Whitehead Enstitüsü&#39;nden Fellows Fonları

Bölüm 1: enfeksiyon ayarlama <ol><li> Matara HeLa hücreleri büyümek ve hücrelerin yaklaşık% 80 konfluent kadar bekleyin. </li><li> % 2 FBS ile düzenli DMEM ve ilavesiz antibiyotikler: deney için yeterli viral büyüme ortamı hazırlayın. </li><li> Enfeksiyon ya ham vaccinia virüs bilinen bir titreye sahip hisse senedi, ya da ev sahibi gen ekspresyonu ile ilgili endişeleriniz varsa, bilinen bir titreye sahip virüs saflaştırılmış sakaroz ile yapılabilir. </li><li> Doğrudan devam, sakaroz saflaştırılmış virüs kullanıyorsanız, bölüm 2. </li><li> Hemen önce enfeksiyonu, ham vaccinia virüs stok kullanıyorsanız, çoğunlukla buz banyosu ve bir miktar su ile bir bardak sonikatör virüsün bir kısım sonikasyon. <ul style="list-style-type:lower-alpha;"><li> 30W yaklaşık 20 saniye boyunca sonikasyon. </li><li> Vorteks tüp ve buz ve soğuk dolu bardağı tutmak için gerekirse daha fazla buz ekleyerek, sonication 3 kez tekrarlayın. </li><li> Her sonication adım arasında vorteksleyin. </li><li> Bölüm 2 ile devam edin. </li></ul></li><li> Alternatif olarak, bir fincan sonikatör sahip değilse, ham virüs stok ve 0.25mg/mL tripsin eşit miktarda karıştırın. <ul style="list-style-type:lower-alpha;"><li> Şiddetle Vortex. </li><li> 37 ° C su banyosunda 30 dakika süreyle virüs stok / tripsin karışımı inkübe. </li><li> 5-10 dakika aralıklarla Vortex. </li><li> Bölüm 2 ile devam edin. </li></ul></li></ol> Bölüm 2: Enfekte hücrelerin <ol><li> , Enfeksiyon istenen çokluğu (İçişleri Bakanlığı) tek tabaka bulaştırmak için gerekli viral parçacıkların miktarını hesaplayın. Tipik olarak, (5 ila 10) İçişleri Bakanlığı, yüksek enfeksiyon timecourses için kullanılır. </li><li> 37 sakaroz saflaştırılmış virüs veya tripsinize ham virüs istenilen miktarda ° C viral büyüme medya ve iyice karıştırın. Sadece balonun alt kapağı için yeterli medyayı kullanmak gerekir. Örneğin, medya bir T-175 şişesinde 10 ml. </li><li> Hücrelerden medyayı çıkartın ve oda sıcaklığında PBS ile durulayın. </li><li> Her balona virüs / viral büyüme ortamı ekleyin. <ul style="list-style-type:lower-alpha;"><li> Nazikçe Swirl, 37 ° eğim plakaları ve inkübe ° C 1 saat süreyle% 5 CO2 inkübatör. </li><li> Tilt ve girdap levhalar her 15 dakikada düzgün virüs yaymak ve hücrelerinin nemli tutmak için. </li><li> Bir 0hr/Mock zaman noktası için, hiçbir virüs, sadece viral büyüme medya ekleyebilirsiniz. </li></ul></li><li> 1 saat inkübasyondan sonra, virüsü içeren medyayı çıkartın ve oda sıcaklığında PBS ile üç kez yıkayın. </li><li> Her balona viral büyüme ortamının maksimum miktarda ekleyin. Örneğin, medya bir T-175 şişesinde 30 ml. 0 saat zaman noktası olarak sayma başlayın. </li></ol> Bölüm 3: Hasat hücreleri <ol><li> Hücrelerin mikroskop altında kontrol edin ve herhangi bir sitopatik etki (CPE) not edin. </li><li> Medyayı çıkartın ve oda sıcaklığında PBS 30 ml ile durulayın hücreleri. </li><li> Tripsin 15 mL hücrelere ekleyin ve 37 2-5dk inkübe ° C. </li><li> Hücre ayrılması için mikroskop altında kontrol edin ve hücreleri çıkarmak için şişeyi hafifçe dokunun. </li><li> Hücreleri, steril bir serolojik pipet yardımıyla 50 ml konik bir tüp içine aktarın. </li><li> Hücre büyümesi medya eşit hacmi ile şişesi yıkayın ve konik tüp tripsinize hücreleri için ortam ekleyin. </li><li> Oda sıcaklığında 5 dakika boyunca 300 x g hücreleri santrifüjleyin. </li><li> Hücre pelet tripsin / medya çıkarın. </li><li> İstediğiniz