Diese Arbeit wurde von der isländischen Research Fund und der Universität von Island Research Fund gefördert.

1. Transfektion Die molekulare Klon ist in zwei Plasmiden, p8XSp5-EGFP und p67r, von 12 kb und 4,5 kb bzw. enthalten. 9,10 <ol><li> Cut äquimolaren Mengen der beiden Plasmide, dh 4,4 pg p8XSp5-eGFP und 1,6 ug p67r mit XbaI und ligieren vor der Transfektion. </li><li> Für die Ligation, verwenden Sie 1 Weiss Einheit Ligase in einem 50 ul Reaktions-und Inkubieren bei 16 ° C über Nacht. </li><li> Wenn Ligation abgeschlossen ist, inkubieren Ligationsmix bei 65 ° C für 15 min zur Inaktivierung der Ligase. Dieser Schritt kann weggelassen werden. </li><li> Zwei Tage vor der Transfektion, Saatgut 10 6 SCP Zellen in einer T25 Zellkulturflasche und wachsen Zellen für zwei Tage in 5 ml DMEM + 10% Lamm Serum und 2 mM Glutamin, ohne Antibiotika, um eine Monoschicht von 90% Konfluenz. Wenn die Zellen nicht 90% Konfluenz nach zwei Tagen erreicht, lassen die Zellen wachsen einem zusätzlichen Tag. </li></ol> Hinweis: Das Lamm Serum kann durch fötales Rinderserum (FBS) ersetzt werden. Wir verwenden routinemäßig Lamm Serum, da einige Stämme von MVV von FBS sind gesperrt; dieser MVV Stamm ist nicht gehemmt durch FBS jedoch. <ol start="5"><li> Wenn die Zellen bereit für die Transfektion sind, ändern Sie mittel-bis DMEM mit 1% Serum (Lamm Serum oder FBS) ohne Antibiotika. Wir verwenden Lipofectamin 2000 (Invitrogen) für die Transfektion. </li><li> Verdünnen Sie die DNA in 500μl Opti-MEM-Medium. </li><li> Verdünnen Sie 20 &amp;mgr; l Lipofectamin 2000 in 500μl Opti-MEM-Medium und lassen bei Raumtemperatur (RT) für 5 min. </li><li> Nach dem 5 min Inkubation, kombinieren die verdünnte DNA mit verdünnter Lipofectamine 2000 (Gesamtvolumen = 1000 ul). Vorsichtig mischen und inkubieren für 20-30 min bei RT. </li><li> Fügen Sie die Transfektion Mix (1 ml) der T25 Kolben mit SCP Zellen und Medium (5 ml). Mix von rocking sanft. </li><li> Bei 37 ° C und 5% CO 2 über Nacht (18 bis 24 h). </li><li> Ändern Medium am folgenden Tag um DMEM mit 1% Serum, 2 mM Glutamin, Penicillin und 100IU 100IU Streptomycin. </li><li> Überwachen der Herstellung von GFP in der SCP-Zellen durch invertierte Fluoreszenz-Mikroskopie. 15% - Niedrige Transfektionseffizienz können mit diesen primären Zellen, in der Regel 5 zu erwarten. </li><li> Inkubieren bei 37 ° C, 5% CO 2 für mehrere Tage, damit die Verbreitung des Virus in den Kolben. Es dauert in der Regel 7-14 Tage für volle Infektion. </li><li> Ernte des Virus durch Aliquotierung der Überstand in Eppendorf-Röhrchen. Spin in einer Mikrozentrifuge bei 3000 rpm für 5 min um Zelltrümmer zu entfernen. Übertragen Sie den Überstand mit dem Virus auf neue Röhren. Lagerung bei -80 ° C. </li></ol> 2. Titration von Viren <ol><li> Seed 3x10 4 SCP-Zellen in 100 ul DMEM Medium mit 10% Serum, 2 mM Glutamin, Penicillin und 100IU/ml 100IU/ml