Bu çalışma İzlandalı Araştırma Fonu ve İzlanda Araştırma Fonu Üniversitesi tarafından finanse edildi.

1. Transfeksiyon Moleküler klon sırasıyla 12 kb ve 4.5 kb&#39;lik iki plazmit, p8XSp5-EGFP ve p67r, içinde yer alır. 9,10 <ol><li> Iki plazmit, yani p8XSp5-EGFP 4.4 ug ve Xbal ile p67r 1.6 ug eşit molar miktarlarda kesme ve transfeksiyondan önce Arter. </li><li> Ligasyon için, 16 ° C&#39;da gece boyunca bir 50 ul reaksiyon ile inkübe ligaz 1 Weiss birimi kullanılır. </li><li> Ligasyon tamamlandığında, ligaz inaktivasyonu için 15 dakika süreyle 65 ° C&#39;de inkübe edilir ligasyon karışımı. Bu adım, ihmal edilebilir. </li><li> İki gün önce transfeksiyon, tohum 10 6 SCP bir T25 doku kültürü şişesi hücreleri, ve% 90 confluency bir tek tabaka antibiyotik olmadan 5 ml DMEM +% 10 kuzu serum ve 2mM glutamin iki gün boyunca hücreler büyür. Hücreler iki gün sonra% 90 confluency ulaşmış değil varsa, hücreler ek bir gün büyümeye bırakın. </li></ol> Not: Kuzu serum subs olabilirfetal bovin serumu (FBS) ile tituted. MVV bazı suşları FBS tarafından inhibe edilir beri rutin, kuzu serum kullanın; Bu MVV suşu Ancak, FBS tarafından inhibe edilmez. <ol start="5"><li> Hücrelerin transfeksiyon için hazır olduğunuzda, antibiyotikler olmadan% 1 serum (kuzu serum veya FBS) ile DMEM orta değiştirin. Bu transfeksiyon için Lipofectamin 2000 (Invitrogen) kullanılır. </li><li> 500μl Opti-MEM ortam maddesi içinde DNA seyreltilir. </li><li> 500μl Opti-MEM ortam maddesi içinde 20uL Lipofectamin 2000 seyreltilir ve 5 dakika boyunca oda sıcaklığında (RT) olarak bırakın. </li><li> 5 dakika inkübasyondan sonra, seyreltilmiş lipofektamin 2000 (toplam hacim = 1000 ul) ile seyreltilmiş DNA birleştirir. İyice karıştırın ve oda sıcaklığında 20-30 dakika inkübe edilir. </li><li> SCP hücreler ve ortam (5 mi) içeren bir şişeye T25 transfeksiyon karışımı (1 ml) ilave edilir. Hafifçe sallayarak karıştırın. </li><li> 37 ° C&#39;de inkübe edin ve gece boyunca% 5 CO 2 (18-24 saat). </li><li> % 1 serumu, 2mM glutamin ile DMEM ile ertesi gün ortam değiştirme100 IU penisilin ve 100 IU, streptomisin. </li><li> Ters flüoresan mikroskop ile SCP hücrelerde GFP üretim izlenmesi. % 15 - Düşük transfeksiyon verimliliği genellikle 5 bu birincil hücreler ile beklenebilir. </li><li> Inkübe 37 daha fazla ° C,% 5 şişe içerisinde virüsün yayılmasını sağlamak için birkaç gün boyunca CO2. Genellikle tam enfeksiyonu için 7-14 gün sürer. </li><li> Eppendorf tüpleri içine süpernatant aliquoting tarafından virüs Harvest. Hücre enkaz kaldırmak için 5 dakika süreyle 3000 rpm&#39;de bir mikrofuge&#39;de yılında Spin. Yeni tüplere virüs içeren süpernatant aktarın. -80 ° C de saklayınız </li></ol> 2. Virüs titrasyonu <ol><li> Bir 96 oyuklu plaka içinde% 10 serumu, 2mM glutamin, penisilin ve 100IU/ml 100IU/ml streptomisin, kuyu başına ile desteklenmiş 100 ul DMEM ortamı içinde Seed 3x10 4 SCP hücreleri. 