RNA izolasyon kiti TRIzol reaktif veya lizis tamponu ya da hücre pelletini tekrar. </li><li> TRIzol kullanıyorsanız, -80 1.5mL etiketli Eppendorf tüpleri ve dondurma 1 ml alikotları örnek bölmek ° C </li><li> Zaman noktası başına bir şişeyi hasat, tüm zamanların noktaları için tekrarlayın. </li></ol> Bölüm 4: TRIzol numune RNA ekstraksiyonu <ol><li> Bu noktada, örnekleri işlenecek hazır TRIzol reaktif yeniden bekletildi ve olmalıdır. </li><li> Her bir tüp içinde TRIzol her 1 mL BCP Faz Ayırma Reaktif 200μL ekleyin. TRIzol büyük bir miktarı ile başlıyor, daha büyük bir tüp örnek aktarmak gerekebilir. </li><li> Vortex veya kuvvetlice çalkalanır ve oda sıcaklığında 2-3 dakika inkübe edin. </li><li> 15 dakika boyunca 12.000 xg&#39;de örnek santrifüjleyin. </li><li> Sulu faz temiz bir tüpe aktarın. Sulu faz açık üst katmanı. Ile başlayan TRIzol her 1 ml için yaklaşık 600μL kurtarmak olmalıdır. </li><li> BCP yerine ikinci bir fenol / kloroform ekstraksiyon, 1 ml TRIzol başına kloroform 500μL kullanarak bu kez yapın. </li><li> Vortex veya kuvvetlice çalkalanır ve oda sıcaklığında 2-3 dakika inkübe edin. </li><li> 15 dakika boyunca 12.000 xg&#39;de örnek santrifüjleyin. </li><li> Sulu faz temiz bir tüpe aktarın. Sulu faz açık üst katmanı. </li><li> Her bir örnek için doğrusal akrilamid 20μg ekleyin. Doğrusal akrilamid bir taşıyıcı gibi davranır ve RNA&#39;yı çöktürmek için yardımcı olur. </li><li> Ekle 500 &amp; micro; izopropanol L TRIzol 1 ml başına her bir örnek ile başladı ve iyice karıştırın. </li><li> 10 dakika boyunca oda sıcaklığında numune inkübe edin. </li><li> 10-15 dakika maksimum hızda bir mikrosantrifüj santrifüjleyin. </li><li> RNA pelet tüpün alt kısmında görünür olacaktır. Çok dikkatli bir şekilde, bir vakum aspiratör ile, ya tüpten süpernatantı kaldırmak veya elle pipet. Pelet rahatsız etmeyin. </li><li> Pelet 1 ml% 70 etanol ile yıkayın. <ul style="list-style-type:lower-alpha;"><li> Pelet için 1 ml% 70 etanol ekleyin. </li><li> En yüksek hızda 5-7 dakika boyunca 14.000 g santrifüjleyin. </li><li> Çok dikkatli bir şekilde, bir vakum aspiratör ile, ya etanol tüpten kaldırmak veya elle pipet. Pelet tüpün alt kısmında görünür olduğunu kontrol edin. </li><li> Süpernatantı atın. </li></ul></li><li> Yıkama adımı yineleyin. Süpernatantı atılır sonra pelet tüp işaretleyin. </li><li> 5 dakikadan daha uzun süre Hava kuru pelet. Overdry, yoksa RNA sulandırmak için zor olacak! </li><li> Kalan herhangi bir DNA numune kaldırmak için isteğe bağlı DNaz tedavisini takip etmek isterseniz, nükleaz içermeyen su 17μL pelet tekrar süspansiyon haline getirin. Aksi takdirde, nükleaz ücretsiz su 20μL pelet tekrar süspansiyon ve 22 adıma geçin. </li><li> (Opsiyonel DNaz tedavi) Qiagen RNaz-free DNaz seti kullanarak, 2μL Tampon RDD ve 1μL RNaz yeniden süspanse RNA ücretsiz DNaz sulandırılmış eklemek. Yavaşça karıştırın. </li><li> (Opsiyonel DNaz tedavi) 37 ° C&#39;de 30 dakika. </li><li> (Opsiyonel DNaz tedavi) 2.5 mM EDTA ve inkübe 2μL 65 ° C 5 dakika DNaz inaktive. Kat daha uzun / yüksek sıcaklıklarda RNA düşmesine neden olarak inaktivasyon zaman veya sıcaklık aşmayın. </li><li> Spektrofotometre tarafından RNA konsantrasyonu kontrol edin. </li><li> -80 ° C&#39;de RNA örnek Mağaza </li><li> (Opsiyonel QC) Agilent BioAnalyzer kullanarak RNA kalitesini kontrol edin, ya da örnek denatüre edici bir jel üzerinde çalışıyor ve OD okumaları kontrol. (260nm ve 260/280 oranı) </li></ol>

