作者希望感谢博士Cliona研究麦克海尔批判性阅读和编辑的稿件。这项工作是由美国国家环境健康科学，国家卫生研究所授予R01ES017452张为L研究所。

1。样品幻灯片准备<ul><li> OctoChrome鱼，旨在探讨在人体细胞，包括中期利差的染色体变化，但不限于外周血细胞培养，干/祖细胞和细胞株。样品应准备后的收获中期3,11-13的标准程序：采用秋水仙胺治疗逮捕中期，低渗处理的细胞肿胀细胞，卡诺的固定液固定在悬挂在约0.5-1×10 6个细胞的细胞每毫升。 </li><li>清理的8平方米的幻灯片（中的OctoChrome的套件，目录＃：大马803 Cytocell有限公司，剑桥，英国，图2）纯乙醇浸渍2分钟。 </li><li>样本幻灯片之前，拖放到幻灯片少量的细胞，细胞密度检查。如果细胞密度过高（太多的重叠细胞），与新鲜固定液稀释暂停。如果细胞的密度太低（太少细胞在幻灯片），自旋向下的细胞和重新暂停了新鲜固定液体积小。移液器5-10μL细胞悬液到8区交替广场1/7的序列模板幻灯片 - 2/8 - 3/5 - 4/6。一旦前两个广场已风干，发现以同样的方式，其余的广场。这将防止相互干扰细胞差。 </li><li>干幻灯片可以储存在-20°C氮气气氛中为杂交大于前5年。 </li></ul> 2。中期使用自动化系统的本地化<ul><li>申请4&#39;,6-diamidino-2-苯基（0.2微克/毫升）20μL中心幻灯片各占一半，然后盖上盖玻片。 </li><li>执行自动扫描的样本幻灯片本地化使用Metafer软件（MetaSystems，Altlussheim，德国）的中期细胞。这有利于所有的中期和未来重新评估所有的细胞图像的位置。 </li><li>手动查看扫描结果，并删除非中期的文物。 </li></ul> 3。杂交<ol><li>制备的样品幻灯片和OctoChrome的设备</li><ul><li>样本幻灯片2X SSC缓冲液在室温下浸泡30分钟洗去的DAPI。 </li><li>通过一系列的乙醇洗涤（每2分钟在70％，85％和100％乙醇）脱水样品的幻灯片，让干燥，放置在37℃5分钟烤盘。 </li><li>在37℃水浴中放置的Chromoprobe多功能的杂交厅（提供在OctoChrome工具包），并允许以平衡至37°（+ / - 1℃）。 </li><li>通过反复的移液和每OctoChrome设备预温25μl的等分混合杂交液（在OctoChrome工具包提供）至37°C。 </li><li>预温暖每个OctoChrome装置（的OctoChrome套件提供，请参阅图2）至37°C的设备标签的一面朝下放在烤盘上。请勿触摸t他的OctoChrome设备的浮雕表面，因为它们含有的探头。 </li></ul><li>样品幻灯片在OctoChrome设备定位（图2） </li><ul><li>加入2μL预热的杂交液前的的温暖OctoChrome设备的八个方面，而它仍然在37°C。 </li><li>仔细颠倒，在OctoChrome设备，如数字1，现在是倒挂，是通过位于顶部的右手区的OctoChrome设备模板的幻灯片。为了帮助找到平方米1，其设备上的地位已被标记为橙色。 </li><li>确保该模板的幻灯片仔细对齐匹配上OctoChrome设备领域。仔细降低在OctoChrome设备的幻灯片，幻灯片接触，使杂交溶液滴。适用于温和，甚至施加压力，以确保杂交液蔓延到每个提出上OctoChrome设备领域的边缘。 </li><li>抬磨砂玻片年底的幻灯片/ OctoChrome的，小心翼翼地捧着，反转，使幻灯片下方OctoChrome设备。确保设备不涂抹整个模板幻灯片，因为这可能导致探针的交叉污染。 </li><li>广场上37°（+ / - 1℃）10分钟的烤盘。 </li></ul><li>变性</li><ul><li>样本幻灯片/ OctoChrome设备转移到烤盘，采取特别的护理，以保持它的水平。确保样本幻灯片均匀接触的烤盘。 </li><li>烤盘上的变性在75°C（+ / - 1℃）5分钟。 </li></ul><li>杂交</li><ul><li>在Chromoprobe多功能杂交商会的地方样品幻灯片/ OctoChrome的设备。 </li><li>替换盖和浮动室在37°（+ / - 1°C）水洗澡（非 - 搅拌）隔夜。请注意：请勿杂交室盖密封，切勿密封盖子上的水洗澡，不要在杂交孵化器。这些步骤是关键控制湿度。 </li></ul><li>杂交后，清洗</li><ul><li>洗液的制备 解决方案1：含0.4倍SSC的准备Coplin / Hellendahl罐子。平衡到72°C间（+ / - 1℃）水浴中，并调整pH值至7.0。 解决方案2：含2X SSC和0.05％吐温20准备Coplin / Hellendahl罐子。在室温下静置。 </li><li>从幻灯片仔细删除OctoChrome设备，并放置2分钟，在方案1的幻灯片。 </li><li> 30秒的幻灯片放入解决方案2。 </li></ul></ol> 4。安装和结果的可视化<ul><li> 20μL的DAPI应用中心幻灯片各占一半，并适用于盖玻片。 </li><li>荧光显微镜下观察，在黑暗中前10分钟，在室温下孵育。 </li></ul> 5。代表结果 figu3A重新显示在正常的中期细胞广场2。 （1） - （3）：8日，21日和12染色体被涂成红色，绿色和蓝色，德州红，FITC标记，和DEAC过滤器，分别是通过可视化;（4）：通过的DAPI / FITC /可视化得克萨斯红三重过滤。一般来说，用蓝色画的染色体不明确，通过三重过滤，需要根据具体DEAC滤波器观看。 图3B显示代表特定的白血病染色体易位和非整倍体异常细胞。 （1）：T（8; 21），一种常见的急性髓细胞性白血病染色体易位;（2）：21三体综合征，21号染色体，在白血病的常见的非整倍体的三个副本。 <img alt="图1" src="/files/ftp_upload/3619/3619fig1.jpg" /> 图1。染色体上的OctoChrome设备和预期的染色体绘画结果组合安排。 <img alt="图2" src=“/ files/ftp_upload/3619/3619fig2.jpg”/&gt; 图2。定位样本幻灯片在OctoChrome设备的。 代表结果如图3。获得OctoChrome鱼（方2）。 <img alt="图3A" src="/files/ftp_upload/3619/3619fig3A.jpg" /> 图3A，正常细胞与染色体8，12，21广场2画。 <img alt="图3B" src="/files/ftp_upload/3619/3619fig3B.jpg" /> 图3B。代表在广场与特定的白血病染色体易位和非整倍体异常细胞。

