Уайтхед Институт стипендиаты фондов

Часть 1: Настройка инфекции <ol><li> Расти HeLa клеток в колбах и ждать, пока клетки приблизительно на 80% вырожденная. </li><li> Подготовка достаточно вирусных питательную среду для эксперимента: регулярные DMEM с 2% ЭТС и без добавления антибиотиков. </li><li> Инфекция может быть выполнена либо с сырой запасов вируса коровьей оспы с известным титром, или сахароза очищенный вирус с известным титром, если вы беспокоитесь о хост экспрессии генов. </li><li> Если вы используете сахарозы очищенный вирус, переходите к части 2. </li><li> Если вы используете сырой вируса коровьей оспы акций, непосредственно перед инфекцией, разрушать ультразвуком аликвоты вируса в чашку sonicator с ванной в основном из льда и небольшого количества воды. <ul style="list-style-type:lower-alpha;"><li> Разрушать ультразвуком около 30 Вт в течение 20 секунд. </li><li> Вихревые трубки и повторить обработку ультразвуком в 3 раза, добавляя больше льда, если это необходимо держать чашку упакован со льдом и охлажденным. </li><li> Vortex между каждым шагом обработки ультразвуком. </li><li> Приступить к части 2. </li></ul></li><li> Или же, если вы не обладаете чашку sonicator, смешать равные объемы сырой запас вируса и 0.25mg/mL трипсина. <ul style="list-style-type:lower-alpha;"><li> Vortex энергично. </li><li> Инкубируйте вирус запасов / трипсин смеси в 37 ° С водяной бане в течение 30 минут. </li><li> Vortex на 5-10 минутным интервалом. </li><li> Приступить к части 2. </li></ul></li></ol> Часть 2: Заражение клетки <ol><li> Рассчитать количество вирусных частиц необходимо, чтобы заразить монослоя на желаемом множественности заражения (МВД). Как правило, высокие МВД (между 5 и 10) используется для инфекции timecourses. </li><li> Добавить необходимое количество сахарозы очищенный вирус или вирус трипсином сырой до 37 ° C вирусных питательной среды и хорошо перемешать. Вы должны использовать достаточно средств массовой информации, чтобы только покрыть дно колбы. Например, 10 мл среды для Т-175 колбу. </li><li> Удаление информации из ячеек и промойте комнатной температуре PBS. </li><li> Добавить вирус / вирусный питательной среды для каждой колбе. <ul style="list-style-type:lower-alpha;"><li> Swirl мягко, наклона пластин и инкубировать при температуре 37 ° С в 5% СО2-инкубатора в течение 1 часа. </li><li> Наклон и вихревые пластин каждые 15 минут распространение вируса равномерно и сохранить клетки влажным. </li><li> Для момента времени 0hr/Mock, добавить вирусные медиа роста только, без каких-либо вирус, добавленный. </li></ul></li><li> После 1 часа инкубации удалить средах, содержащих вирус, и полоскать в три раза при комнатной температуре PBS. </li><li> Добавить максимальное количество вирусных питательную среду для каждого флакона. Например, 30 мл среды для Т-175 колбу. Начало подсчета этом качестве точки 0 раз в час. </li></ol> Часть 3: Уборочная клетки <ol><li> Проверьте клетки под микроскопом и отметить любые цитопатический эффект (ЦПЭ). </li><li> Выньте материал и промойте клетки с 30 мл комнатной температуре PBS. </li><li> Добавьте 15 мл трипсина в клетки и инкубировать в течение 2-5мин при температуре 37 ° C. </li><li> Проверьте под микроскоп для мобильного отряда и нажмите колбе осторожно, чтобы выбить клеток. </li><li> Передача клеток с стерильных серологические пипетки в 50 мл коническую трубку. </li><li> Промыть колбу с равным объемом средств массовой информации роста клеток, а также добавлять СМИ трипсинизировали клеток в конической трубе. </li><li> Центрифуга клетки при 300 х г в течение 5 минут при комнатной температуре. </li><li> Удалить трипсина / СМИ от осадок клеток. </li><li> Ресуспендируют осадок клеток в любом TRIzol реагента или лизис буфера от желаемого комплект изоляции РНК. </li><li> Если вы используете TRIzol, разделить образца в 1 мл аликвоты в 1,5 мл меченых труб Эппендорф и заморозить при -80 ° C. </li><li> Повторите эти действия для всех временных точках, сбор урожая в одном флаконе момент времени. </li></ol> Часть 4: РНК добыча образцов в TRIzol <ol><li> На данный момент, ваши образцы должны быть ресуспендировали в TRIzol реагента и готовы к обработке. </li><li> Добавить 200 мкл реагента Разделение фаз BCP на каждый 1 мл TRIzol в каждой трубке. Возможно, вам придется перенести образец больше трубку, если вы начинаете с большим количеством TRIzol. </li><li> Vortex или энергично встряхивают и инкубируют при комнатной температуре в течение 2-3 минут. </li><li> Центрифуга образца при 12 000 мкг в течение 15 минут. </li><li> Передача водной фазы в новую пробирку. Водную фазу прозрачный слой на самом верху. Вы должны быть в восстановление примерно 600μL на каждый 1 мл TRIzol вы начали с. </li><li> У второго фенол / хлороформ добычи, на этот раз используя 500 мкл хлороформа на 1 мл TRIzol, вместо того, BCP. </li><li> Vortex или энергично встряхивают и инкубируют при комнатной температуре в течение 2-3 минут. </li><li> Центрифуга образца при 12 000 мкг в течение 15 минут. </li><li> Передача водной фазы в новую пробирку. Водную фазу прозрачный слой на самом верху. </li><li> Добавить 20 мкг линейных акриламида для каждого образца. Линейные акриламида выступает в качестве носителя и помогает осадок РНК. </li><li> Добавить 500-миCRO, L изопропанола на 1 мл TRIzol вы начали с для каждого образца и хорошо перемешать. </li><li> Инкубируйте образцы при комнатной температуре в течение 10 минут. </li><li> Центрифуга в микроцентрифужных на максимальной скорости в течение 10-15 минут. </li><li> РНК гранулы будут видны в нижней части трубы. Очень осторожно, удалить супернатант из трубки, либо с вакуумным аспиратором, или вручную с помощью пипетки. Не беспокоить гранул. </li><li> Вымойте гранул с 1 мл 70% этанола. <ul style="list-style-type:lower-alpha;"><li> Добавить 1 мл 70% этанола, пеллет. </li><li> Центрифуга с максимальной скоростью 14 000 г в течение 5-7 минут. </li><li> Очень осторожно, удалить этанола из трубки, либо с вакуумным аспиратором, или вручную с помощью пипетки. Убедитесь, что гранулы видна в нижней части трубы. </li><li> Удалите супернатант. </li></ul></li><li> Повторите мыть шаг. После надосадочную отбрасывается, отметить, где осадок на трубке. </li><li> Воздух сухой шарик на срок не более 5 минут. Не пересушивать или РНК будет трудно восстановить! </li><li> Если вы хотите следовать необязательный лечения ДНКазы для удаления остатков ДНК из образца, ресуспендируют осадок в 17μL нуклеазы без воды. В противном случае, ресуспендируют осадок в 20 мкл нуклеазы без воды и перейдите к шагу 22. </li><li> (Дополнительно лечение ДНКазы) Использование Qiagen РНКазы без ДНКазы установлен, добавить 2μL буфера RDD и 1 мкл РНКазы восстановленный без ДНКазы я к ресуспендировали РНК. Осторожно перемешать. </li><li> (Дополнительно лечение ДНКазы) Инкубировать при 37 ° С в течение 30 минут. </li><li> (Дополнительно лечение ДНКазы) Добавить 2μL в 2,5 мМ ЭДТА и инкубировать при температуре 65 ° С в течение 5 минут для инактивации ДНКазы. Не превышайте время инактивации или температуры как раз больше / более высоких температурах может привести к деградации РНК. </li><li> Проверьте концентрацию РНК спектрофотометр. </li><li> Храните образцы РНК при температуре -80 ° C. </li><li> (Дополнительно КК) Проверьте качество РНК с использованием Agilent BioAnalyzer, или путем запуска образца на денатурирующих гель и проверки OD чтениях. (260nm и 260/280 отношение) </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>

