Whitehead Institute Fellows Fondi

Parte 1: Impostare l&#39;infezione <ol><li> Crescere le cellule HeLa in fiaschi e aspettare che le cellule sono circa l&#39;80% confluenti. </li><li> Preparare abbastanza virale medio di crescita per l&#39;esperimento: regolare DMEM con il 2% FBS e senza antibiotici aggiunti. </li><li> L&#39;infezione può essere effettuata sia con uno stock di greggio virus vaccinia con un titolo noto, o saccarosio virus purificato con un titolo noto se sei preoccupato per ospitare espressione genica. </li><li> Se si utilizza virus saccarosio purificato, procedere direttamente alla parte 2. </li><li> Se si utilizza un magazzino greggio virus vaccinico, appena prima l&#39;infezione, sonicare un&#39;aliquota del virus in un sonicatore tazza con un bagno di ghiaccio e per lo più un poco d&#39;acqua. <ul style="list-style-type:lower-alpha;"><li> Sonicare a circa 30 W per 20 secondi. </li><li> Vortex il tubo e ripetere la sonicazione 3 volte, aggiungendo più ghiaccio se necessario per mantenere la coppa piena di ghiaccio e freddo. </li><li> Vortex tra un passo e sonicazione. </li><li> Procedere alla parte 2. </li></ul></li><li> In alternativa, se non si possiede un sonicatore tazza, mescolare un uguale volume dello stock del virus greggio e tripsina 0.25mg/mL. <ul style="list-style-type:lower-alpha;"><li> Energicamente. </li><li> Incubare il brodo / tripsina mix di virus a 37 ° C bagnomaria per 30 minuti. </li><li> Vortex ad intervalli di 5-10 minuti. </li><li> Procedere alla parte 2. </li></ul></li></ol> Parte 2: le cellule Infezione <ol><li> Calcolare la quantità di particelle virali necessari per infettare il monostrato alla molteplicità desiderato di infezione (MOI). Tipicamente, un alto MOI (tra 5 e 10) è utilizzato per timecourses infezione. </li><li> Aggiungere la quantità desiderata di virus saccarosio purificato o trypsinized virus greggio a 37 ° C terreni di crescita virale e mescolare bene. Si dovrebbe usare mezzi sufficienti a coprire appena il fondo del pallone. Ad esempio, 10 ml di media per un T-175 pallone. </li><li> Rimuovere i supporti dalle cellule e lavare con PBS temperatura ambiente. </li><li> Aggiungere il virus / media della crescita virale ad ogni pallone. <ul style="list-style-type:lower-alpha;"><li> Agitare delicatamente, piastre inclinazione e incubare a 37 ° C in un incubatore a CO2 del 5% per 1 ora. </li><li> Piastre di inclinazione e turbolenza ogni 15 minuti per diffondere virus in modo uniforme e mantenere le cellule umido. </li><li> Per un punto temporale 0hr/Mock, aggiungere i terreni di coltura virale solo, senza virus aggiunto. </li></ul></li><li> Dopo l&#39;incubazione 1 ora, rimuovere il supporto contenente il virus, e lavare tre volte con PBS temperatura ambiente. </li><li> Aggiungere la quantità massima di terreno di coltura virale ad ogni pallone. Per esempio, 30 ml di media per un T-175 pallone. Comincia a contare questa come punto 0 tempo hr. </li></ol> Parte 3: Raccolta delle cellule <ol><li> Controllare le cellule al microscopio e nota alcun effetto citopatico (CPE). </li><li> Togliere la carta e lavare le cellule con 30 ml di PBS temperatura ambiente. </li><li> Aggiungere 15 mL di tripsina alle cellule e incubare per 2-5min a 37 ° C. </li><li> Controllare al microscopio per il distacco delle cellule e toccare fiasco delicatamente per staccare le cellule. </li><li> Trasferire le cellule con una pipetta sterile sierologica in una provetta da 50 ml. </li><li> Lavare il pallone con un uguale volume di mezzi di crescita delle cellule, e aggiungere la media per le cellule trypsinized nel tubo conico. </li><li> Centrifugare le cellule a 300 x g per 5 minuti a temperatura ambiente. </li><li> Rimuovere la tripsina / supporti dal pellet. </li><li> Risospendere il pellet cellulare in entrambi i reagenti TRIzol o il tampone di lisi dal kit desiderato isolamento del RNA. </li><li> Se