<ol>
	<li>Levsky, J. M., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization:+past,+present+and+future.">Fluorescence in situ hybridization: past, present and future.</a> J. Cell. Sci. 116, 2833-2838 (2003).</li><li>Zhang, L., Eastmond, D. A., Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+of+chromosomal+aberrations+detected+in+humans+exposed+to+benzene.">The nature of chromosomal aberrations detected in humans exposed to benzene.</a> Crit. Rev. Toxicol. 32, 1-42 (2002).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrations+in+chromosomes+associated+with+lymphoma+and+therapy-related+leukemia+in+benzene-exposed+workers.">Aberrations in chromosomes associated with lymphoma and therapy-related leukemia in benzene-exposed workers.</a> Environ. Mol. Mutagen. 48, 467-474 (2007).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzene+increases+aneuploidy+in+the+lymphocytes+of+exposed+workers:+a+comparison+of+data+obtained+by+fluorescence+in+situ+hybridization+in+interphase+and+metaphase.">Benzene increases aneuploidy in the lymphocytes of exposed workers: a comparison of data obtained by fluorescence in situ hybridization in interphase and metaphase.</a> Environ. Mol. Mutagen. 34, 260-268 (1999).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+aneusomy+and+long+arm+deletion+of+chromosomes+5+and+7+in+the+lymphocytes+of+Chinese+workers+exposed+to+benzene.">Increased aneusomy and long arm deletion of chromosomes 5 and 7 in the lymphocytes of Chinese workers exposed to benzene.</a> Carcinogenesis. 19, 1955-1961 (1998).</li><li>Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+translocations+and+aneusomy+in+chromosomes+8+and+21+among+workers+exposed+to+benzene.">Increased translocations and aneusomy in chromosomes 8 and 21 among workers exposed to benzene.</a> Cancer. Res. 58, 2176-2181 (1998).</li><li>Bayani, J., Squire, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+detection+of+chromosomal+aberrations+using+spectral+karyotyping.">Advances in the detection of chromosomal aberrations using spectral karyotyping.</a> Clin. Genet. 59, 65-73 (2001).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome-wide+aneuploidy+study+(CWAS)+in+workers+exposed+to+an+established+leukemogen,+benzene.">Chromosome-wide aneuploidy study (CWAS) in workers exposed to an established leukemogen, benzene.</a> Carcinogenesis. 32, 605-612 (2011).</li><li>Rowley, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+chromosome+translocations+in+leukemogenesis.">The role of chromosome translocations in leukemogenesis.</a> Semin. Hematol. 36, 59-72 (1999).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+OctoChrome+fluorescence+in+situ+hybridization+to+detect+specific+aneuploidy+among+all+24+chromosomes+in+benzene-exposed+workers.">Use of OctoChrome fluorescence in situ hybridization to detect specific aneuploidy among all 24 chromosomes in benzene-exposed workers.</a> Chem. Biol. Interact. , 153-154 (2005).</li><li>Barch, M. J., Lawce, H. J., Arsham, M. S., Barch, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In The ACT cytogenetics laboratory. , 17-30 (1991).</li><li>Zhang, L., Yang, W., Hubbard, A. E., Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonrandom+aneuploidy+of+chromosomes+1,+5,+6,+7,+8,+9,+11,+12,+and+21+induced+by+the+benzene+metabolites+hydroquinone+and+benzenetriol.">Nonrandom aneuploidy of chromosomes 1, 5, 6, 7, 8, 9, 11, 12, and 21 induced by the benzene metabolites hydroquinone and benzenetriol.</a> Environ. Mol. Mutagen. 45, 388-396 (2005).</li><li>Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroquinone,+a+benzene+metabolite,+increases+the+level+of+aneusomy+of+chromosomes+7+and+8+in+human+CD34-positive+blood+progenitor+cells.">Hydroquinone, a benzene metabolite, increases the level of aneusomy of chromosomes 7 and 8 in human CD34-positive blood progenitor cells.</a> Carcinogenesis. 21, 1485-1490 (2000).</li><li>Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+understanding+benzene+health+effects+and+susceptibility.">Advances in understanding benzene health effects and susceptibility.</a> Annu. Rev. Public. Health. 31, 133-148 (2010).</li></ol>

