这项工作是由人类前沿科学计划授予RGP0024。

1。 DNA模板制备及体外转录<ol><li>感兴趣的基因被克隆在任何T7和/或如SP6启动基于质粒。为了获得利益的RNC，C-末端的多肽的目标是扩大加入逮捕诱导序列的SECM FXXXXWIXXXXGIRAGP 7。为了确保感兴趣的多肽片段，将挤出的核糖体隧道，目标蛋白C-末端的一部分被延长至少30个氨基酸12-14。可以推出一个灵活的富含甘氨酸，丝氨酸连接器之间的蛋白质和SECM逮捕序列，以避免任何可能的构象约束。 </li><li> 在体外转录，模板DNA应限制酶切割的ORF下游线性。一个需要验证完整的线性质粒DNA的琼脂糖凝胶电泳上运行酶切产品。 </li><li>线性血浆ID被进一步用于体外转录反应。不同浓度的DNA模板可以进行测试，以确定最佳的DNA浓度在体外转录。一般用Ambion公司的MEGAscript高产转录试剂盒（Ambion公司/生命科技，大岛，NY），1微克的线性的DNA产生40-60微克的mRNA 在体外转录完成后，制造商的指示（Ambion公司/生命科技，大岛，NY） 。 </li><li>继在体外转录，mRNA的氯化锂沉淀纯化，根据制造商的指令（Ambion公司的MEGAscript高产转录试剂盒，Ambion公司/生命科技，大岛，NY）。 </li><li>使用丙烯酰胺或琼脂糖凝胶电泳进一步证实mRNA的完整性。 </li></ol> （2） 在体外翻译 在体外翻译使用的RTS 100大肠杆菌兴业套件（5总理，盖瑟sburg，MD）按照以下步骤： <ol><li>准备100μL 体外翻译反应下列制造商的指示。简言之，添加核酸自由水24μL氨基酸混合物减蛋氨酸（1毫米），10个单位的核糖核酸酶抑制剂（Invitrogen公司/生命科技，大岛，NY），20微居里的放射性[35]-蛋氨酸（MP的Biomedicals梭伦，俄亥俄州），20μL反应混合物，24μL重组缓冲区和24μL 大肠杆菌大肠杆菌裂解物。 </li><li>在30°C孵育5分钟预先的翻译反应热烈反应。新增1-2微克的mRNA的翻译反应，并在30°C孵育10-15分钟。 </li><li>停止反应，放置在冰的翻译反应。 </li></ol> 3。分离新生多肽在体外翻译反应<ol><li>为了隔离的RNC，翻译反应是分层4.5毫升5-30％蔗糖梯度上，在20毫米肝素钠，氢氧化钾pH值7.5，15毫米氯化镁 ，醋酸钾100毫米，1毫米数码地面离心41,000 RPM使用Beckman Coulter公司的SW55钛转子，4℃2小时Ç。 </li><li>蔗糖密度梯度离心后，分馏来隔离不同的核糖体的人口和相关的新生链。分离使用连续录音使用职业分类UA-6吸光度探测器在254纳米的职业分类可编程密度梯度系统监测。 </li><li>分数（S），含70S核糖体根据实验的目的，进一步收集和分析。 </li></ol> 4。观察蛋白质势必核糖体的Tris-甘氨酸SDS-PAGE 要进行检查，，SECM扩展蛋白是否仍然连接到70S核糖体，梯度馏分收集如下处理： <ol><li>在每个部分的蛋白加入终浓度10％三氯乙酸（TCA）沉淀和培养在4°ÇoverniGHT。 </li><li>过夜培养后，离心15分钟沉淀蛋白质14,000 XĞ。以下离心弃上清和沉淀两次洗涤液中含有丙酮：1毫米的Tris-HCl pH值7.6（4:1）。