responsables chargés positivement ions Ca 2 + de l&#39;hydroxyapatite et le squelette phosphate chargé négativement des sous-types d&#39;acides nucléiques, il est possible d&#39;éluer préférentiellement chaque sous-type acide nucléique indépendant des autres. Nous avons récemment utilisé cette stratégie de manière indépendante fractionner les génomes de ADNsb, et de l&#39;ARN-ADN double brin contenant des virus dans la préparation du séquençage de l&#39;ADN 12. Ici, nous présentons une méthode pour le fractionnement et la récupération des ADNsb, ADNdb et l&#39;ARN viral à partir d&#39;acides nucléiques viraux assemblages mixtes utilisant chromatographie d&#39;hydroxyapatite.

Watch this Scientific Journal Video about Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography at JoVE.com

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Nous décrivons une méthode efficace pour séparer l&#39;ADN simple brin, double brin de l&#39;ADN et des molécules d&#39;ARN à partir de l&#39;environnement communautés virales. Les acides nucléiques sont fractionnés par chromatographie d&#39;hydroxyapatite avec des concentrations croissantes de phosphate contenant du tampon. Cette méthode permet l&#39;isolement de tous les types d&#39;acides nucléiques viraux dans des échantillons environnementaux.

Séparation de l&#39;ADN simple brin, double brin de l&#39;ADN et l&#39;ARN à partir d&#39;un environnement communautaire viral en utilisant chromatographie d&#39;hydroxyapatite

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

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31.4K vues.  The J. Craig Venter Institute. Nous décrivons une méthode efficace pour séparer l'ADN simple brin, double brin de l'ADN et des molécules d'ARN à partir de l'environnement communautés virales. Les acides nucléiques sont fractionnés par chromatographie d'hydroxyapatite avec des concentrations croissantes de phosphate contenant du tampon. Cette méthode permet l'isolement de tous les types d'acides nucléiques viraux dans des échantillons environnementaux.

Vidéo: Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Génération de virus recombinants de la grippe des ADN plasmidique

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

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Immunologie et infection

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

Isolement des variantes des virus à ARN de Fidelity et caractérisation de la fréquence de mutation de virus

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

Développement d&#39;un type de cellule spécifique anti-VIH gp120 aptamères pour la livraison de siRNA

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Préparation de l&#39;ADN viral à partir des nucléocapsides

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Un protocole Chelex simple pour l&#39;extraction d&#39;ADN à partir de<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Mise en place d&#39;un<em&gt; In vitro</em&gt; Système d&#39;étudier le comportement intracellulaire de<em&gt; Candida glabrata</em&gt; En droits macrophages THP-1

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Utilisation de Click Chemistry pour mesurer l&#39;effet de l&#39;infection virale sur Host-Cell Synthèse des ARN

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Moyenne de la glycoprotéine d&#39;enveloppe virale Pointes de Electron Cryotomography reconstructions utilisant Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Développement d&#39;un ADN Système économique Livraison par « Acufection » et son application à la recherche de la peau

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.