梅哈里医学院，詹姆斯EK Hildreth，我们感谢他的意见和对手稿的审查。我们也感谢戴安娜Marver的手稿的编辑。这项工作是支持梅哈里范德比尔特艾滋病研究中心（美国国立卫生研究院授予AI054999 P30 - 05）;艾滋病健康差距研究中心，美国国立卫生研究院授予5U54 RR019192 - 05;梅哈里临床研究中心，美国国立卫生研究院批准P20RR011792的;由NIH授予P01 CA113239。 DJA部分资金由试点补助艾滋病研究的范德比尔特梅哈里中心（CFAR）和梅哈里临床研究中心（CRC）。

1。 KSVH培养和感染DMVEC <ol><li> BCBL - 1细胞系，从人体腔基于淋巴瘤原本孤立的，是培养完整的RPMI 1640媒体（Gibco公司，大岛，纽约州）。 BCBL - 1细胞从液氮，干冰运输，1分钟快速解冻在37 ° C。一个BCBL - 1细胞的50μL分装密度1 × 10 4细胞/ mL，加入500毫升的RPMI 1640媒体辅以10％小牛血清，青霉素1X /链霉素，并分发到175厘米烧瓶，在50毫升/瓶。 </li><li> BCBL - 1细胞的烧瓶，然后在37 ° C直到细胞密度达到3x106细胞/毫升5％的CO 2的气氛。加入20ng /毫升和钠在0.3纳克/毫升丁酸TPA诱导裂解周期病毒复制。 </li><li>二十四小时后诱导细胞在Sorvall传奇RT离心机1500 RPM（删除丁酸）一个40毫升的离心体积pH7.4的PBS中洗两次，并与TPA诱导20毫微克/毫升在继续RPMI 1640培养5天以上。 </li><li>无细胞病毒是孤立的，集中差速离心。简言之，细胞和细胞碎片首先在Sorvall RC - 6加离心机在4 °彗星颗粒30分钟以10,000 rpm。上清含有的病毒被删除和细胞游离病毒颗粒在4 ° C在Beckman Coulter公司的Optima L - 90K超速离心机在25,000 rpm时为1小时。 </li><li>无病毒上清被删除，病毒颗粒悬浮在冻结媒体（10％的二甲基亚砜和90％胎牛血清），并保存于-80℃直至需要。 KSHV的基层DMVEC细胞的感染。 </li><li>通过一级2-5 DMVEC细胞接种于初始密度为1 × 10 5 100x20mm组织培养皿或玻片，并保持完整的EMB - 2媒体（龙沙，巴塞尔，瑞士），在37 ° C在5％的大气中的CO 2 。在80％汇合，DMVEC被感染MOI的0.01;模拟受感染的细胞（媒体）作为对照。新媒体被添加到细胞中，每2天。 </li><li> DMVEC单层细胞玻片检查，每天由梭形细胞形成指出感染的证据。梭形细胞形成后的第一个迹象（7-14天），我们停止媒体的变化。然而，在超过15-21天，而不会提供新媒体的潜伏期较长时间可以妥协DMVEC细胞，导致细胞死亡或解除培养皿的完整性。 </li><li>在感染过程中的最大spindling点，单层细胞蛋白质或RNA提取的细胞沉淀的准备收获。感染DMVEC细胞玻片洗两次，用1 mL /预热PBS pH值7.4的窗口。玻片上的单元格的收获洗涤后用PBS pH 7.4的2倍。让他们完全晾干后方可卸下商会和放置在免疫荧光法（IFA）的需要，在-20 ° C，直到100％甲醇的幻灯片。 </li></ol> 2。总RNA提取和cDNA的生产从模拟和KSHV感染DMVEC <ol><li>感染KSHV的DMVEC和使用Qiagen公司RNesay的试剂盒（Qiagen公司，瓦伦西亚，CA）模拟受感染的细胞中提取总RNA。制造商的协议处理动物组织采用，但没有同质化的步骤，如果用于RNA分离培养的细胞源。 </li><li>选择提供治疗上，使用Qiagen公司RNA免费DNA酶试剂盒的列DNAseI的RNA，根据制造商的建议在说明书的附录位于的选项。 DNAaseI降低任何污染的基因组RNA制备或病毒DNA。 DNAseI治疗是下游应用，包括RT - PCR定量RT - PCR和基因芯片分析的关键。 </li><li> RNA是根据洗涤缓冲液中的说明和任何残留的乙醇是由离心列更多的时间比制造商建议删除洗净。残留的乙醇洗脱后的RNA的纯度妥协。 </li><li>最后一次洗涤后，一个额外的离心步骤是执行之前的RNA可以从列洗脱。 RNA是从30μL的RNase自由水列洗脱。然后量化分析和纯度的RNA是一个的Eppendorf AG - 6131生物光度计。光密度比值（260/280 nm）的应between1.8 - 2.0这是典型的高纯度的RNA。 </li><li> RNA的基因芯片分析，安捷伦2100生物分析仪上进行分析，使用RIN的软件算法。生物分析仪的分析提供了与RNA的完整性数量从1-10（RIN），但是你的RIN从7-10号码已被证明产生芯片或定量RT - PCR分析相一致，更可靠的结果。 RIN的人数平均为RNA隔离特德在我们手中的Qiagen公司RNAeasy套件范围从7.5-9。 </li><li> RNA定量是通过加入1μL洗脱RNA列99μL10MM的Tris - EDTA pH值7.6。 RNA和缓冲区吸管混合，并添加到一个干净的比色皿的Eppendorf生物光度计光度分析。 </li><li>信使RNA是在每个样品微克底漆使用由制造商提供的随机六聚体，扭转前与一个高容量的cDNA反转录试剂盒（应用生物系统公司的Foster City，CA。）的转录。 cDNA合成 </li><li>合成的单链从我们使用高容量的cDNA反转录试剂盒由美国应用生物系统公司，并继续根据制造商的指示总RNA的cDNA。 </li><li> 2X主混合的试剂和0.5至1微克的RNA在10μL的体积是10μL2X主试剂混合。 1X解决方案，轻轻混合，并根据制造商的说明进行逆转录是在一个BIORAD MJ迷你热敏纸料循环仪。 </li><li>两微克总RNA可用于每20μL的反应。一个的Eppendorf AG - 6131生物光度计在260 nm的是定量的cDNA，并保存于-80℃直至QRT - PCR所需。 </li></ol> 3。芯片模拟和KSHV感染DMVEC细胞的比较分析我们的实验室进行微阵列分析，在美国范德比尔特大学医学中心的微阵列共享资源上的有偿服务的基础上的核心设施。我们比较KSHV感染DMVEC细胞模拟感染对照细胞的基因转录失调在输出文件。感染的细胞在KSHV的基因转录失调，血管生成，细胞增殖相关的，或代表转移的生物标志物使用的KSHV感染的实时PCR，Western印迹和双独立财务顾问 DMVEC细胞在体外进行验证，结果相比，结果在档案金水肿瘤组织。 4。定量RT - PCR的转录失调基因分析发现在模拟和感染KSHV的DMVEC <ol><li>实时PCR是使用逆转录酶产生的基因来自纯化的RNA样品，并使用一个MyiQ单色实时PCR检测系统（Bio - Rad公司实验室，大力士，CA），96光板（索伦森生物科学公司） 25μL反应体积。这个过程是在生物安全柜，以避免可能的污染，多余的DNA /基因，可能是在房间里。 </li><li>一个主混合是准备在1.5毫升管每个引物设置使用SYBR GREEN Supermix（Bio - Rad公司实验室，大力士，CA）和选定的提出和感兴趣的基因的反向引物（根据制造商的指示），精神高度集中每口井的250纳米，在RNA酶DNAase免费H 2 O每个样品应做一式三份。 </li><li>宿主细胞基因QRT - PCR引物序列包括KSHV的LANA的，这是高度的KSHV感染的DMVEC细胞和KS肿瘤表达的病毒潜伏期相关核抗原的引物序列。 </li><li>为10 ng / UL股票从模拟感染和KSHV感染DMVEC细胞的cDNA稀释1:3使用RNA酶DNAase免费H 2 O;3μL此稀释，每孔加入。控制井的cDNA代替水。 </li><li> 96孔板是密封的光封箱胶带（Bio - Rad公司实验室），轻轻混合，并在1000 rpm，室温，反应混合物在井的底部收集离心2分钟。在Bio - Rad MyiQ检测系统和灯被打开，此时允许在开始运行前一个10分钟的预热期。 </li><li>离心96孔板中，然后将放在一个中号Bio - Rad公司IQ单色实时PCR检测系统，计划周期序列的输入和保存，并开始运行。 </li><li>循环顺序如下：95℃3分钟，95 ° C 15秒，60 ° C为1分钟，95 ° C为1分钟，55 ° C为1分钟，55℃，30秒共81个周期。作为底漆效率的检查，包括在这一方案中熔解曲线。一个GAPDH的引物扩增，包括正常化。 </li><li>数据分析是在Bio - Rad iQ5光学系统软件版本2。 </li></ol> 5。 KSHV感染DMVEC细胞的双标记免疫荧光分析<ol><li>玻片（商会删除），其中包含两者融合的感染和未感染DMVEC洗净，用PBS pH值7.4的两倍，空气干燥，-20℃预冷10分钟的绝对甲醇固定，并在-20 ° C举行</li><li>与细胞的幻灯片是日EN空气干燥15分钟，成Coplin瓶放在冰含5分钟，三液（0.05M的Tris pH值7.4）。细胞，然后1小时在37 ° C在湿盒与KSHV的LANA的混合单克隆抗体在1:50稀释在PBS pH值7.4（矢量实验室，伯林格姆，CA），加上一个山羊多克隆抗体（1： 100 pH值7.4的PBS稀释）对宿主因子蛋白被发现使用的KSHV感染的DMVEC细胞芯片dyregulated的。 </li><li>细胞，然后洗净3X三生理盐水（决不允许干细胞），然后孵育30分钟次要的驴抗小鼠IgG抗体与罗丹明红色X和驴抗山羊抗体结合的混合共轭FITC（杰克逊ImmunoResearch，西树丛，PA），在PBS 1:100稀释。 </li><li>细胞后洗净3X三生理盐水在Coplin JAR，而幻灯片仍然潮湿，幻灯片Vectashield安装媒体（媒介实验室，伯林格姆，CA）含有1.5克/毫升的DAPI和盖玻片上。 </li><li>荧光观察，并安装一个电荷耦合器件（CCD）照相机尼康TE 2000S荧光显微镜拍摄的图像。照片是使用了200X的总放大倍率为20倍目标。 </li><li>采取单独的照片，使用相同的放大倍率的DAPI（200X）usinge紫外光照射，与罗丹明绿色的过滤器和FITC标记的蓝色过滤。使用尼康的免费软件，以后合并的图像。 </li></ol> 6。 KSHV的拉娜和内皮细胞标记Pecam-1/cd31 KS组织和KS组织芯片上的双标记免疫组化<ol><li>双标记免疫组化DAKO公司strepavidin ABC对唱KSHV的LANA的宿主细胞，发现病毒感染后改变蛋白质染色试剂盒。 IHC执行档案艾滋病KS福尔马林固定组织，石蜡包埋。 </li><li> 5微米的部分被切掉一个切片，置于凯美幻灯片，以免疫前。 </li><li>石蜡包埋的幻灯片deparaffinzed二甲苯通风柜下一个10分钟，然后细胞水合了100％，95％，每五分钟和70％的乙醇梯度系列。幻灯片，然后放置在水中，10分钟前抗原揭露（又称抗原修复）。 </li><li>免疫，抗原揭露是在微波炉中的50 mM柠檬酸pH值6.0在95 ° C 15分钟的缓冲区。简言之，幻灯片含有细胞被淹没在95 ° C的柠檬酸缓冲液为15分钟，然后立即放置在室温下举行的蒸馏水。 </li><li>淬火内源性过氧化物酶是由孵化淬火溶液中30分钟的幻灯片。简单地说，添加47毫升95％乙醇50 mL锥形管;添加11.2毫升PBS pH 7.4的共60毫升和1.8毫升3％的双氧水。 淬火液在使用前。 </li><li> pH值7.4的PBS洗涤细胞在室温下每5分钟的3倍。准备一个封闭液（12.5毫升95％的乙醇，正常山羊血清1.4毫升，140毫克牛血清白蛋白，和2滴吐温20），以防止非特异性结合的抗体。使用前置于冰上。 （此阻塞的解决方案可以被保存在4 ° C和1个星期）。 </li><li>封闭液添加到幻灯片上的标本，再覆盖地带的石蜡（以避免干燥标本），置于湿盒，并在室温下1小时孵育。 </li><li>病毒抗原的单克隆或多克隆抗体的主要是准备堵在1:50稀释液，用吸管混合，置于冰上，直到需要。五十至100μL抗体溶液，将需要根据标本的大小和数量的幻灯片，每个组织标本。 </li><li>孵化后立即封闭液摆脱幻灯片和50-100μL抗体加入到的组织样本，然后用石蜡地带覆盖。标本在室温下湿盒中孵育1-2小时。 </li><li>幻灯片包含标本，然后洗3倍，pH值7.4的PBS每添加一个中学的生物素标记的羊抗鼠抗体在pH值7.4的PBS 1:100稀释，并在在室温下湿盒30孵育5分钟 - 45分钟。 </li><li>使用前约30分钟，pH值7.4的PBS过氧化物酶标记的strepavidin准备。使用DAKO公司Cytomation二重奏套件，50μL的解决方案添加一个5毫升PBS pH值7.4，旋涡，然后添加50μL溶液B混合再次。 </li><li>一步完成6.10抗体孵育后，幻灯片洗3倍的PBS pH值7.4，然后在新鲜公关epared辣根过氧化物酶标记的strepavidin解决方案的补充，并在室温下孵育30-45分钟在湿盒。 </li><li>孵育后，幻灯片pH值7.4的PBS洗涤3倍于室温。洗涤前5分钟完成，3，3 - 二氨基联苯胺（DAB，SIGMA）底物溶液（20毫升PBS pH值7.4，3％的双氧水25μL，和1民建联平板）准备。民建联片完全溶解后，棕颜色的解决方案是通过一个0.45微米注射器过滤器过滤。过滤DAB溶液，然后加入到标本后，最后一次洗涤和KSHV的LANA的颜色在感染细胞的发展是实时明照明，使用尼康的Eclipse E200显微镜下观察。一旦实现发展所需的颜色，反应是淬火在蒸馏水的潜伏期为5分钟。 </li><li>对于双标记免疫组化，孵化后在水中含有标本的幻灯片通过IHC协议第二抗原检索步骤（6.4）开始。然而，在过氧化物酶底物一步（6.11）的协议的变化，而不是采用辣根过氧化物酶的共轭strepavidin，碱性磷酸酶strepavidin共轭</li><li>第二个标签是通过一个额外的抗原在微波的检索步骤，如上所述，加上与单克隆抗体免疫细胞蛋白被发现失调（矢量实验室，伯林格姆，CA）。随后洗细胞，然后培养与生物素化山羊anti-mouse/rabbit IgG抗体（DAKO公司），碱性磷酸酶strepavidin共轭（矢量实验室，伯林格姆，CA）在1:500稀释1:100稀释。色彩的发展是实现培育细胞与基底向量，根据制造商的说明使用一个Vector红碱性磷酸底物试剂盒（媒介实验室，伯林格姆，CA）红色。反染色，如果需要的话，可以进行与orecin或苏木。组织标本，然后永久地安装一个玻璃盖，与permount安装媒体。明照明用尼康TE - 2000S与CCD相机安装在显微镜下的图像。 </li></ol> 7。 DMVEC细胞腺病毒转导的基因传递<ol><li>电镀密度2.5 X10的5％的细胞室EBM - 2完整的媒体师范大学DMVEC细胞培养玻片。 </li><li> DMVEC细胞增长到80％汇合（服用3-5天）后5 UL /腺病毒表达EGFP室滑动窗口在滴度1X 10颗粒/毫升。 </li><li>现场DMVEC细胞培养，以腺病毒添加EGFP荧光18小时后加入病毒研究。 </li><li>个人相和荧光灯的照片被采取了200X的总放大倍率，和一台尼康TE 2000S荧光显微镜与CCD相机安装合并。 </li></ol> 8。代表性的成果<img src="/files/ftp_upload/2078/2078fig1.jpg" alt="图1" /> 图1。龙沙公司的主DMVEC细胞均保持在EBM - 2介质为0.01教学语言。BCBL - 1病毒被感染后感染DMVEC的细胞单层日常检查。十天后感染模拟感染的细胞表现出鹅卵石如形态和KSHV感染的细胞表现出一个梭形细胞型主轴形血管内皮细胞的形态特征，发现感染KSHV的在堪萨斯州的肿瘤组织。照片是在100X的总放大倍率的显微镜与CCD相机安装了尼康TE 2000S。 <img src="/files/ftp_upload/2078/2078fig2.jpg" alt="图2" /> 图2。 KSHV的感染，在感染后10天DMVEC细胞进行双标记间接免疫荧光染色 。KSHV的LANA的一个山羊多克隆抗体的发现改变后的KSHV感染特定的宿主细胞蛋白的单克隆抗体的双重感染的细胞染色。中学的驴抗小鼠和驴抗山羊罗丹明和FITC标记的抗体混合物。装有CCD相机与尼康TE2000S荧光显微镜研究细胞。用于可视化细胞核用DAPI安装媒体。所有的照片是在一个200X的总放大倍率。受感染的细胞表达的LANA的病毒蛋白（罗丹明染色）的本地化，以核降低宿主细胞的特定蛋白质（FITC染色）局部细胞质表达。 <img src="/files/ftp_upload/2078/2078fig3.jpg" alt="图3" /> 图3。 KSHV的LANA和PECAM - 1 /艾滋病KS肿瘤组织单和双标记免疫组化染色在KS肿瘤组织的免疫组化的LANA和PECAM - 1 / CD31的 。D31。 A）显示，用单克隆抗体的LANA KS组织染色与苏木素复染。 LANA的阳性细胞是确定由白色箭头。 B）。金水组织染色的单克隆抗体PECAMI/CD31，并具体到周围白色箭头描绘血管内皮染色出现。 c）显示了堪萨斯州组织的LANA（载体红色）和CD31（棕色）双染色为CD31阳性细胞染色LANA的一个白色的黑色箭头描绘。 d）显示面积从同一幻灯片面板C.明场图像放大倍率总放大倍率600X交流TE2000S在尼康显微镜拍到面板和200X D。 <img src="/files/ftp_upload/2078/2078fig4.jpg" alt="图4" /> 图4。 KSHV的LANA的时空表达的QRT - PCR在感染后10天相比，模拟感染对照细胞。KSHV感染DMVEC的细胞的所有值，以GAPDH的正常化。没有证据的LANA在模拟感染细胞的cDNA扩增。 <img src="/files/ftp_upload/2078/2078fig5.jpg" alt="图5" /> 图5。 KS肿瘤组织芯片 ，组织芯片（TMA）的代表摆着一张幻灯片上的0.6毫米的组织核心。每个组织的核心代表了从不同病人的KS的肿瘤标本。财资市场公会可通过艾滋病癌症标本资源利昂娜艾尔斯在俄亥俄州立大学博士（ASCR）。随着匹配的正常和积极的控制组织KS肿瘤组织石蜡包埋，分别置于载玻片上。堪萨斯州组织标本从口腔，皮肤，软腭，舌和颈部群众。所有患者均HIV阳性。 <img src="/files/ftp_upload/2078/2078fig6.jpg" alt="图6" /> 图6。组织芯片采用免疫组织化学染色KSHV的LANA明显微镜分析颜色的LANA发展与民建联作为过氧化物酶底物和阳性细胞LANA的可视化是一个激烈的棕黄色染色格局。可以观察到不同病毒的负担和梭形细胞区域。 a）表示局灶性在定义区域的肿瘤病毒感染。二）传播感染，大致与目前梭形细胞数目相关的具有高病毒负荷。彩绘的组织核心，在200X的放大倍率观察。 <img src="/files/ftp_upload/2078/2078fig7.jpg" alt="图7" /> 图7。 EBM - 2完整的媒体在2.5 X10的5％的细胞密度栽培DMVEC细胞转5μL腺病毒滴度为1.0 × 10。细胞EGFP荧光18小时后传导检查。尼康TE 2000S显微镜的总放大倍率200X相位和荧光图像。初级DMVEC细胞已被证明是问题，因此，我们已经确定，将使我们能够在小学DMVEC fibulin - 2和galetin - 3非常有效的病毒载体转染。我们发现，表达EGFP的腺病毒能有效地转导的主要DMVEC。我们还发现，我们从Lentigen公司获得的慢病毒载体转导的主要DMVEC细胞（数据未显示）也同样高效。