沉淀进一步风干，在SDS-PAGE上样缓冲液溶解。 </li><li>处理后的样品被解决的Tris-甘氨酸SDS-PAGE电泳分析。 </li><li>电泳后，凝胶固定，干燥，采用真空凝胶代尔受到显影。新生多肽的分布观察使用phosphoimaging。 </li></ol> 5。代表结果在这里，我们提出了一个实验描述全长牛- B伽玛稳定约束的70S核糖体的晶体的隔离。 图1描述了隔离牛γ-B晶体的RNC中涉及的步骤。 γ-B晶体的C-末端延长整体ţ由32个氨基酸Ø确保核糖体隧道，全长蛋白挤出出，这也包括放置在C-末端多肽的融合非常的的SECM拖延序列。继在体外翻译，70S核糖体分离蔗糖梯度离心法（图2.1）。为了确保保持稳定绑定到核糖体蛋白质，含有分数的70S汇集，交换脱盐，缓冲和受到额外蔗糖密度梯度离心轮（图2.2）。在图2的结果清楚地表明，SECM能有效地诱导转化逮捕的γ-B晶体的RNC。 <img alt="图1" src="/files/ftp_upload/4027/4027fig1.jpg" /> 图1。原理解释隔离的RNC所涉及的步骤。牛γ-B晶体的C-末端延伸，由32个氨基酸序列。这个扩展C-端区域，还包括SECM逮捕序列。 γ-B与晶状体SECM序列克隆在pIVEX 2.3（T7启动基于）质粒。 在体外转录模板DNA线性XbaI位。进一步用于体外转录线性模板。在DNA模板的T7 T7 RNA聚合酶转录的伽马 - B晶体蛋白基因的T7启动子下游，位于确认。使用氯化锂沉淀法纯化表达。然后用纯化的mRNA在体外翻译。以下孵化，翻译反应5-30％蔗糖梯度上呈层状和Beckman Coulter公司的SW55钛转子离心41000转，4℃2小时。 <sp一个类=“的pdflinebreak”&gt; <img alt="图2" src="/files/ftp_upload/4027/4027fig2.jpg" /> 图2。牛γ-B晶体隔离RNCS稳定约束的70S核糖体。1微克的mRNA混合100μl反应E.在协议部分中提到的20微居里[35 S的 大肠杆菌 S30的提取物系统-蛋氨酸，并在30°C孵育15分钟。以下孵化，翻译反应5-30％蔗糖梯度上呈层状，在20毫米肝素钠，氢氧化钾pH值7.5，MgCl 2的 15毫米，100毫米醋酸钾，1毫米数码地面电视和在贝克曼SW55钛转子离心41,000转速，4℃2小时。渐变的速度沉降，卸载和核糖体亲乐获得。由PeakTrak计划（职业分类梯度密度梯度分离系统）录得的数据。馏分收集，蛋白三氯乙酸沉淀和16.5％T的解决，6％C处的Tris-T的ricine PAGE凝胶15。凝胶干燥和为显影暴露。暴露后的凝胶台风9410成像扫描仪扫描。 图2.1显示了全长伽马-B的晶体是目前在70S核糖体组分。因此，SECM拖延序列允许稳定的牛γ-B晶体的RNC的隔离。 图2.2中的数据清楚地表明，孤立的RNC确实稳定。在目前的实验蔗糖密度梯度离心后的第一轮70S组分汇集和使用Amicon超4离心过滤单元（Millipore公司），由交换缓冲区中含有肝素钠氢氧化钾pH值7.5，15毫米20毫米的解决方案，其次蔗糖被删除MgCl 2的 100毫米醋酸钾，1毫米的DTT。这个范例是进一步受到离心通过额外的5-30％蔗糖梯度和分馏一轮。像图2.1中，每个部分的处理和分析。