我们什么都没有透露。

这种新颖的OctoChrome鱼法允许同时在一张幻灯片上的单一杂交研究所有人类染色体24。 8平方的染色体组合的安排，已设计，以方便识别中最常见的白血病和淋巴瘤的非随机的染色体重排。因此，该法可检测数值（非整倍体）以及结构（易位）染色体的改变同时进行。 在这个实验中最关键的一步是控制湿度在过夜杂交在水浴浮动商会。 FISH探针和杂交缓冲容易干燥，如果湿度低?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td><tr><tr><td> OctoChrome套件</td><td> cytocell有限公司，英国剑桥</td><td>大马803 </td><td></td></tr><tr><td>植物血凝素（PHA的） </td><td> Invitrogen公司</td><td> 10576-015 </td><td></td></tr><tr><td>秋水仙胺</td><td> Invitrogen公司</td><td> 15212-012 </td><td></td></tr><tr><td>卡诺的固定液</td><td></td><td></td><td>甲醇：冰醋酸= 3：1 </td></tr><tr><td>荧光公里croscope </td><td></td><td></td><td>配备过滤器，以查看德州红，FITC标记，香豆素光谱单独和的DAPI / FITC /得克萨斯州红三重过滤的DAPI，同时查看不同颜色</td></tr><tr><td> metafer软件</td><td> MetaSystems，Altlussheim，德国</td><td></td><td>方便找到所有中期和重新评估异常</td></tr></tbody></table>

chromosomics: detection of numerical and structural alterations in all 24 human chromosomes simultaneously using a novel octochrome fish assay

