第1部分：荧光标记的cDNA Cy3标记的芯片实验中使用的染料是敏感的光降解。该程序应采取少量的光的地方。改编自BioPrime阵列比较基因组杂交基因标记系统手册1标签协议。 <ol><li>标记反应试剂存放于-20 ° C。从BioPrime阵列比较基因组杂交基因标记系统解冻2.5X随机引物缓冲液，10X dCTP混合，无核酸酶水。此外解冻染料Cy3标记的dCTP。解冻后，所有试剂使用前短暂离心。 </li><li>一个单独的标记反应将需要为每个基因样本进行分析完成。添加到0.6毫升厚壁管500毫微克的cDNA，40μL的2.5倍随机引物缓冲和无核酸酶水总量达到86μL。 </li><li>充分混匀，短暂离心。孵育5分钟的反应在100℃热循环仪上一个彗星。 </li><li>立即转移到10分钟，冰浴浆反应。 </li><li>每管中，再加入10μL10X dCTP混合，加入2μl1毫米Cy3标记的dCTP和2μL埃克索的Klenow DNA聚合酶。埃克索的Klenow DNA聚合酶应留在冰箱，直到需要，应始终保持在冰上。 </li><li>混合反应良好，短暂离心。 </li><li>在37 ° C孵育16小时的热循环反应。 </li></ol>第2部分：标记的cDNA降水<ol><li>在继续之前，NimbleGen的杂交系统的协议转让系统平衡至42 ° C。 </li><li>对于每一个反应，放入1.5 ml管中的一个单片机列。加入300μL0.1X SSC的标记反应（100微升）的内容每一列。 </li><li>离心管列，其中包含10分钟，在16100 XG 4 ° C。 </li><li>放弃通过的流量，这可能是略带粉色的颜色，和另外300μL0.1X SSC添加到列。 </li><li>重复离心10分钟，在16100 XG在4 ° C。 </li><li>丢弃流量通过添加一个额外的300μL0.1X SSC列。 </li><li>离心12分钟，在16100 XG 4 ° C。 </li><li>丢弃流量通过。通过流动应该清楚这最后一次洗涤后，你应该可以看到粉红色标记的cDNA列。 </li><li> 100μL无核酸酶水的应用列。删除列从1.5毫升管和翻转到一个新的1.5 ml管的列。 </li><li>离心2分钟，在2300 XG在4 ° C。标记的cDNA应水化水收集在试管底部。 </li><li>根据制造商的指示使用一个NanoDrop ND - 1000分光光度计，计算DNA浓度和评估计算基染 ​​料的比例，其中核苷酸Cy3标记的比例等于260 / 550的比例由23.15乘以的染料掺入。基染料的比例应在50和80之间。 </li><li>如果标有足够的cDNA如果等分6微克的cDNA到一个新的1.5 ml管的标记。 </li><li>混合在0.1倍体积的3 M NaOAc，pH值5.2 1X体积异丙醇。允许混合物在黑暗中孵育30分钟，在室温下。 </li><li>下一步离心机16100 XG样品在室温下20分钟。 </li><li>小心丢弃，而不会干扰DNA沉淀，应在粉红色的上清。 </li><li>执行加入500μL70％乙醇，轻轻颠倒混合乙醇洗。 </li><li>离心样品在室温下5分钟XG 16100。 </li><li>去除上清，逆变管，并允许颗粒干燥5分钟。在这一步沉淀可在-20℃保存° C，如果需要，或该协议可以继续杂交步骤。 </li></ol>第3部分：杂交杂交过程是改编自罗氏NimbleGen阵列用户指南“的基因表达分析2。 <ol><li> 5μL无核酸酶水溶解沉淀。 </li><li>一旦沉淀溶解添加表1中所列的杂交组件。杂交组件从罗氏NimbleGen杂交试剂盒。所有组件都添加后总体积现在应该共18μL。拌匀，并简要旋管底部收集的内容。 </li><li>下一步是变性在95℃放置5分钟热块标记的cDNA。 </li><li>立即传送管NimbleGen的杂交系统，而数组在以下步骤准备。 </li><li>微阵列应小心处理和存储根据制造商的指示。罗氏NimbleGen的建议，在黑暗中干燥剂在室温下储存表达阵列。 </li><li>装入精密混合器调整工具提供的NimbleGen阵列处理配件（PMAT）阵列。 </li> &lt;李&gt;加载到PMAT X1的调音台，用镊子取出胶条。