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在这份报告中所采用的方法可以进行各种实验室设置。他们需要一些特殊的设备或核心设施。涉及感染性病毒如KSHV的病原体，这可能需要一个biosaftey 3级（BSL - 3）设施的使用做实验时，必须采取适当的预防措施。组织收购，也必须由适当的机构审查委员会（IRBs）的批准。 这些方法可以提供确定基因， 在体外表达失调，并提供定义的方法，以及改变基因的表达谱基因?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>05-0001DJ</td>
<td>&nbsp;</td>
<tr>
<td>TPA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1585</td>
<td>&nbsp;</td>
<tr>
<td>PBS pH 7.4</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10010-023</td>
<td>&nbsp;</td>
<tr>
<td>Sodium butyrate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>19364 (FLUKA)</td>
<td>&nbsp;</td>
<tr>
<td>EBM-2 media</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC-3156</td>
<td>+ bullet kits supplement </td>
</tr>
<tr>
<td>DMVEC cells (HMVEC)</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>CC2543</td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30066.03</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Chamber slides</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>154461</td>
<td>&nbsp;</td>
<tr>
<td>KSHV LANA Mab</td>
<td>&nbsp;</td>
<td>Vector Labs</td>
<td>VP-H913</td>
<td>1:50 dilution/IFA</td>
</tr>
<tr>
<td>Galectin-3 goat pAb</td>
<td>&nbsp;</td>
<td>R&D Systems</td>
<td>AF1154</td>
<td>&nbsp;</td>
<tr>
<td>Donkey-anti-goat FITC</td>
<td>&nbsp;</td>
<td>Jackson ImmunoRessearch</td>
<td>705-095-1</td>
<td>&nbsp;</td>
<tr>
<td>Donkey-anti-goat Rho</td>
<td>&nbsp;</td>
<td>Jackson ImmunoRessearch</td>
<td>705-295-003</td>
<td>1:100 dilution/IFA</td>
</tr>
<tr>
<td>Mounting media</td>
<td>&nbsp;</td>
<td>Vector Labs</td>
<td>H 1500</td>
<td>Vectashield with DAPI</td>
</tr>
<tr>
<td>BCA Protein Assay Kit</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>23235</td>
<td>&nbsp;</td>
<tr>
<td>Nitrocellulose </td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>161-0112</td>
<td>&nbsp;</td>
<tr>
<td>Donkey-anti-goat-conj. </td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>SC 2020</td>
<td>&nbsp;</td>
<tr>
<td>Western substrate</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>34075</td>
<td>&nbsp;</td>
<tr>
<td>Biotin-rabbit-anti goat</td>
<td>&nbsp;</td>
<td>DAKO</td>
<td>305-065-045</td>
<td>&nbsp;</td>
<tr>
<td>AP-Strepavidin conj.</td>
<td>&nbsp;</td>
<td>DAKO</td>
<td>K 0492 (kit)</td>
<td>&nbsp;</td>
<tr>
<td>RNase DNase Free Set</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>79254</td>
<td>&nbsp;</td>
<tr>
<td>MJ Mini Cycler</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>PTC-1148C</td>
<td>&nbsp;</td>
<tr>
<td>Bio-Photometer</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>952000006</td>
<td>&nbsp;</td>
<tr>
<td>RC 6 Plus Centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>46910</td>
<td>&nbsp;</td>
<tr>
<td>Coulter Optima L-90K Ultracentrifuge</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>392052</td>
<td>&nbsp;</td>
<tr>
<td>MYiQ iCycler Real Time PCR Detection System</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>170-9770</td>
<td>&nbsp;</td>
<tr>
<td>TE 2000S Fluorescent microscope</td>
<td>&nbsp;</td>
<td>NIKON</td>
<td>TE2000S</td>
<td>&nbsp;</td>
<tr>
<td>2100 Bioanalyzer</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>G2938C</td>
<td>&nbsp;</td>
<tr>
<td>Eclipse E 200</td>
<td>&nbsp;</td>
<td>NIKON</td>
<td>E 200</td>
<td>&nbsp;</td>
<tr>
<td>Sorvall Legend RT Centrifuge</td>
<td>&nbsp;</td>
<td>Thermo</td>
<td>75004377</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

