Name: rescue of recombinant newcastle disease virus from cdna
Uploaded: 2013-10-11T23:30:00
Description: Watch this Scientific Journal Video about Rescue of Recombinant Newcastle Disease Virus from cDNA at JoVE.com

Autoren möchten Vergangenheit und Gegenwart Mitglieder in den Labors von Dr. danken. Peter Palese und Adolfo García-Sastre für die Entwicklung von NDV Reverse Genetik Techniken und für die technische Unterstützung. Research in Newcastle-Disease-Virus in AG-S Labor wird teilweise finanziert durch NIAD R01AI088770 gewähren und durch das Department of Homeland Security Science &amp; Technology Center of Excellence für Schwellen-und Tierkrankheiten Zoonosen (CEEZAD, Verleihungsnummer 2010-ST-061-AG001). Forschung in der LM-S-Labor wird von den NIH RO1 Zuschüsse AI077719, R21NS075611-01, R03AI099681-01A1, die NIAID Centers of Excellence für Influenza-Forschung und Überwachung (HHSN266200700008C), und der University of Rochester Zentrum für Biodefense Immune Modeling (HHSN272201000055C) gefördert .

1. Herstellung von Säugerzellen (4A, Tag 1) Split HEp-2-Zellen oder A549 am Tag vor der Transfektion in 6-Well-Platten. Dichte der Zellen sollte 80-90% Konfluenz am nächsten Tag zu erreichen. Normalerweise kann eine konfluente 100-mm-Schale in 8 Vertiefungen aufgeteilt werden (ca. 1 x 10 6 Zellen pro Vertiefung). Für jedes Virus, gerettet zu werden, sollte 2-4 verschiedenen Vertiefungen enthalten sein, sowie 2 zusätzliche Bohrungen für die Kontrollen pCAGGS-GFP und GFP-pCITE <s...

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Mehrere Faktoren sind zu berücksichtigen, um gute Ergebnisse zu erzielen, während die Rettung NDV werden. Zuerst wird die Volllängen-cDNA-Konstrukt verwendet werden muss, ausgelegt werden, um die funktionelle Einbindung der neuen Transgene / Änderungen in dem NDV-Genom zu ermöglichen. Dies bedeutet, wie oben angegeben, dass (i) entsprechende Gen Ende (GE), intergenen (IG) und Gen-Start (GS)-Sequenzen hinzugefügt werden, falls erforderlich, (ii) es gibt keine mutmaßlichen GE oder GS-Sequenzen in das Fremd Gen, und...