Критические Шаги Часть 1 и 2 Есть несколько важных шагов для создания синхронной инфекции вакцины, первая из которых осторожны ультразвуком (или trypzinizing) вируса, чтобы разбить вирусных частиц. Vaccinia очень склонны к агрегирования, и нарушение вирусных частиц им...

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<td>TRIzol Reagent</td>
<td>Reagent</td>
<td>Invitrogen</td>
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<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>
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vaccinia virus infection &amp; temporal analysis of virus gene expression: part 1

Семья<em&gt; Poxviridae</em&gt; Состоит из больших двухцепочечной ДНК, содержащие вирусы, которые копируют только в цитоплазме инфицированных клеток. Члены<em&gt; Orthopox</em&gt; Рода включают натуральной оспы, возбудитель человеческого оспа, обезьяньей, и вакцины (VAC), прототипных член вирус семьи. В течение относительно большого (~ 200 кб) вакцины генома, три класса гены закодированы: ранние, средние и поздние. Хотя все три класса транскрибируются вирусно-закодированных РНК-полимеразы, каждый класс выполняет различные функции в жизненном цикле вируса. Poxviruses использовать несколько стратегий для модуляции хост клеточного окружения во время инфекции. Для того чтобы понять регулирования хозяина и выражение гена вируса, мы использовали генома подходы к анализу стенограммы изобилии от обоих вирусов и клеток-хозяев. Здесь мы демонстрируем время курс инфекции клеток HeLa с вирус коровьей оспы и отбора проб РНК в нескольких точках времени после заражения. Оба хозяина и вирусных общей РНК выделяют и усиливаются для гибридизации микрочипов для анализа экспрессии генов.

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

Протокол Vaccinia заражения клеток HeLa и анализ хозяина и вирусных экспрессии генов. Часть 1 из 3.

Vaccinia вирусной инфекции и временной анализ вируса экспрессия генов: часть 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. Протокол Vaccinia заражения клеток HeLa и анализ хозяина и вирусных экспрессии генов. Часть 1 из 3.

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

Bacterial Gene Expression Analysis Using Microarrays

Бактериальный анализ экспрессии генов Использование Microarrays

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.

Подготовка первичных гемопоэтических клеточных культур из костного мозга мышей для Электропорация

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 вирусной инфекции и временной анализ вируса экспрессия генов: часть 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 вирусной инфекции и временной анализ вируса экспрессия генов: часть 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.

Вирус-индуцированные генов (VIGS) в Nicotiana benthamiana И томатный

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.

Использование генной Pulser MXcell Электропорация системы для трансфекции первичных элементов с высоким КПД

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.

Использование автоматизированных счетчиков сотовый для упрощения исследования экспрессии генов: миРНК Нокдаун ИЛ-4 зависимости экспрессии генов в клетках 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.

В пробирке восстановление активность Т. castaneum Теломераза

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.

Агробактериальной вирус-индуцированного Пробирной Отключение гена В Хлопок

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.

MultiBac белкового комплекса добывающей платформы на EMBL

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