identifying dysregulated genes induced by kaposi's sarcoma-associated herpesvirus (kshv)

目前，KS是在南部非洲的艾滋病毒/艾滋病的最主要相关的恶性肿瘤，因此世界。1,2 angioproliferative肿瘤血管内皮细胞的特点，并产生罕见的B细胞淋巴组织增生性疾病的形式在胸腔积液淋巴瘤（PEL）和3-5只有1-5％的细胞在KS病变的多中心Castleman病的某些形式。积极支持的卡波西氏肉瘤相关疱疹病毒（KSHV），与堪萨斯州相关的病原体裂解复制，很明显，细胞因子必须病毒因素在癌变和肿瘤的进展过程中进行交互 。6,7确定新的宿主因素决定因素有助于金水病理是必不可少的发展为肿瘤进展和转移预后指标以及开发新型疗法治疗的KS 8影随行的细节我们使用的方法来识别宿主细胞基因表达的KSHV感染后的方案，在KS肿瘤组织真皮微血管内皮细胞（DMVEC）改变。一旦失调基因芯片分析鉴定，蛋白表达的变化免疫印迹和双标记免疫荧光证实。失调基因的转录表达的变化实时定量聚合酶链反应（QRT - PCR）在体外证实。使用档案KS肿瘤组织在体外研究结果进行验证也是双标记免疫组化和组织芯片 8,10我们的方法来确定在KS肿瘤组织的微环境失调的基因， 将允许在体外和体内模型系统随后发展为发现和评估潜在的新的治疗，治疗的KS。