关闭安全PMAT。应用到调音台的压力，并通过在PMAT孔缓缓打开，使阵列和混频器的PMAT脱离。 </li><li>紧紧申请brayer与周围外部混频器的压力，以确保密封的阵列完成，并删除任何气泡。广场上的杂交系统阵列混合器复杂温暖。 </li><li>使用位移吸管，慢慢加入15μl标记样品的填充端口。这是在混频器的中心位于孔。应用缓慢而稳定的压力，吸液管，使样品均匀，无任何气泡通过调音台传播。从端口孔擦去多余的样品，并用胶带密封的端口孔。 </li><li>关闭闩锁并开始使用上的NimbleGen杂交系统解决方案B的杂交混合。允许芯片杂交16-20小时。 </li></ol>第4部分：杂交后的洗涤和微阵列扫描洗涤和扫描过程是改编自罗氏NimbleGen阵列用户指南2的基因表达分析。 <ol><li>一旦杂交的准备，如表2所列的洗缓冲区已接近完成。预温洗涤缓冲液执行机构至42 ° C水浴中。 </li><li> GenePix 4000B阵列扫描仪打开，让扫描仪在使用前约15分钟的热身。 </li><li>倒入一盘预热洗涤缓冲液执行机构。 </li><li>从NimbleGen的杂交系统和幻灯片夹具（提供的NimbleGen阵列处理配件），删除阵列的混频器复杂。浸大会进入预热洗涤缓冲液执行机构，并仔细清除剥落从幻灯片搅拌机。 </li><li>在洗涤缓冲液执行机构为15秒，取出夹具和鼓动阵列。 </li><li>快速传输到阵列在室温洗涤缓冲IB的滑动机架。大力幻灯片机架搅拌2分钟。 </li><li>转移到洗涤缓冲液II幻灯片机架，搅拌1分钟。 </li><li>转移洗涤缓冲液III和搅拌15秒的幻灯片机架。 </li><li>阵列转移到干燥的离心机幻灯片和自旋为2分钟，以除去所有水分。继续扫描。 </li><li>这是建议立即扫描阵列，因为染料是既轻又臭氧敏感。 </li><li>负载的阵列，条码下来，到GenePix 4000B阵列扫描仪。 </li><li>打开GenePix Pro软件进行扫描数组。罗氏NimbleGen基因表达阵列，利用一个彩色杂交战略。结果只有一个波长进行扫描。扫描Cy3标记的荧光信号，使用532 nm激光。打开硬件设置对话框，设置的532 nm激光的光电倍增管的价值500的值。 </li><li>在硬件设置对话框中，设置100％，平均为1至5微米的像素尺寸，线，电源和焦点位置为0微米。 </li><li>执行预览扫描并设置扫描区域。 </li><li>执行扫描。 </li><li>一旦扫描完成后检查图像的直方图。理想的情况下，1E - 5正常化数应在65000〜1-2％的特点是饱和的饱和极限的意义。如果规范化计数小于1E - 5，提高光电倍增管的增益和重新扫描的幻灯片。如果规范化计数大于1E - 5，降低光电倍增管的增益和重新扫描的幻灯片。 </li><li>一旦扫描完成后保存图像。建议选择在便于参考的图像标准命名公约。图像可以被装载到数据分析程序的数据规范化和强度值计算的偏好。 </li></ol>代表性的成果开始与0.1X SSC洗时，你可以先开始评估Cy3标记荧光标记的cDNA反应的效率。在其后每洗流过，应该成为粉红色，最后通过明确流。此外，Cy3标记的cDNA产物应列上可见，在这些洗涤步骤。如果通过的流量是在色彩明亮的粉红色，或有没有列上的粉红色产品，这是一个很好的迹象，没有正确地进行标记反应。标记反应可定量使用NanoDrop ND - 1000分光光度计，以确定标记的DNA和基染料的比例（图1）的浓度进行评估。标记反应，经常产生至少10微克标记的DNA，在我们的实验室。染料掺入可以通过使用一个简单的基地/染料比率核苷酸Cy3标记的比例等于260 / 550的比例由23.15乘以计算。值从50-80不等，表明足够的染料掺入标记反应。如果标记反应产生到话筒低浓度的DNA或较差的染料掺入，样品的杂交roarray不建议应反复标记反应。 可以通过计算图像的直方图和微阵列扫描图像（图2）进行杂交的质量评估。可接受的杂交将有1-2％的饱和特性（即高产1E - 5正常化计数65000饱和极限）近500集的PMT值。如果PMT值进行调整，从500大幅1E - 5正常化数达到65000饱和极限，这通常表示差标记的cDNA微阵列杂交。