怀特黑德研究所的研究员基金

第1部分：设置感染<ol><li> HeLa细胞生长在烧瓶，等到约80％融合的细胞。 </li><li>准备足够的实验病毒的生长介质：含2％FBS的DMEM定期和不添加抗生素。 </li><li>感染可以进行原油库存的牛痘病毒与已知的滴度，或蔗糖纯化与一个已知滴度的病毒，如果你对宿主基因表达有关。 </li><li>如果您使用的是蔗糖纯化的病毒，请直接进入第2部分。 </li><li>如果您使用的是一个粗略的牛痘病毒的股票，只是之前的感染，超声一杯sonicator病毒等分的大多是冰的浴缸和一点点水。 <ul style="list-style-type:lower-alpha;"><li>在20秒左右30W超声。 </li><li>涡管和重复的超声3次，加入更多的冰，如果需要保持与冰和冷冻包装的杯子。 </li><li>涡之间的每个超声一步。 </li><li>进入第2部分。 </li></ul></li><li>另外，如果你不具备一个杯子sonicator，等体积混合的原油病毒库存和0.25mg/mL胰蛋白酶。 <ul style="list-style-type:lower-alpha;"><li>涡大力。 </li><li>股票/胰蛋白酶在37℃水浴孵育病毒混合30分钟。 </li><li>涡在5-10分钟一班。 </li><li>进入第2部分。 </li></ul></li></ol>第2部分：感染细胞<ol><li>计算病毒感染所需的感染复数（MOI）的单层颗粒的数量。通常情况下，高MOI（5和10之间），是用于感染timecourses。 </li><li>添加蔗糖纯化的病毒或胰酶消化原油病毒所需的金额，以37 ° C病毒的生长媒体和拌匀。您应该使用足够的介质，以刚好盖住烧瓶的底部。例如，10毫升的T - 175烧瓶媒体。 </li><li>从细胞中移除的媒体和室温PBS冲洗。 </li><li>添加到每个烧瓶中的病毒/病毒的生长介质。 <ul style="list-style-type:lower-alpha;"><li>涡流轻轻倾斜板和37 ° C为1小时5％CO2培养箱。 </li><li>每15分钟倾斜和旋流板，传播病毒的统一，保持细胞的湿润。 </li><li>对于0hr/Mock时间点，只添加病毒的生长介质，添加没有病毒的。 </li></ul></li><li> 1小时的潜伏期后，取出含有病毒的媒体，并与室温PBS冲洗三次。 </li><li>添加到每个烧瓶中的病毒的生长介质的最高金额。例如，30毫升的T - 175烧瓶媒体。开始计数为0小时的时间点。 </li></ol>第3部分：收获细胞<ol><li>细胞在显微镜下检查，并注意任何细胞病变效应（CPE）。 </li><li>取出介质，并与30毫升室温PBS冲洗细胞。 </li><li>添加15毫升的胰蛋白酶的细胞和2 - 5分钟，在37 ° C。 </li><li>在显微镜下检查细胞脱落和挖掘烧瓶中，轻轻地打跑细胞。 </li><li>血清50 mL锥形管，用无菌吸管转移细胞。 </li><li>洗细胞生长介质等体积的容量瓶中，并添加媒体在锥形管的胰酶消化细胞。 </li><li>离心机在室温下5分钟的300 ×克细胞。 </li><li>取出细胞​​沉淀的胰蛋白酶/媒体。 </li><li>无论你所需的RNA提取试剂盒TRIzol试剂或细胞裂解液重悬细胞沉淀。 </li><li>如果你是用TRIzol，分为1毫升分装在1.5ml的标记Eppendorf管中，并冻结的样品在-80 ° C。 </li><li>重复所有的时间点，每一个时间点瓶收获。 </li></ol>第4部分：在TRIZOL RNA提取样品<ol><li>在这一点上，您的样品应悬浮于Trizol试剂，并准备进行处理。 </li><li>新增的Trizol每1毫升每管BCP相分离试剂200μL。您可能需要将样品转移到一个较大的管，如果你开始与大量的Trizol。 </li><li>涡或用力振摇，并在室温下孵育2-3分钟。 </li><li>离心12,000 15分钟XG的样本。 </li><li>水相转移到一个新的管。水相是在顶部的层次清晰。你应该收回大约为每毫升TRIZOL你开始600μL。 </li><li>做第二个酚/氯仿抽提，而不是口岸这次使用的氯仿每1 ml TRIZOL500μL，。 </li><li>涡或用力振摇，并在室温下孵育2-3分钟。 </li><li>离心12,000 15分钟XG的样本。 </li><li>水相转移到一个新的管。水相是在顶部的层次清晰。 </li><li>每个样品中添加20μg线性丙烯酰胺。线性丙烯酰胺作为载体，有助于沉淀的RNA。 </li><li>新增500＆MICRO;异丙醇大号，每1毫升的Trizol，你开始了与每个样品拌匀。 </li><li>样品在室温下孵育10分钟。 </li><li>离心机在一个最高速度为10-15分钟，离心。 </li><li>的RNA沉淀在试管底部可见。非常仔细，从管中移除上清液，使用真空吸引器，或手动移液器。请勿打扰的颗粒。 </li><li> 1毫升70％的乙醇洗涤沉淀。 <ul style="list-style-type:lower-alpha;"><li>加入1毫升70％的乙醇沉淀。 </li><li>以最快的速度离心14000克为5-7分钟。 </li><li>非常仔细，从管中删除乙醇，使用真空吸引器或手动移液器。检查，沉淀在试管底部可见。 </li><li>上清液。 </li></ul></li><li>重复洗涤步骤。后取上清液丢弃，标记颗粒管。 </li><li>空气干燥颗粒不超过5分钟。不要overdry，或RNA的重组将难以！ </li><li>如果你想按照可选的DNA酶处理，以去除任何剩余的DNA样品，无核酸酶水悬浮颗粒在17μL。否则，重悬沉淀，无核酸酶水20μL，继续到步骤22。 </li><li> （可选的DNA酶处理）使用Qiagen公司的无RNA酶的DNA酶设置，添加2μL缓冲区的RDD和1μL重组无RNase的悬浮RNA的DNA酶我。轻轻混匀。 </li><li> （可选的DNA酶处理）37 ° 30分钟。 </li><li> （可选的DNA酶处理）添加2μL2.5毫米EDTA和孵化，在65 ° C至5分钟灭活DNA酶。不要超过灭活时间或温度，更长的时间/温度较高，可能会导致RNA的降解。 </li><li>检查由分光光度计的RNA浓度。 </li><li> RNA样品存放在-80 ° C </li><li> （可选的QC）使用安捷伦生物分析仪检查RNA的质量，或通过运行一个变性凝胶上的样品和检查的OD值。 （260nm和260/280的比例） </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>