Streptomycin pro Well in einer 96-Well-Platte ergänzt. Bei 37 ° C, 5% CO 2. </li><li> Wenn die Zellen konfluent sind am folgenden Tag, ändern mittel-bis DMEM mit 1% Serum, 2 mM Glutamin, Penicillin und 100IU/ml 100IU/ml Streptomycin, 100 ul in jede Vertiefung. </li><li> Verdünnen Sie das Virus in folgender Weise: <ol><li> Add 180 ul DMEM mit 1% Serum, 2 mM Glutamin, Penicillin und 100IU/ml 100IU/ml Streptomycin pro Vertiefung zu 4 x 10 Wells in einer 96-Well-Rundboden-oder V-Bodenplatte. </li><li> Für die serielle Zehnfachverdünnungen in vierfacher Ausfertigung, mit 20 ul des Virus auf jeden 4wells in der ersten Spalte der Platte, und mit einem Vier-Kanal-Pipette Pipette auf und ab, um sicherzustellen, dass die Proben gründlich gemischt werden. Ändern Pipettenspitzen und Transfer 20 pl auf die nächsten 4 Brunnen und so weiter. Lassen Sie die letzten Brunnen ohne Virus für eine negative Kontrolle. </li></ol></li><li> 100 l des Virus Verdünnungen der SCP-Zellen und Medium. </li><li> Bei 37 ° C und 5% CO 2. Beachten Sie für die GFP-Fluoreszenz und zytopathischen Effekte für zwei Wochen. Zytopathischen Effekte bestehen aus Zellen immer gerundet und weniger transparent als normale Zellen mit Prozessen ausstreckt, endend mit mehrkernigen Zellen erscheinen und Zelltod. </li><li> Berechnen Virustiter (TCID50) mit der Reed Muench-Methode 11. </li></ol> 3. FACS-Analyse von infizierten Zellen Hinweis: Für die schnelle Kontrolle der Replikation des Virus, infizierten Zellen können mittels Durchflusszytometrie untersucht werden. <ol><li> Trypsinize infizierten Zellen (ca. 10 5), waschen 1x mit PBS und Resuspendieren in 500μl PBS. </li><li> Add 500 ul 4% Paraformaldehyd-Lösung (2% Endkonzentration) bei RT für 30 min inkubieren. </li><li> Pellet-Zellen in einer Mikrozentrifuge bei 3000 rpm für 5 min. </li><li> Waschen mit PBS 2x5 min und zu analysieren, auf einem FACScan-Analysator. Count 10.000 Veranstaltungen. </li></ol> 4. Repräsentative Ergebnisse: Dieser Virus sollte voll ansteckender und zu replizieren, um einen Titer von 10. Juni - 10 Juli TCID 50 / ml. Infizierte Zellen werden Fluoreszenz und kann det werdenected mittels Durchflusszytometrie und Fluoreszenzmikroskopie. In der Regel können GFP in mehr als 60% der Zellen in die infizierten Zellkulturen mittels Durchflusszytometrie (Abb. 1) festgestellt werden. Ebenso, wenn visualisiert durch Fluoreszenz-Mikroskopie, sind ein Großteil der Zellen fluoreszierende (Abb. 2A und B). <img src="/files/ftp_upload/3483/3483fig1.jpg" alt="Abbildung 1" /> Abbildung 1. FACScan Analyse der SCP-Zellen mit KV1772-eGFP Viren nach 7 Tagen der Infektion infiziert. <img src="/files/ftp_upload/3483/3483fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Phasenkontrast-Mikroskopie von SCP-Zellen mit KV1772-egfp nach 7 Tagen Inkubation (A) infiziert. Gleiche Zellen visualisiert mittels Fluoreszenzmikroskopie (B).