如果这个数据是继续通过随后的分析将包括数据质量。杂交质量还可以通过查看图像扫描阵列（图3）定性评估。罗氏NimbleGen杂交试剂盒包含对齐寡核苷酸的混合物Cy3标记和Cy5标记的48mer寡核苷酸阵列杂交对齐功能。对齐寡核苷酸的强度的定性评估，以阵列上的其他功能允许到阵列上的荧光标记的cDNA杂交效率的质量检查。经过进一步的分析，分析数据的散点图可以用来确定基因表达的相对水平，并确定您的样品（图4）之间的差异表达基因。 <img src="/files/ftp_upload/1471/1471figure1thumb.jpg" alt="图1的缩略图" /> 图1。从用Cy3标记的cDNA标记的cDNA产量和染料掺入NanoDrop的频谱可以评估使用NanoDrop ND - 1000分光光度计。请<a href="https://www.jove.com/files/ftp_upload/1471/1471figure1.jpg">点击这里</a>看到图1的放大版本。 <img src="/files/ftp_upload/1471/1471figure2thumb.jpg" alt="图1的缩略图" /> 图2。一个GenePix直方图可以扫描芯片杂交质量进行评估，通过评估图像的直方图。可接受的杂交将有1-2％1E - 5正常化计数饱和的PMT值近500 65,000饱和极限的功能。请<a href="https://www.jove.com/files/ftp_upload/1471/1471figure2.jpg">点击这里</a>看大图图2。 <img src="/files/ftp_upload/1471/1471figure3.jpg" border="0" alt="图3" /> 图3。通过查看扫描图像的芯片扫描芯片图像质量进行评估。杂交。应检查图像的存在灰尘或其他的干扰因素，禁止每个功能的可视化，从而防止功能的荧光强度的精确计算。对齐寡核苷酸的强度可以比阵列上的其他功能，以评估杂交效率。 <img src="/files/ftp_upload/1471/1471figure4.jpg" border="0" alt="图4" /> 图4。一个散点图分析芯片实验。此过程的目标是要确定，在您的样品的差异表达基因。微阵列数据可以进一步分析和散点图来看，可视化，差异表达基因。 表1。罗氏NimbleGen杂交试剂盒杂交第2步要添加的组件。 <table border="1"><tbody><tr><td> 组件 </td><td> 卷 </td></tr><tr><td>样品（溶解于水） </td><td> 5μL </td></tr><tr><td> 2X杂交缓冲液</td><td> 9μL </td></tr><tr><td> HYB一个</td><td> 3.6μL </td></tr><tr><td>对齐寡核苷酸</td><td> 0.4μL </td></tr><tr><td>总成交量</td><td> 18μL </td></tr></tbody></table> 表2。洗净准备以下杂交洗涤阵 ​​列的缓冲区。 <table border="1"><tbody><tr><td> 组件 </td><td> 洗涤缓冲液IA * </td><td> 洗涤缓冲液。 B </td><td> 洗涤缓冲液II </td><td> 洗涤缓冲液III </td></tr><tr><td>水</td><td> 225毫升</td><td> 225毫升</td><td> 225毫升</td><td> 225毫升</td></tr><tr><td>我10X洗涤缓冲液，第二，或第三</td><td> 25毫升</td><td> 25毫升</td><td> 25毫升</td><td> 25毫升</td></tr><tr><td> 1中号数码地面电视</td><td> 25μL </td><td> 25μL </td><td> 25μL </td><td> 25μL </td></tr><tr><td>总成交量</td><td> 250毫升</td><td> 250毫升</td><td> 250毫升</td><td> 250毫升</td></tr></tbody></table> *预温至42 ° C