关键步骤第1部分和2 有几个关键步骤设置同步的牛痘感染，小心病毒（或trypzinizing）超声，以分解病毒颗粒。牛痘是极易发生聚合，重要的是确保即使感染细胞的病毒颗粒的破坏。为了实现一个同步的感染，高MOI（大于2）应使用，以确保每一个细胞都感染。受感染和未受感染的细胞的混合物，会导致感染，多轮异构混合物的时间点，和异步的病毒与宿主转录...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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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>
</tr>
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<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

家庭<em&gt; Poxviridae</em&gt;由大型双链DNA含有病毒在感染细胞的细胞质中完全复制。成员<em&gt;痘</em&gt;属包括天花，人类的小痘病原体，猴痘，牛痘（VAC）的病毒家族的原型成员。在比较大（约200 KB）痘苗病毒基因组中，3类基因编码的早期，中期，和后期。维拉利编码RNA聚合酶转录虽然所有这三个类，每一类有一个病毒的生命周期中的不同功能。痘病毒利用调制主机在感染的细胞环境的多种策略。为了了解主机和病毒基因表达的调控，我们利用全基因组的方法，从病毒和宿主细胞的转录丰度分析。在这里，我们展示日久感染的HeLa细胞在几个时间点后感染牛痘病毒和采样RNA。主机和病毒总RNA分离和扩增为杂交微阵列基因表达分析。

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

协议牛痘感染的HeLa细胞和宿主和病毒基因的表达分析。

牛痘病毒感染及病毒基因表达的时空分析：第1部分

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 次观看.  MIT - Massachusetts Institute of Technology. 协议牛痘感染的HeLa细胞和宿主和病毒基因的表达分析。

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

Bacterial Gene Expression Analysis Using Microarrays

使用微阵列的细菌基因表达分析

科研

生物学

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.

主电穿孔的小鼠骨髓造血细胞培养制备

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.

牛痘病毒感染及病毒基因表达的时空分析：第2部分

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.

牛痘病毒感染及病毒基因表达的时空分析：第3部分

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.

病毒诱导的基因沉默（VIGS）烟草benthamiana和番茄

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.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

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.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

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.

在体外重组活跃T。谷盗端粒酶

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.

农杆菌介导的病毒诱导的基因沉默含量在棉花

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.

在EMBL的MultiBac蛋白质复杂的生产平台

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