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没有利益冲突的声明。

对于重复性的结果，使用的组件， 在体外转录和翻译的质量和浓度是关键。我们已经用市售的体外转录和翻译提取的，他们给高效率和可重复性的结果，如果小心处理。质量可以影响mRNA的翻译，所以它是最重要的测试之前使用它在体外翻译的mRNA的完整性。 体外翻译的孵化时间随长度的蛋白质。此外，离心时间/速度可相应调整的情况下，70S核糖体的分数没有得?...

<table border="1"><tbody><tr><td> 试剂名称/盒 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> MEGAscript T7高产转录试剂盒</td><td> Ambion公司</td><td> AM1333 </td></tr><tr><td>转运站100大肠杆菌兴业套件</td><td> 5总理</td><td> 2401100 </td></tr><tr><td>核糖核酸酶抑制剂</td><td> Invitrogen公司</td><td> 15518012 </td></tr><tr><td>反式[35]标签</td><td> MP的Biomedicals </td><td> 0151006 </td></tr><tr><td> amicon超4离心过滤单元</td><td> Millipore公司</td><td> UFC801008 </td></tr><tr><td>存储荧光粉显影</td><td> GE医疗集团</td><td>台风9410可变模式成像</td></tr><tr><td>密度梯度分离系统</td><td> Teledyne公司ISCO​​公司</td><td>职业分类可编程密度梯度系统</td></tr><tr><td>蔗糖梯度岑trifugation </td><td>贝克曼</td><td>舰L - 90 K制备超速离心机</td></tr></tbody></table>

using secm arrest sequence as a tool to isolate ribosome bound polypeptides

广泛的研究提供了充足的证据表明，细胞中蛋白质折叠是合作转化过程1-5。不过，确切的途径来实现其功能的形式在合作转化折叠多肽链的如下仍然是一个谜。为了了解这个过程，以确定确切的翻译合作的折叠中间体构象，它是必不可少的开发技术，允许携带预定大小的新生链的RNC，让他们进一步的结构分析的隔离。 SECM（分泌显示器）是一个170个氨基酸E.大肠杆菌蛋白，调节下游SECA（SECM SECA操纵6分泌驾驶）在ATP酶的表达。 nakatogawa和伊藤最初发现了17个氨基酸组成的长序列中的SECM蛋白的C端区域（150-FSTPVWISQA QGIRAĞ的P-166）是足够的和必要的造成拖延在Gly165 SECM伸长，从而产生肽，甘氨酰-tRNA的稳定势必核糖体P位7-9。更重要的是，它被发现，这17个氨基酸的长序列可以融合到几乎所有的全长和/或截断，从而使生产的RNC携带新生链的预定大小7蛋白C-末端。因此，当融合或插入到目标蛋白质，SECM拖延序列产生的多肽链延伸逮捕，并在体内产生稳定在大肠杆菌中的RNC 大肠杆菌细胞和无细胞系统在体外 。进一步利用蔗糖密度梯度离心法分离的RNC。 孤立的RNC可以用来分析合作翻译的折叠中间体的结构和功能特点。最近，这项技术已成功地用于获得多个核糖体结合10,11新生链的结构见解。在这里，我们描述了隔离牛γ-B晶体RNCS融合SECM和在体外翻译系统生成的。

Watch this Scientific Journal Video about Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides at JoVE.com

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

我们在这里描述，现在经常使用的孤立稳定结合的核糖体新生肽链复合物（RNC）中的技术。这种技术发现了17个氨基酸长SECM“逮捕序列”可以停止在原核翻译延伸的优势（<em&gt; E。大肠杆菌</em&gt;）系统，几乎所有的蛋白质（或C-末端融合）插入时。

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

using-secm-arrest-sequence-as-tool-to-isolate-ribosome-bound

Research

JoVE Journal

Biology

12.3K 次观看.  Cleveland State University. 我们在这里描述，现在经常使用的孤立稳定结合的核糖体新生肽链复合物（RNC）中的技术。这种技术发现了17个氨基酸长SECM“逮捕序列”可以停止在原核翻译延伸的优势（ E。大肠杆菌）系统，几乎所有的蛋白质（或C-末端融合）插入时。

视频: Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

微阵列后探测器（mPADs）：机械力的方法来隔离

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

科研

生物学

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

作为寄主植物挥发物的筛选工具electroantennographic生物活性

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

非洲爪蟾作为一种模型来确定翻译减值

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

考试蛋白在哺乳动物细胞中绑定到新生的DNA使用的BrdU芯片插槽西方技术

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

激光辅助显微切割（LAM）作为个体细胞类型的转录谱的工具

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

一种从小鼠中分离大脑围取体的高输出方法

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...