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> BioPrime Array CGH Genomic Labeling System Manual. , (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Roche NimbleGen Arrays User's Guide for Gene Expression Analysis. , (2008).</li></ol>

许多芯片的协议，其中包括一个详细介绍，使用荧光染料，作为测量和量化杂交手段。 Cy3标记的荧光染料，是敏感的照片和臭氧降解。这是建议的程序，在最短的照明进行。此外，细小的灰尘颗粒可以干扰与杂交和扫描。应给予特别关注，以确保工作场所清洁无尘。 有许多替代品的标签协议，在此过程中介绍。建议各种标签协议进行测试，以确定每一个具体的表达微阵列平?...

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		<th>Catalogue Number</th>
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<td>Array Microcentrifuge Dryer</td>
<td>&nbsp;</td>
<td>ISC Bioexpress</td>
<td>C-1303-T</td>
<td>&nbsp;</td>
<tr>
<td>BioPrime Array CGH Genomic Labeling System</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18095-011</td>
<td>&nbsp;</td>
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<td>Cyanine 3-dCTP</td>
<td>&nbsp;</td>
<td>PerkinElmer</td>
<td>NEL576</td>
<td>&nbsp;</td>
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<td>GenePix 4000B</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>GENEPIX 4000B</td>
<td>&nbsp;</td>
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<td>Microcon YM-30</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>42411</td>
<td>&nbsp;</td>
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<td>NimbleGen Array Processing Accessories</td>
<td>&nbsp;</td>
<td>Roche NimbleGen</td>
<td>5223539001</td>
<td>&nbsp;</td>
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<td>NimbleGen Hybridization Kit</td>
<td>&nbsp;</td>
<td>Roche NimbleGen</td>
<td>5223474001</td>
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<td>NimbleGen Hybridization System 4</td>
<td>&nbsp;</td>
<td>Roche NimbleGen</td>
<td>5223652001</td>
<td>&nbsp;</td>
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<td>NimbleGen Wash Buffer Kit</td>
<td>&nbsp;</td>
<td>Roche NimbleGen</td>
<td>5223504001</td>
<td>&nbsp;</td>
<tr>
<td>X1 Mixers</td>
<td>&nbsp;</td>
<td>Roche NimbleGen</td>
<td>5223725001</td>
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global gene expression analysis using a zebrafish oligonucleotide microarray platform