Newcastle Disease Virus (NDV), ein Vogel-Paramyxovirus zu der Gattung Avulavirus 1 gehört, ist eine wirtschaftlich relevante und damit weit erforscht und überwachten Zoonoseerreger, die schwerwiegende Auswirkungen auf die Geflügelzucht in der ganzen Welt. Obwohl nicht ein menschlicher Krankheitserreger, NDV wurde auch gründlich über den Tierarzt Feld sowohl als Modell Paramyxovirus und aufgrund seiner höchst interessanten, Natur onkolytischen Eigenschaften 6 untersucht. Forschung auf NDV stark von der Entwicklung der Techniken reverser Genetik für einzelsträngige, nicht-segmentierten Negativ-Strang-RNA-Viren, zunächst für Tollwutvirus von Conzelmann und coleagues 2 beschrieben profitiert. Eine Vielzahl von gentechnisch veränderten NDVs, Trage fremde Gene oder Änderungen ihrer Wildtyp-Genoms wurden weitgehend seit sucht. Die Arbeit mit diesen rekombinanten Viren war entscheidend für verschiedene Virulenzfaktoren nicht nur von NDV, sondern auch von anderen relevanten huma charakterisierenn Krankheitserreger wie Influenza-A-Virus 7 - oder 8 emergent Nipah-Virus. Weiterhin wurden eine Reihe von verschiedenen Studien die Verwendung dieser Techniken, um die Antitumoraktivität von angeborenen NDV 6,9,10 verbessern, vor allem durch die Verstärkung der immunstimulierenden Eigenschaften des Virus untersucht. Andere relevante Bereich der Forschung auf rekombinante NDVs war die Erzeugung von Impfstoffkandidaten gegen andere Viruserkrankungen, wie Influenza 5,11,12, 13 HIV, Masern 14, SARS 15 oder die durch die Atem sincytial Virus (RSV) 16 verursacht wird. Unter den verschiedenen bemerkenswerten Vorteile, die durch NDV bereitgestellt sind der Mangel an bereits existierenden Immunität in menschlichen Populationen, die Stabilität der fremden genetischen Einsätze, einen rekombinatorischen Mangel an Aktivität und einer hohen Gesamtwirkungsprofil in Kombination mit den zuvor genannten natürlichen immunstimulierenden Eigenschaften 17. Es ist auch bemerkenswert, die mögliche Verwendung von rekombinanten Impfstoffen in bivalenten poultry, Schutz sowohl gegen NDV und hochpathogene aviäre Influenzaviren 11,12. Dies kann eine hervorragende Möglichkeit, um die Chancen des letzteren Verbreitung von wild bis domestizierte Tiere, also auch helfen, eine mögliche interspezifische Sprung der gefürchteten Vogelgrippe auf den Menschen zu verhindern, zu verringern. Schließlich Reportergen exprimierenden NDV Zur Beurteilung der angeborenen Immunantwort sowie der Identifizierung von Interferon-Antagonist durch mehrere Viren codiert 18-27 verwendet. Das Rettungsverfahren eines rekombinanten, nicht-segmentierten, besteht Negativstrang-RNA-Virus im wesentlichen zu erzwingen künstlich einen viralen Replikationszyklus in einer erzeugenden Zelle durch Transfektion von cDNA, die die minimale infektiöse molekulare Maschinerie, wie Ribonukleoprotein oder RNP bekannt (Fig. 2) codiert. Die RNPs aus der viralen Polymerase (P-und L-Proteine), das Nucleoprotein (NP) und das Volllängen-antigenomischen RNA des Virus. Diese RNA + Anti ist thzur Erzeugung der komplementären RNA-Genomen, die auch mit dem Rest der Proteine ​​der viralen RNP erforderliche zugehörigen E-Vorlage, rekapituliert die gleiche Infektionskomplex eine natürliche Virus würde auf das Zytoplasma der Zelle nach der Infektion (Fig. 2A frei ). Von diesem Schritt ab, kann das virale Zyklus selbst fort und rekombinanten Virionen enkapsidieren die modifizierten Genom, erzeugt (Fig. 2B). Bemerkenswert ist, Transfektion der genomischen cDNA anstelle der cDNA antigenomische stark beeinträchtigt oder ganz abschafft Rettungs Effizienz 2,28-30. Selbst wenn antigenomischen cDNA transfiziert ist die Effizienz der Einkapselung der rekombinanten RNA in RNPs in transfizierten Zellen vermutlich sehr gering. Dadurch Rettungs Protokolle für NDV umfassen häufig verschiedene Schritte zur Amplifikation der wenigen Viruspartikel von den ursprünglich transfizierten Zellen durch Co-Kultivierung mit ihnen permissiven Zellen und / oder das freiInfektion von bebrüteten Eiern. Vor der Rettungs kann die cDNA durch Standard Klonierungsverfahren, um die gewünschten Änderungen erzeugen manipuliert werden. Während spezifische Mutationen der verschiedenen Genprodukte und regulatorischen Sequenzen des Virus kann ohne weiteres auf diese Weise erreicht werden kann, hat viele der veröffentlichten Arbeiten mit rekombinanten NDV das Hinzufügen einer neuen Transkriptionseinheit in das NDV-Genoms erforderlich ist. Wie andere Mitglieder der Familie der Paramyxoviren, kodiert die NDV Genom acht verschiedene Proteine ​​in sechs Transkriptionseinheiten, die differentiell exprimiert werden je nach ihrer Lage in Bezug auf das 3&#39;-Ende in einer abnehmenden Gradienten kritisch für den viralen Lebenszyklus ein. Aus diesem Grund muss die Lage des neuen Transkriptionseinheit, innerhalb des Genoms sorgfältig ausgewählt werden, um ein Gleichgewicht zwischen der Expression des Transgens und die Beeinträchtigung der viralen Replikation zu erreichen. Einsetzen zwischen P-und M-Gene verwendet wurde den meisten, though anderen Websites wurden ebenfalls getestet 13,31. Was auch immer der Einsatz, die Klonierung in NDV cDNA muss einige Regeln zu befolgen, um eine rescuable Konstrukt zu generieren: (i) ein neues Gen in das Genom NDV aufgenommen werden muss unter der Kontrolle der entsprechenden Signale für die virale RNA-abhängigen RNA-sein Polymerase. Diese Sequenzen sind stromaufwärts des neuen offenen Leserahmen (ORF) zugegeben werden, so kann die Polymerase das Ende der vorherigen Gen (GE) und dem Beginn des neuen Transgen (GS) zu erkennen, um ein einziges Nukleotid intergenischen Sequenz (IG) beabstandet . Zugabe eines gültigen Kozak (K)-Sequenz an eukaryontische Ribosomen Übersetzung zu verbessern, wird auch eine bessere Fremdproteinexpression 32 empfohlen, (ii) eine effiziente Replikation von NDV, wie für die meisten Mitglieder der Familie Paramyxoviridae, ist abhängig von der Genomlänge, die ein mehr von sechs 33, daher das Einstecken in die NDV hat, um dieses &quot;rule of six&quot; zu folgen. Wenn nötig, required zusätzliche Nukleotide stromabwärts des ORF neue hinzugefügt werden, und (iii) die Sequenz des Transgens überprüft werden, um zu finden möglich GE und GS wie Rettungssequenzen, die Effizienz, die Transgen-Expression und / oder die Lebensfähigkeit des Virus beeinträchtigt. Falls vorhanden, müssen diese Sequenzen durch stille Mutagenese entfernt werden. Die Erzeugung von rekombinanten cDNA voller Länge folgenden genannten Regeln ist der erste Schritt, um effizient zu produzieren, eine genetisch modifizierte NDV wie hier beschrieben. In dem System sind alle DNA-Konstrukte unter der Kontrolle des T7-RNA-Polymerase-Promotor (Fig. 3). Das Zytoplasma-Polymerase in trans durch Koinfektion mit einem rekombinanten Vaccinia Ankara Virus verändert (MVA-T7) 34 vorgesehen. 3A zeigen die pNDV-B1 Plasmid, das in voller Länge cDNA antigenomische 5 kodiert. 3B zeigt pTM1 Plasmide NP kodiert, P und L ORFs. Plasmide pCITE-GFP, die codiert, under der T7-Promotor, dem Green Fluorescent Protein (GFP) und GFP pCAGGS 18, die die gleiche ORF unter dem Huhn beta-Aktin-Promotor 35 kodiert werden als Kontrollen verwendet. In diesem Protokoll zeigen wir das Verfahren zur rekombinanten NDV aus der cDNA der lentogenen NDV-Stamm Hitchner B1 5 (Abbildung 4) zu retten.