si utilizza TRIzol, dividere il campione in 1 mL aliquote in provette etichettate Eppendorf 1,5 ml e congelare a -80 ° C. </li><li> Ripetere l&#39;operazione per tutti i tempi, la raccolta un pallone per ogni punto del tempo. </li></ol> Parte 4: estrazione di RNA di campioni in TRIzol <ol><li> A questo punto, i campioni devono essere risospesi in reagente TRIzol e pronto per essere lavorato. </li><li> Aggiungere 200μL di reagente separazione di fase BCP per ogni 1 ml di TRIzol in ciascun tubo. Potrebbe essere necessario trasferire il campione ad un tubo più grande se si sta partendo con una grande quantità di TRIzol. </li><li> Vortex o agitare vigorosamente e incubare a temperatura ambiente per 2-3 minuti. </li><li> Centrifugare il campione a 12.000 xg per 15 minuti. </li><li> Trasferire la fase acquosa in una nuova provetta. La fase acquosa è lo strato più chiaro sulla parte superiore. Si dovrebbe essere il recupero di circa 600μL per ogni 1 ml di TRIzol hai iniziato con. </li><li> Fare una seconda estrazione fenolo / cloroformio, questa volta usando 500μL di cloroformio per 1 ml TRIzol, invece di BCP. </li><li> Vortex o agitare vigorosamente e incubare a temperatura ambiente per 2-3 minuti. </li><li> Centrifugare il campione a 12.000 xg per 15 minuti. </li><li> Trasferire la fase acquosa in una nuova provetta. La fase acquosa è lo strato più chiaro sulla parte superiore. </li><li> Aggiungi 20μg di acrilamide lineare per ogni campione. L&#39;acrilamide lineare agisce come un vettore e aiuta a precipitare l&#39;RNA. </li><li> Aggiungere 500 e micro; L di isopropanolo per 1 ml di TRIzol hai iniziato con a ciascun campione e mescolare bene. </li><li> Incubare i campioni a temperatura ambiente per 10 minuti. </li><li> Centrifuga in una microcentrifuga a velocità massima per 10-15 minuti. </li><li> Il pellet di RNA sarà visibile nella parte inferiore del tubo. Con molta attenzione, rimuovere il surnatante dal tubo, o con un aspiratore a vuoto, o manualmente pipettatore. Non disturbare il pellet. </li><li> Lavare il pellet con 1 ml di etanolo al 70%. <ul style="list-style-type:lower-alpha;"><li> Aggiungere 1 ml di etanolo al 70% al pellet. </li><li> Centrifugare alla massima velocità 14.000 g per 5-7 minuti. </li><li> Con molta attenzione, rimuovere l&#39;etanolo dal tubo, o con un aspiratore a vuoto, o manualmente pipettatore. Controllare che il pellet è visibile nella parte inferiore del tubo. </li><li> Scartare il surnatante. </li></ul></li><li> Ripetere la fase di lavaggio. Dopo il supernatante viene scartato, marchio dove pellet è sul tubo. </li><li> Aria secca a pellet per non più di 5 minuti. Non asciugare eccessivamente, o l&#39;RNA sarà difficile da ricostruire! </li><li> Se volete seguire il trattamento opzionale DNasi per rimuovere qualsiasi residuo di DNA dal campione, risospendere il pellet in 17μL di acqua priva di nucleasi. Altrimenti, risospendere il pellet in 20μL di acqua priva di nucleasi e continuare al punto 22. </li><li> (Opzionale trattamento DNasi) Utilizzando la RNasi-free Qiagen set DNasi, aggiungere 2μL Buffer RDD e 1ml ricostituito RNasi-free ho DNasi per l&#39;RNA risospeso. Mescolare delicatamente. </li><li> (Opzionale trattamento DNasi) Incubare a 37 ° C per 30 minuti. </li><li> (Opzionale trattamento DNasi) Aggiungere 2μL di 2,5 mM EDTA e incubare a 65 ° C per 5 minuti per inattivare la DNasi. Non superare il tempo di inattivazione o la temperatura, come più volte / alte temperature possono provocare la degradazione dell&#39;RNA. </li><li> Controllare la concentrazione di RNA da spettrofotometro. </li><li> Conservare il campione di RNA a -80 ° C. </li><li> (QC Opzionale) Verificare la qualità dell&#39;RNA utilizzando un Bioanalyzer Agilent, o eseguendo il campione su un gel denaturante e controllando le letture OD. (260 nm e 260/280 ratio) </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>