Watch this Scientific Journal Video about Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus KSHV at JoVE.com

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

宿主细胞因子发挥卡波西氏肉瘤（KS）的建立和维护的关键作用。我们概述的方法来识别宿主细胞在KSHV感染的DMVEC细胞改变的因素，在堪萨斯州的肿瘤组织。由病毒改变的细胞基因，将作为潜在目标（S）的新型疗法。

确定失调卡波西氏肉瘤相关疱疹病毒（KSHV）诱导的基因

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

identifying-dysregulated-genes-induced-kaposi-s-sarcoma-associated

Research

JoVE Journal

Immunology and Infection

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

12.5K 次观看.  Meharry Medical College. 宿主细胞因子发挥卡波西氏肉瘤（KS）的建立和维护的关键作用。我们概述的方法来识别宿主细胞在KSHV感染的DMVEC细胞改变的因素，在堪萨斯州的肿瘤组织。由病毒改变的细胞基因，将作为潜在目标（S）的新型疗法。

视频: Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus KSHV

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development 1. From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in 2). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice 3,4. We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.

Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.

酸修饰的寡核苷酸锁定核酸转染和蚊虫细胞的靶基因突变

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

科研

免疫与感染

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

解剖宿主 - 病毒相互作用模型疱疹病毒裂解性复制

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3.
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase &eta;)4-10. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18.
Ramos &#x2013; a Burkitt lymphoma cell line that constitutively undergoes SHM &#x2013; has been a popular cell-line model to study SHM18-24. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. 
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.

评估拉莫斯B细胞的特定基因的过表达或击倒后的体细胞超突变

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

在纯化造血干/祖细胞识别DNA突变

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

细胞与分子极化的快速和强大的分析趋化因子诱导信号

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. 
Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. 
This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.

Here, we present a protocol for the development and validation of a quantitative PCR method used for the detection and quantification of EHV-2 DNA in equine respiratory fluids. The EHV-2 qRT-PCR validation protocol involves a three-part procedure: development, characterization of qRT-PCR assay alone, and characterization of the whole analytical method.

为马科动物疱疹病毒2诊断的呼吸道液体定量PCR检测方法的开发和验证

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

T淋巴细胞有丝分裂原诱导芽生的快速定量辨识免疫调节药物

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

甲醛暴露对大鼠新生辣椒素致过敏性皮炎模型的影响

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...