<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>

Keine Interessenskonflikte erklärt.

Die GFP-exprimierenden molekularen Klon des MVV hier vorgestellten sollte für die Analyse der Host-Zell-Interaktionen und Pathogenität von MVV sowohl in vitro und in vivo. Das Virus nach 7-14 Tagen der Replikation erhalten hat zweifellos einige Mutationen aufgrund der Ungenauigkeit der reversen Transkriptase erworben. Allerdings gibt es Einschränkungen bei der Verwendung dieser Klon als Single-Cycle-Vektor. Einer ist, dass die Verwendung der Klon-Zellen von Schafen und Ziegen Herkunft beschränkt ist. Die Transfektio...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </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) ist ein Lentivirus von Schafen, was langsam fortschreitende interstitielle Pneumonie und Enzephalitis 1. Die primären Zielzellen von MVV in vivo gelten als der Monozyten-Linie 2 sein. Bestimmte Stämme von MVV können in andere Zelltypen zu replizieren, jedoch 3,4. Das grün fluoreszierende Protein ist ein häufig verwendeter Marker für die Untersuchung Lentiviren in lebenden Zellen. Wir haben die EGFP-Gen in das Gen für dUTPase der MVV eingefügt. Die dUTPase Gen ist in den meisten Lentivirus Stämme von Schafen und Ziegen erhalten und hat sich gezeigt, dass bei der Replikation von CAEV 5 wichtige. Allerdings hat dUTPase konnte gezeigt werden, entbehrlich für die Replikation des molekularen Klon des MVV in dieser Studie sowohl in vitro und in vivo 6 verwendet. MVV-Replikation ist streng auf Zellen von Schafen oder Ziegen Herkunft beschränkt. Wir verwenden eine primäre Zelllinie, die aus dem Plexus von Schafen (SCP-Zellen) für die Transfektion und Vermehrung der Virus 7. Die fluoreszierenden MVV ist voll Infektions-und EGFP-Expression über mindestens 6 Durchgänge 8 stabil. Es ist eine gute Korrelation zwischen den Messungen der TCID 50 und EGFP. Dieses Virus ist daher sinnvoll sein, für die schnelle Erkennung von infizierten Zellen in Studien Zelltropismus und Pathogenität in vitro und 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

Wir beschreiben einen molekularen Klon des Maedi-Visna Virus, das GFP exprimiert und ist voll ansteckend. Die Replikation von diesem Virus kann durch die Verwendung der Fluoreszenzmikroskopie und Durchflusszytometrie nachgewiesen werden.

Propagierung und Erkennen einer ansteckenden Molecular Clone von Maedi-Visna Virus, das Green Fluorescent Protein exprimiert

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

propagating-detecting-an-infectious-molecular-clone-maedi-visna-virus

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

9.8K Aufrufe.  University of Iceland. Wir beschreiben einen molekularen Klon des Maedi-Visna Virus, das GFP exprimiert und ist voll ansteckend. Die Replikation von diesem Virus kann durch die Verwendung der Fluoreszenzmikroskopie und Durchflusszytometrie nachgewiesen werden.

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

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

Visualizing Zell-Zell-Transfer von HIV unter Verwendung von fluoreszierenden Klone von HIV und Live-konfokale Mikroskopie

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Forschung

Immunologie und Infektion

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

Spezielle Kennzeichnung von HIV-1 positiven Zellen mit Hilfe eines Rev-abhängigen Lentivirale Vector mit dem Ausdruck des Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

Viral Tracing von genetisch definierten Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Neurowissenschaften

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

Methodik zur effizienten Erzeugung fluoreszierender Vaccinia-Virusproteine

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Vaccinia-Viren Reporter zur Quantifizierung von Viral-Funktion auf allen Stufen der Genexpression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

Intracerebroventricular und intravaskuläre Injektion von Viruspartikeln und Fluorescent Microbeads in die neonatale Gehirn

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

Überwachung des Ubiquitin-Proteasom-Aktivität in lebenden Zellen unter Verwendung eines Degron (DGN)-destabilisiert Green Fluorescent Protein (GFP)-basierte Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Biologie

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

Live-Bildgebung und Quantifizierung von Virusinfektionen in transgenen K18-hACE2-Mäusen mit Reporter-exprimierendem rekombinantem SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-basierte Screening Expression von Membranproteinen in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Molecular Cloning

Molecular cloning is a set of methods, which are used to insert recombinant DNA into a vector - a carrier of DNA molecules that will replicate recombinant DNA fragments in host organisms. The DNA fragment, which may be a gene, can be isolated from a prokaryotic or eukaryotic specimen. Following isolation of the fragment of interest, or insert, both the vector and insert must be cut with restriction enzymes and purified. The purified pieces are joined together though a technique called ligation. The enzyme that catalyzes the ligation reaction is known as ligase.
This video explains the major methods that are combined, in tandem, to comprise the overall molecular cloning procedure. Critical aspects of molecular cloning are discussed, such as the need for molecular cloning strategy and how to keep track of transformed bacterial colonies. Verification steps, such as checking purified plasmid for the presence of insert with restrictions digests and sequencing are also mentioned.

Molecular Cloning: Principle, Procedure, and Applications

Molekulare Klonierung

Molecular cloning is a set of methods, which are used to insert recombinant DNA into a vector - a carrier of DNA molecules that will replicate recombinant ...