荧光原位杂交（FISH）是一种技术，使特定的DNA序列，中期或相间染色体检测细胞核1。该技术采用独特的序列杂交，整条染色体或特定的染色体区域的DNA探针，作为一个强大的辅助经典遗传学。例如，许多早期的研究报告中增加相关的白血病患者接触苯，苯中毒患者，接触苯的健康工人的染色体畸变的频繁检测，采用经典遗传学分析2。使用鱼，已观察到白血病特定染色体改变升高，显然健康的工人暴露于苯3-6，说明在苯致白血病细胞遗传学改变的关键角色。 一般来说，一条鱼检测只检查一个或几个整条染色体或每张幻灯片的特定基因，使多个杂交需要进行多张幻灯片，涵盖所有人类染色体。光谱核型分析（SKY）允许可视化的整个基因组的同时，但要求特殊的软件和设备的限制及其应用7。在这里，我们描述了一种新的鱼类试验，OctoChrome鱼，可用于Chromosomics的应用，这是我们定义为所有人类染色体24上的一个广泛的染色体非整倍体的研究，人类的研究，如幻灯片同步分析（CWAS） 8。作为Chromoprobe多功能系统由Cytocell上市的方法，基础是OctoChrome的的设备，被划分成8个广场，每个带有三种不同的整条染色体涂染探针（图1）。三个探头都直接标有不同颜色的荧光，绿色（FITC标记），红（得克萨斯红），蓝（香豆素）。染色体组合的安排上OctoChromE设备已设计，以方便识别中最常见的白血病和淋巴瘤中发现的非随机的染色体结构改建（易位），实例t（9; 22），T（15 17），T（8; 21 ），T（14 18）9。此外，在染色体上的数值变化（非整倍体）可以同时检测。相应的模板幻灯片，也分为8到中期利差是约束（图2），定位在OctoChrome设备的平方。在高温和潮湿室杂交探针和目标DNA变性，然后所有人类染色体24同时可以可视化。 OctoChrome FISH技术是一种很有前途的技术为白血病和淋巴瘤的临床诊断和所有染色体的非整倍体的检测。我们已申请在研究苯暴露中国工人8,10 CWAS这个新Chromosomic方法。

Watch this Scientific Journal Video about Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay at JoVE.com

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

一种新型的荧光<em&gt;原位</em&gt;杂交（FISH）的方法，同时检查数值和染色体结构的改变，尤其是特定的染色体易位的白血病和淋巴瘤相关的所有24人类染色体上的一​​个杂交的单设备，描述。

在所有人类染色体24 chromosomics：检测的数值模拟和结构上的改动同时使用一种新型OctoChrome鱼含量

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

chromosomics-detection-numerical-structural-alterations-all-24-human

Research

JoVE Journal

Biology

18.7K 次观看.  University of California, Berkeley. 一种新型的荧光原位杂交（FISH）的方法，同时检查数值和染色体结构的改变，尤其是特定的染色体易位的白血病和淋巴瘤相关的所有24人类染色体上的一​​个杂交的单设备，描述。

视频: Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

荧光原位杂交（FISH）的议定书“在人类精子

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

科研

生物学

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

检测信号效应复合物BMP4的下游使用原位解放军，接近结扎分析

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

评估具体物种的贡献颅面开发利用鹌鹑，鸭嵌合体

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

重组蛋白表达的结构生物学在HEK 293F悬浮细胞：一种新的和可访问方法

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

研究核苷酸合并: 酵母基因组 Ribonucleotides 的链特异检测及核苷酸诱变的检测

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...