<table border="1">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>CORNING Cellgro</td>
			<td>10-013-CV</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>GIBCO</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (LPF2000)</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>35% Bovine Albumin (BA)</td>
			<td>Sigma</td>
			<td>232-936-2</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>CORNING Cellgro</td>
			<td>25-052-CI</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (PS) 100x</td>
			<td>CORNING Cellgro</td>
			<td>30-002-CI</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			Cell lines 
			A549 cells (catalogue number CRL-185), HEp-2 cells (catalogue number CRL-185), chicken embryo fibroblasts (catalogue number CRL-12203) and duck embryo fibroblasts (catalogue number CCL-141) are available from the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA. 20110-2209 USA). All cell lines are maintained in a 37 &#176;C incubator with 5% CO2 in DMEM 10% FBS, 1% PS. 
			Embryonated chicken eggs 
			Embryonated chicken eggs can be obtained from Charles River Laboratories, Specific Pathogen Fee Avian Supply (SPAFAS) Avian Products and Services (Franklin Commons, 106 Route 32, North Franklin, CT 06254, USA) and are maintained at 37 &#176;C. Viability of the embryos is assessed with an egg candler. Eggs are infected when they reach 8-10 days old. Both infection and harvest of the allantoic fluid takes place under sterile conditions. All eggs are autoclaved and discarded following standard laboratory biosafety protocols. 
			Turkey red blood cells (RBC) 
			Turkey RBC can be purchased from Truslow Farms (201 Valley Road, Chestertown, Md 21620, USA)and stored at 4 &#176;C. To prepare RBC for HA assay, wash 5 ml of the commercial stock with 45 ml of PBS 1x in a 50 ml conical tube. Centrifuge for 5 min at 1,000 rpm and carefully discard the supernatant. Dilute pelleted RBC 1:1,000 in PBS 1x for a final 0.5-1.0% concentration. Washed RBC can be stored at 4 &#176;C for several days. 
			Plasmids 
			Plasmid preparations are obtained with any commercially available maxiprep kit following manufacturer&#39;s instructions, diluted in ddH20 to a final concentration of 1 &#956;g/&#956;l and stored at -20 &#176;C. DNA concentration and purity are assessed by spectrophotometry at 260 and 280 nm. Preparations with a 260/280 ratio higher than 1.8 are considered of acceptable quality for the rescue. Plasmid DNA quality is also routinely double-checked by agarose gel chromatography. 
			Viruses 
			The described protocol for rescue of the lentogenic NDV strain Hitchner B1 can be performed under biosafety level (BSL) 2 conditions. The Modified Vaccinia Ankara expressing the T7 RNA polymerase (MVA-T7) was described34 and obtained from Dr. Bernard Moss. This virus is growth in confluent monolayers of chicken embryo fibroblasts and titrated in mammalian (A549 or HEp-2) cells. NDV stocks are grown in embryonated chicken eggs and titrated by IFA using polyclonal serum raised against purified virions. All contaminated material should be safely sterilized and disposed according to standard biosafety procedures. 
			Tissue culture media and solutions: 
			DMEM 10% FBS 1% PS: Dulbecco&#39;s modified Eagle&#39;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Penicillin/Streptomycin (P/S): 445 ml of DMEM, 50 ml of heat-inactivated FBS, 5 ml of 100x commercial P/S solution. Store at 4 &#176;C. 
			10x Phosphate buffered saline (PBS): 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 L. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature. 
			1x PBS: Dilute 10x PBS 1:10 with ddH2O. Sterilize by autoclave and store at room temperature. 
			100x Ca/Mg : 1.327 g CaCl2.2H2O, 2.133 g MgCl2&#8226;6H2O and add ddH2O up to 100 ml. Autoclave and store at room temperature. 
			1x PBS/BA/PS: 50 ml of 10x Phosphate buffered saline (PBS) in 437 ml ddH2O. Autoclave and when cooled down to room temperature, add 5 ml 100x Penicillin/Streptomycin 3 ml 35% Bovine and 5 ml of 100x Ca/Mg. Store at 4&#176;C.
			</td>
		</tr>
	</tbody>
</table>