Passaggi critici Part 1 &amp; 2 Ci sono diversi passaggi critici della costituzione di una infezione sincrona vaccinia, il primo sonicazione facendo attenzione (o trypzinizing) del virus, in modo da disaggregare le particelle del virus. Vaccino è fortemente incline alla aggregazione e disgregazione di particelle del virus è importante per garantire anche l&#39;infezione delle cellule. Al fine di ottenere una infezione sincrona, un alto MOI (maggiore di 2) deve esser...

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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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<td>BCP Phase Separation Reagent</td>
<td>Reagent</td>
<td>Molecular Research Center</td>
<td>BP151</td>
<td>&nbsp;</td>
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<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

La famiglia<em&gt; Poxviridae</em&gt; È costituito da grandi DNA a doppia elica che contiene virus che si replicano esclusivamente nel citoplasma delle cellule infette. I membri del<em&gt; Orthopox</em&gt; Genere includono vaiolo, l&#39;agente eziologico del vaiolo umano, vaiolo delle scimmie, e vaccinico (VAC), il membro prototipo della famiglia di virus. All&#39;interno del relativamente grande (~ 200 kb) vaccinia genoma, tre classi di geni sono codificati: precoce, intermedio e in ritardo. Anche se tutte e tre le classi sono trascritte dai virale codifica RNA polimerasi, ogni classe svolge una funzione diversa nel ciclo di vita del virus. Poxvirus utilizzano strategie multiple di modulare l&#39;ambiente host cellulare durante l&#39;infezione. Per capire regolamentazione sia di accoglienza e di espressione genica del virus, abbiamo utilizzato tutto il genoma approcci per analizzare abbondanza trascrizione sia da virus e cellule ospiti. In questo studio dimostriamo infezioni corso a tempo di cellule HeLa con il virus vaccinico e RNA di campionamento in diversi momenti dopo l&#39;infezione. Sia host e virale RNA totale è isolato e amplificato per l&#39;ibridazione di microarray per l&#39;analisi dell&#39;espressione genica.

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

Protocollo per vaccinia infezione di cellule HeLa e analisi di accoglienza e l&#39;espressione genica virale. Parte 1 di 3.

Vaccino infezione da virus e analisi temporale di espressione genica del virus: Parte 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

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10.2K Views.  MIT - Massachusetts Institute of Technology. Protocollo per vaccinia infezione di cellule HeLa e analisi di accoglienza e l'espressione genica virale. Parte 1 di 3.

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

Bacterial Gene Expression Analysis Using Microarrays

Batterica analisi di espressione genica mediante microarray

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Biologia

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.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

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.

Vaccino infezione da virus e analisi temporale di espressione genica del virus: 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.

Vaccino infezione da virus e analisi temporale di espressione genica del virus: Parte 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.

Virale silenziamento genico (VIGS) in Nicotiana benthamiana E Pomodoro

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.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

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.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle Namalwa

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.

Ricostituzione in vitro di Active T. castaneum telomerasi

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.

Mediata da Agrobacterium Virus-Induced tacere Assay Gene In Cotone

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.

Il MultiBac Protein Complex Piattaforma di produzione al EMBL

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