<ol>
	<li>KH, R. u. b. i. n. s., LE, H. e. n. s. l. e. y., GW, B. e. l. l., Wang, C., EJ, L. e. f. k. o. w. i. t. z., PO, B. r. o. w. n., DA, R. e. l. m. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+viral+gene+expression+programs+during+poxvirus+infection:+a+transcriptional+map+of+the+vaccinia+and+monkeypox+genomes.">Comparative analysis of viral gene expression programs during poxvirus infection: a transcriptional map of the vaccinia and monkeypox genomes.</a> PLoS ONE. 3 (7), e2628-e2628 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+a+complete+poxvirus+transcriptome+reveals+an+immediate-early+class+of+genes.">Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes.</a> Proc. Natl. Acad. Sci. 105 (6), 2140-2145 (2008).</li><li>Satheshkumar, P. S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poxvirus+transcriptome+analysis.">Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E62-E62 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A.+Reply+to+Satheshkumar+and+Moss:+Poxvirus+transcriptome+analysis.">A. Reply to Satheshkumar and Moss: Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E63-E64 (2008).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., Mu&#241;oz, M., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+gene+expression+survey+of+vaccinia+virus+infection+of+human+HeLa+cells.">Cellular gene expression survey of vaccinia virus infection of human HeLa cells.</a> J Virol. 77 (11), 6493-6506 (2003).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Conde, R., Pascual-Montano, A., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+reveals+characteristic+changes+of+host+cell+gene+expression+in+response+to+attenuated+modified+vaccinia+virus+Ankara+infection+of+human+HeLa+cells.">Microarray analysis reveals characteristic changes of host cell gene expression in response to attenuated modified vaccinia virus Ankara infection of human HeLa cells.</a> J Virol. 78 (11), 5820-5824 (2004).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., N&#225;jera, J. L., Zaballos, A., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+response+to+the+attenuated+poxvirus+vector+NYVAC:+upregulation+of+apoptotic+genes+and+NF-kappaB-responsive+genes+in+infected+HeLa+cells.">Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.</a> J Virol. 80 (2), 985-998 (2006).</li><li>Guerra, S., N&#225;jera, J. L., Gonz&#225;lez, J. M., L&#243;pez-Fern&#225;ndez, L. A., Climent, N., JM, G. a. t. e. l. l., Gallart, T., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+gene+expression+profiling+after+infection+of+immature+human+monocyte-derived+dendritic+cells+by+the+attenuated+poxvirus+vectors+MVA+and+NYVAC.">Distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors MVA and NYVAC.</a> 81 (16), 8707-8721 (2007).</li></ol>

Kritik Adımlar Part 1 &amp; 2 Senkron vaccinia enfeksiyonu kurma pek çok kritik adımlar vardır, virüsün ilk olmanın dikkatli sonication (veya trypzinizing), virüs parçacıkları parçalara ayırma için. Vaccinia toplayarak için son derece eğilimli ve virüs parçacıkları bozulması hücre bile enfeksiyon sağlamak için önemlidir. Senkron bir enfeksiyon ulaşmak için (2&#39;den fazla), İçişleri Bakanlığı, her bir hücre enfekte yüksek sağlamak ...