基因芯片技术允许全基因组的基础上的转录水平和基因表达分析的定量测量。基因芯片技术是使用在众多的生物学科中的各种应用，包括全球的发育阶段的基因表达分析，一种疾病状态，并在毒性反应。在此，我们使用一个全面的斑马鱼的特异性寡核苷酸芯片平台的全球基因表达分析演示。斑马鱼的表达微阵列平台包含385000探头，60个碱基对，长度可达12％目标探头的审讯37157目标。对于这个平台上，所有的cDNA和斑马鱼的基因组信息收集各种基因​​组数据库，包括Ensembl（http://www.ensembl.org），天威（http://vega.sanger.ac.uk），加州大学圣克鲁兹分校（ http://genome.ucsc.edu），ZFIN（http://www.zfin.org）。因此，这种表达阵列提供完全覆盖目前斑马鱼的转录。斑马鱼表达微阵列是由罗氏NimbleGen（麦迪逊，威斯康星州）印刷。这种技术示范，包括荧光标记的cDNA产品，杂交标记的cDNA产品的芯片平台，使用一种颜色的分析策略的信号采集和阵列扫描。

Watch this Scientific Journal Video about Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform at JoVE.com

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

基因芯片在基因表达分析的有力工具，在全基因组水平。这项技术已经在各种生物学科包括发育生物学和毒理学中的应用。在这段视频中，我们详细介绍一个使用斑马鱼为一个全面的寡核苷酸芯片平台，全球的基因表达分析的协议。

全球基因表达分析，使用斑马鱼的寡核苷酸芯片平台

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

global-gene-expression-analysis-using-zebrafish-oligonucleotide

Research

JoVE Journal

Biology

11.6K 次观看.  Purdue University. 基因芯片在基因表达分析的有力工具，在全基因组水平。这项技术已经在各种生物学科包括发育生物学和毒理学中的应用。在这段视频中，我们详细介绍一个使用斑马鱼为一个全面的寡核苷酸芯片平台，全球的基因表达分析的协议。

视频: Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

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 ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

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 ...

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

白蜡窄吉丁的基因表达分析（ Agrilus planipennis）使用实时定量PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

确定基因表达概况 C。线虫使用微阵列和实时PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

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 ...

Gene Trapping Using Gal4 in Zebrafish

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional mutagenesis using fluorescent reporters to tag expression of mutated genes. Several laboratories have constructed and tested enhancer- and gene-trap vectors in zebrafish, using fluorescent proteins, Gal4- and lexA- based transcriptional activators as reporters 1-7. These vectors had two potential drawbacks: suboptimal stringency (e.g. lack of ability to differentiate between enhancer- and gene-trap events) and low mutagenicity (e.g. integrations into genes rarely produced null alleles). Gene Breaking Transposon (GBTs) were developed to address these drawbacks 8-10. We have modified one of the first GBT vectors, GBT-R15, for use with Gal4-VP16 as the primary gene trap reporter and added UAS:eGFP as the secondary reporter for direct detection of gene trap events. Application of Gal4-VP16 as the primary gene trap reporter provides two main advantages. First, it increases sensitivity for genes expressed at low expression levels. Second, it enables researchers to use gene trap lines as Gal4 drivers to direct expression of other transgenes in very specific tissues. This is especially pertinent for genes with non-essential or redundant functions, where gene trap integration may not result in overt phenotypes. The disadvantage of using Gal4-VP16 as the primary gene trap reporter is that genes coding for proteins with N-terminal signal sequences are not amenable to trapping, as the resulting Gal4-VP16 fusion proteins are unlikely to be able to enter the nucleus and activate transcription. Importantly, the use of Gal4-VP16 does not pre-select for nuclear proteins: we recovered gene trap mutations in genes encoding proteins which function in the nucleus, the cytoplasm and the plasma membrane.

This protocol describes the method of gene trap insertional mutagenesis using Gal4-VP16 as the primary reporter and GFP/RFP as secondary reporters in zebrafish. Approximately one in ten high-expressing F0 fish yield gene trap progeny co-expressing GFP and RFP. The screening procedure can be readily scaled to adapt to the size of the laboratory performing the insertional mutagenesis screen.

基因捕获斑马鱼中使用的Gal4

Large clutch size and external development of optically transparent embryos make zebrafish an exceptional vertebrate model system for in vivo insertional ...