rescue of recombinant newcastle disease virus from cdna

Newcastle-Disease-Virus (NDV), dem Prototyp Mitglied der Avulavirus Gattung der Familie Paramyxoviridae 1, ist ein nicht-segmentierten Negativ-Sense-Einzelstrang, umhülltes RNA-Virus (Abbildung 1) mit potentiellen Anwendungen als Vektor zur Impfung und Behandlung von menschlichen Krankheiten. In eingehende Erforschung dieser Anwendungen hat erst nach der Gründung der reversen Genetik Techniken, um rekombinante Viren aus Plasmiden ihre vollständige Genome als cDNA 2-5 kodiert möglich zu retten. Virale cDNA bequem in vitro unter Verwendung von Standard-Klonierungsverfahren, um den Genotyp des Virus zu verändern und / oder neue Transkriptionseinheiten modifiziert werden. Rettung von genetisch modifizierten Viren stellt ein wertvolles Werkzeug, um Faktoren, die mehrere Stadien der Infektion, sowie ermöglicht die Entwicklung und Verbesserung von Vektoren für die Expression von Antigenen und Liefer verstehenImpfung und Therapie. Hier beschreiben wir ein Protokoll für die Rettung von rekombinanten NDVs.

Rettung von NDV ist ein gut etabliertes Verfahren, die routinemäßig in den Laboratorien, die den Zugriff auf die vollständige cDNA des Virus durchgeführt. Die intrinsische stochastischen Natur des Verfahrens macht es jedoch schwierig, 100% Rettungseffizienz. Die Überwachung der frühen Schritte des Prozesses, speziell die Transfektionseffizienz und die Infektion mit MVA-T7, hilft, mögliche Probleme. 5A zeigt Standard-Transfektion und Transfektion / Infektion Wirkungsgrade, die genug für einen erf...

Watch this Scientific Journal Video about Rescue of Recombinant Newcastle Disease Virus from cDNA at JoVE.com

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle Disease Virus (NDV) wurde ausgiebig in den letzten Jahren, um neue Vektoren für Impfung und Therapie zu entwickeln, unter anderem untersucht. Diese Studien wurden durch Techniken, um rekombinante Virus aus cDNA zu retten, wie die, die wir hier beschreiben, möglich.

Rettung von rekombinanten Newcastle Disease Virus aus cDNA

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

rescue-of-recombinant-newcastle-disease-virus-from-cdna

Research

JoVE Journal

Immunology and Infection

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.

Erzeugung von rekombinanten Influenza Virus von 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 ...

Forschung

Immunologie und Infektion

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

Rekombinante Expression von Funktions Hämagglutinin und Neuraminidase-Proteine ​​aus dem Roman H7N9 Influenza-Virus mit dem Baculovirus-Expressionssystem

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Skalierbare High Throughput Auswahl aus Phagen-Antikörperbibliotheken angezeigt Synthetische

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Erzeugung von rekombinanten humanen IgG monoklonalen Antikörpern aus Immortalisierte Sortiert B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophilen-Isolierung und Analyse zur Bestimmung ihrer Rolle in der Lymphom-Zellempfindlichkeit therapeutische Mittel

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Rettung und Charakterisierung des rekombinanten Virus aus einer neuen Welt Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

Erkennung und Entfernung von Nuklease Kontamination während der Reinigung von rekombinanten Prototyp schaumig Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Schnelle, sichere und einfache manuelle am Krankenbett Nukleinsäure-Extraktion für den Nachweis des Virus in Vollblut-Proben

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Bewertung der Zika-Virus-spezifischen T-Zell-Reaktionen in Organe der Immunabwehr von infizierten Ifnar1- / - Mäusen

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Identifizierung der Codierung und nicht-kodierende RNA-Klassen ausgedrückt bei Schweinen Vollblut

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...