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<td>TRIzol Reagent</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15596-026</td>
<td>Similar reagents, such as TriPure from Roche, will also work.</td>
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<tr>
<td>BCP Phase Separation Reagent</td>
<td>Reagent</td>
<td>Molecular Research Center</td>
<td>BP151</td>
<td>&nbsp;</td>
<tr>
<td>RNase-Free DNase Set</td>
<td>Reagent</td>
<td>Qiagen</td>
<td>79254</td>
<td>DNase treatment is an optional step.</td>
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vaccinia virus infection &amp; temporal analysis of virus gene expression: part 1

Aile<em&gt; Poxviridae</em&gt; Sadece enfekte hücrelerin sitoplazmasında çoğaltmak virüsler içeren büyük çift iplikli DNA oluşur. Üyeleri<em&gt; Orthopox</em&gt; Cinsine dahil variola, insan küçük çiçeği etkeni, monkeypox ve vaccinia (VAC), virüs aile prototipik üyesi. Göreceli olarak büyük (~ 200 kb) vaccinia genom içinde gen üç sınıfa kodlanır: erken, orta ve geç. Her üç sınıf, viral kodlanmış RNA polimeraz tarafından transkripsiyonu olsa da, her sınıf, virüsün yaşam döngüsü içinde farklı bir fonksiyon sunar. Poxviruses enfeksiyon sırasında ev sahibi hücresel ortamı modülasyonu için çoklu stratejiler kullanmaktadır. Hem ev sahibi hem virüs gen ekspresyonu düzenlenmesi anlamak için, biz, hem virüs ve konak hücrelerine transkript bolluk analiz etmek için genom yaklaşımlar kullanılmaktadır. Burada, birkaç saat sonrası enfeksiyon Vaccinia virüs ve örnekleme RNA HeLa hücrelerinde zaman ders enfeksiyonları göstermektedir. Her iki konak ve viral toplam RNA gen ekspresyonu analizi için mikroarray&#39;ler hibridizasyon için izole edilmiş ve yükseltilir.

Watch this Scientific Journal Video about Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 1 at JoVE.com

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

Vaccinia enfeksiyonu HeLa hücrelerinde ve host ve viral gen ekspresyon analizi için protokol. Part 1 3.

Vaccinia Virüs Enfeksiyonu &amp; Virus Gen İfadesi Zamansal Analizi: Bölüm 1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

vaccinia-virus-infection-temporal-analysis-virus-gene-expression-part

Research

JoVE Journal

Biology

10.2K Görüntüleme.  MIT - Massachusetts Institute of Technology. Vaccinia enfeksiyonu HeLa hücrelerinde ve host ve viral gen ekspresyon analizi için protokol. Part 1 3.

Video: Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 1

Bacterial Gene Expression Analysis Using Microarrays

Bakteriyel Gen İfadesi Analizi Mikroarray&#39;ler Kullanımı

Biyoloji

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

Vaccinia Virüs Enfeksiyonu &amp; Virus Gen İfadesi Zamansal Analizi: Part 2

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

Vaccinia Virüs Enfeksiyonu &amp; Virus Gen İfadesi Zamansal Analizi: Bölüm 3

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

Virüs indüklenen gen susturma (VIGS) Nicotiana benthamiana Ve Domates

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Yüksek Verimli Primer Hücre Transfect Gen Verici MXcell Elektroporasyon Sistemi

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Gen İfadesi Çalışmaları basitleştirin Otomatik Hücre Sayıcı kullanma: Namalwa Hücreler IL-4 Bağımlı Gen İfadesi siRNA demonte

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Active T. in vitro Sulandırma castaneum Telomeraz

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Pamuk olarak Agrobacterium Virüs Bağlı Gen susturma Testi

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBL de MultiBac Protein Complex Üretim Platformu

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...