37 inkübe ° C,% 5 CO2. </li><li> Ertesi gün hücreler confluent ise,% 1 serum ile DMEM ortamı ile değiştirmek, 2mM glutamin, 100IU/ml penisilin ve 100IU/ml streptomisin, her bir kuyunun 100 ul. </li><li> Aşağıdaki gibi virüs inceltilir <ol><li> 2mM glutamin, 100IU/ml penisilin ve 96 yuvarlak alt veya V-alt plaka 4 x 10 kuyu için kuyu başına 100IU/ml streptomisin,% 1 serum ile 180 ul DMEM ekleyin. </li><li> Dörderli seri on kat dilüsyonları için, numunelerin iyice karışmalarını sağlamak için plaka ilk sütununda 4wells her virüs 20 ul ekleyin ve dört-kanallı pipet kullanarak, ve aşağı yukarı pipetle. Pipet uçları değiştirin ve sonraki 4 kuyu ve böylece 20 ul aktarın. Negatif kontrol için, bir virüs olmadan son kuyu bırakın. </li></ol></li><li> SCP hücreleri ve orta virüs seyreltmeleri 100 ul ekle. </li><li> 37 inkübe ° C ve% 5 CO2. GFP flöresanı ve iki hafta için sitopatik etkiler için dikkate alınmalıdır. Sitopatik etkiler yuvarlak ve normal hücrelere göre daha az şeffaf hale hücre oluşur wITH süreçleri multinükleer görünen hücreleri ve hücre ölümü ile biten, dışarı uzanan. </li><li> Reed Muench yöntemi 11 kullanarak virüs titresi (TCID50) hesaplayın. </li></ol> 3. Enfekte hücreler FACS analizi Not: virüs çoğaltma hızlı izlenmesi için enfekte hücreler flow sitometri ile incelenebilir. <ol><li> Enfekte hücreler (ca 10 5) Trypsinize, 500μl PBS PBS ve tekrar süspansiyon ile 1x yıkayın. </li><li> 30 dakika boyunca oda sıcaklığında inkübe 500 ul% 4 paraformaldehid çözeltisi (% 2 nihai konsantrasyon) ekleyin. </li><li> 5 dakika için 3000 rpm&#39;de bir mikrofüj Pelet hücreler. </li><li> PBS 2x5 dakika ile yıkayınız ve bir FACScan analizöründe analiz. 10,000 olaylar sayın. </li></ol> 4. Temsilcisi sonuçları: 10 7 TCID 50 / ml - Bu virüs tamamen bulaşıcı ve 10 6 titrede çoğaltılması olmalıdır. Enfekte hücrelerin floresan ve det olabilirflow sitometri ve floresans mikroskobu tarafından etkilenen. Tipik olarak, GFP akış sitometresi kullanılarak hücre kültürlerinde enfekte olmuş (Şekil 1) içinde hücrelerin% 60&#39;ından daha fazla olarak tespit edilebilir. Aynı şekilde, floresan mikroskopisi ile analiz zaman, hücrelerin bir kısmı (Şekil 2A ve B) floresan vardır. <img src="/files/ftp_upload/3483/3483fig1.jpg" alt="Şekil 1" /> Şekil 1. Enfeksiyon 7 gün sonra KV1772-EGFP virüs ile enfekte olan hücrelerin SCP FACScan analizi. <img src="/files/ftp_upload/3483/3483fig2.jpg" alt="Şekil 2," /> 7 günlük inkübasyon (A) sonra KV1772-EGFP ile enfekte SCP hücrelerin Şekil 2. Faz kontrast mikroskobu. Aynı hücreler floresan mikroskobu (B) gözlemlenmiştir.

<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>

Çıkar çatışması ilan etti.

Burada sunulan MVV moleküler klon GFP ifade eden ev sahibi hücre etkileşimleri ve in vitro ve in vivo olarak MVV patojenitesi analiz etmek için yararlı olacaktır. Çoğaltma 7-14 gün sonra elde edilen virüs hiç şüphesiz ters transkriptaz hata nedeniyle bazı mutasyonlar sahip olmuştur. Bununla birlikte, tek bir döngü vektör olarak bu klon kullanımı için bir sınırlama yoktur. Bir klon kullanımı koyun ve keçi kaynaklı hücreler ile sınırlı olmasıdır. Bizim yapısı kullanılarak bu hücrelerden transfeksiyon verimi sadece% 5-15 ve bir pEGFP-N3 vektörü kullanılarak transfeksiyon etkinliği 293-T-hücreleri kullanılarak üzerinden% 90 ile karşılaştırıldığında% 15-20. 293-T-hücreleri MVV vektörleri için paketleme hücreleri gibi test edilmiş, ancak elde edilen virüs partiküllerinin, koyun hücreleri ve tanımlanamayan kısıtlama faktörlere bağlı olarak muhtemelen 293-T-hücreleri, 12 her ikisi de enfeksiyöz olmayan ispat edilmiştir. Bulaşıcı klonu başka eksikliktir iki plazmit, yer almaktadır. Bu gerçeği nedeniyletam uzunluktaki klon E. coli içinde dengesiz olduğunu. Ancak, bu plasmid, bir düşük kopya halinde tam uzunluktaki klon klonlamak için mümkün olacaktır. Plazmidler ve hücreler yazarlar elde edilebilir.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </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> Lipofektamin 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 virüsü (MVV) yavaş ilerleyen interstisyel pnömoni ve ensefalit 1 neden, koyun lentivirüs olduğunu. In vivo olarak MVV birincil hedef hücrelerin monosit 2 olduğu kabul edilir. MVV Bazı suşları ancak 3,4, diğer hücre tiplerinde çoğaltabilirsiniz. Yeşil floresan protein canlı hücrelerde lentiviruses incelemek için yaygın olarak kullanılan bir göstergedir. Biz MVV dUTPase için geninin içine EGFP gen taktınız. DUTPase geni de çoğu lentivirüs koyun suşu ve keçi korunduğu ve CAEV 5 çoğaltma önemli olduğu gösterilmiştir. Bununla birlikte, dUTPase in vitro ve in vivo 6 hem de bu çalışmada kullanılan MVV moleküler klon çoğaltılması için vazgeçilebilir olduğu gösterilmiştir. MVV çoğaltma kesinlikle koyun veya keçi kaynaklı hücreler ile sınırlıdır. Biz transfeksiyon ve virüsün 7 yayılması için koyun koroid pleksus (SCP hücreleri) bir birincil hücre hattı kullanın &lt;/&gt; Sup. Floresan MVV tamamen bulaşıcıdır ve EGFP ifadesi en az 6 pasajlar üzerinde 8 sabittir. TCID 50 ve EGFP ölçümleri arasında iyi bir korelasyon vardır. Bu nedenle, virüs hücre tropizm ve in vitro ve in vivo 8 patojen çalışmalarda enfekte hücrelerin hızlı tespit için yararlı olacaktır.

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

Biz, GFP ifade eden ve tam olarak bulaşıcı olan Maedi-visna virüs bir moleküler klon tanımlamak. Bu virüs çoğaltma floresan mikroskopisi ve akış sitometrisi ile tespit edilebilir.

Çoğaltım ve Yeşil floresan proteini ifade Maedi-visna Virüs bir Enfeksiyöz Moleküler Clone algılama

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.8K Görüntüleme.  University of Iceland. Biz, GFP ifade eden ve tam olarak bulaşıcı olan Maedi-visna virüs bir moleküler klon tanımlamak. Bu virüs çoğaltma floresan mikroskopisi ve akış sitometrisi ile tespit edilebilir.

Video: 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.

HIV ve Live Konfokal Mikroskop Floresan Klonlar kullanılarak HIV görselleştirme Hücre-hücre Transferi

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

İmmünoloji ve Enfeksiyon

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.

Yeşil Floresan Protein ifade Rev-bağımlı lentiviral Vector kullanarak HIV-1 Olumlu Hücreler Özgül İşareti

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.

Genetiği Tanımlı Nöral Devre arasında Tracing Viral

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

Nörobilim

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.

Floresan Etiketli Vaksinia Virüs Proteinlerinin Verimli Üretimi için Metodoloji

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.

Gen İfade Tüm Dönemlerinde Viral Fonksiyonu sayısallaştırabilmek için vaksiniya Reporter Virüsler

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.

Yenidoğan Beyin içine Viral Parçacıklar ve Floresan mikro boncuklar intraserebroventriküler ve İntravasküler Enjeksiyon

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.

Yaşayan Hücreler ubikitin-proteozom Faaliyet Monitoring Degron (DGN)-destabilize Yeşil floresan proteini (GFP) tabanlı Muhabir Protein Kullanımı

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

Biyoloji

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.

K18 hACE2 Transgenik Farelerde Viral Enfeksiyonun Canlı Görüntülenmesi ve Raporlayıcı İfade Eden Rekombinant SARS-CoV-2 Kullanılarak Ölçülmesi

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.

Membran proteinlerinin Yeşil floresan proteini temelli Ekspresyon elemesinde<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

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 ...