我们感谢批判性阅读的手稿，罗宾博士兰迪埃尔金Kastenmayer，特德托里，丹帕Tovi雷曼。这项工作是由院内研究司院内研究计划，国家过敏和传染病，美国国立卫生研究院研究所，由中国国家973基础研究项目，＃2007CB513103支持。我们感谢援助NIAID的壁间编辑布伦达李博马歇尔。

无菌技术必须适用于所有材料将动物给药，以避免无意中引入到小鼠体内，可以混淆实验结果外源性感染制剂。 1。血液阶段的疟疾寄生虫感染的实验室小鼠<ol><li>在室温下解冻，需要跨越两个小瓶冷冻血液阶段疟疾寄生虫。在这个例子中，两个鼠疟原虫株氏疟原虫约氏疟原虫和氏疟原虫nigeriensis。 </li><li>配合一个22-30号（G）的针头注射器，并绘制了400μL无菌，医药级PBS液（pH 7.4）。 </li><li>使用同一注射器，吸取100μL解冻受感染的血液;倒置注射器和内容，直到均匀混合。 （可选：可以使用吸管转移到一个PBS和感染者的血液收集管前转移到一个注射器）。 </li><li>注入200μL含有寄生虫，直接进入腹膜鼠标解决方案。当完成后，丢弃在一个锐器斌的注射器。 （见维修实验室老鼠的补充材料）。在一般情况下，标准尺寸的接种之间100-500μL，在原有的冷冻材料寄生虫感染水平而定。 </li><li>注入的两个寄生虫两只老鼠越过株。小鼠将成为积极的微观注射后3至5天。当parasitemias达到1-5％之间，继续下一步。 </li></ol> 2。混合克隆小鼠感染<ol><li>混合克隆感染之前，收集从两个捐助者与Giemsa染色薄血涂片镜检（图3）不同的疟原虫株感染小鼠尾血，红血细胞密度的测量（见补充材料的详细程序） 。 </li><li>计算了生理盐水（1.5％W / V二水枸橼酸三，0.85％W / V氯化钠），并须出示接种混合物的小鼠血液量，获得早期使用的原虫和红血细胞密度测量。 </li><li>调整到最后一百万受感染的红细胞每100μL浓度的寄生虫解决方案。 </li><li>从两个捐助者以1:1的比例合并感染者的血液，获得混合克隆接种。当父母的两个克隆增长速度的不同，一个调整的比例增长较快的父母到父母克隆一个增长较慢的1:2或1:5。 </li><li>每个被感染的小鼠的腹膜注入100μL混合样品。 （见步骤3.9，以确定被注入小鼠） </li><li>使用这种方法，每个单身父母株感染两只老鼠。单克隆的感染将使两个亲株的成功传输的决心。 </li></ol> 3。喂食蚊子<ol><li>准备三个笼子里含有300-500成人（男性和女性）按蚊蚊子每5-7天，年龄从蛹后出现。扣除安全由塑料或金属盖的容器都覆盖着。两个笼子饲养父母克隆;第三笼是一种遗传交叉的准备。 </li><li>开始克隆混合感染后4天，准备从被感染的小鼠的姬姆萨染色的薄血尾巴涂片检查。 </li><li>检查涂片在显微镜下，在1000倍的放大倍率扫描至少50微观领域，并确定是否存在男性和女性配子体。 </li><li>由于大液泡和大颗粒的存在（图3），男性和女性配子体可识别。 ，男性和女性配子体有粉色和蓝色的细胞质中，分别。如果配子体存在，请按照下列步骤3.6。 </li><li>如果配子体均缺席，保持日常监测，直到它们出现。如果配子体存在，继续以下步骤。 </li><li>注入50μL含有33毫克/毫升氯胺酮和3毫克/毫升的甲苯噻嗪鼠标麻醉每个感染小鼠（每20克鼠标体重）的腹膜的麻醉药物鸡尾酒。最终剂量的氯胺酮和甲苯噻嗪，分别为82.5和7.5毫克/公斤。 </li><li>将小鼠正面朝下放在含有成年按蚊的蚊子已饿死的食糖供给24小时，让蚊子喂30分钟无间断的笼子的顶部。 </li><li>标准条件下保持之间保持温度是24-26 ° C，还可以看到维护实验室蚊子的补充方法insectary蚊子。 </li><li>每50-100雌性蚊子感染小鼠。立即安乐死颈椎脱位后喂养的老鼠，而老鼠仍然是在麻醉状态下。 </li></ol> 4。解剖蚊MidgUTS和计数卵囊<ol><li> 9天的喂养后，蚊子将研究昆虫的中肠卵囊。单克隆感染小鼠的饲料，表明两个亲株配子体在蚊子的卵囊存在传染性。 </li><li>解剖一只蚊子的中肠，使用吸引器连接到一个小容器，以收集从笼子里受感染的蚊子5至10。 </li><li>使用CO 2气体，麻醉的蚊子，并转移到玻璃培养皿中，他们在冰上。独立和丢弃雄性蚊子。触角的男性与女性相比，明显bushier。 </li><li>用PBS填写两个注射器和26计半英寸（G ½）针配合。存款约100μL的PBS从注射器到载玻片。 </li><li>转嫁的PBS下降5至10麻醉雌性蚊子，并将其放置在解剖显微镜幻灯片。 </li><li>使用相同的针，删除每个蚊子的翅膀和腿，然后，在昆虫的胸部和后腹部，拉除了身体，直到肠，一个白色的囊样的身体，被释放。分离的肠和丢弃身体的其余部分。 </li><li>如果是干的幻灯片，添加更多的PBS中肠。中肠会变质，如果让干。放置一个盖在肠滑。 </li><li>检查下一个在100倍放大倍率的光学显微镜下中肠。计数每肠卵囊的数量（图3）。卵囊厚壁的圆形结构，外表面的肠谎言识别。 </li><li>完成后，处理的注射器，针头，和锐器斌在一个幻灯片。 </li><li>如果卵囊在肠缺席，饲料蚊子较多的配子体感染小鼠，并给予较长的哺乳时间。温度是这一步的关键。确保设置的温度是在24日至26 ° C。 </li></ol> 5。收集离心蚊虫孢子<ol><li>孢子收集管准备如下：削减一个干净的0.5 mL离心管的盖子，并拳打在使用一个火烧19 G针管底部的一个孔（直径0.1-0.2毫米）。 </li><li>填充玻璃棉管的1 / 3。过滤管插入1.5 - 2ml收集管（一管就足够了〜100蚊子收获孢子） </li><li>在后17天喂养，从笼子里收集到一个小容器使用一个抽吸的蚊子（步骤4）。 </li><li>使用CO 2气体麻醉蚊子。 </li><li>独立女性的雄蚊。将麻醉雌性蚊子从容器玻璃的Petri冰盘。 </li><li>准备用26克半充满针头，用PBS两个注射器。存款约100μL的PBS从注射器到载玻片。 </li><li>转移到载玻片的蚊子。在解剖显微镜下取出蚊子的头部，翅膀，腿和腹部。 </li><li>用细尖镊子转移到0.5 mL的PBS中的1.5 ml匀浆器（研钵）thoraxes。商店冰thoraxes（孢子仍然为2-3小时感染）。 </li><li>均质冰thoraxes从唾液腺释放孢子体（见图3），并转移到填充玻璃棉使用玻璃吸管（步骤5.1-5.2）管的细胞裂解液（匀浆）。 </li><li>离心机在1000转速在室温为10至20秒。 </li><li>收集的流量通过（纯化的孢子悬浮液）使用与22-30 G针注射器。 （可选：掉落10-50μL流通过一个微小的幻灯片上，将盖玻片和一个标准的光学显微镜，400X的放大倍率下可视化的活孢子，请参阅图3） </li><li>注入到小鼠体内（高达200,000寄生虫可以从一个单一的蚊子收获;见文献12）的孢子悬浮IP 0.1-0.5毫升。在一个成功的案例，将成为72小时注射后感染的动物。 </li></ol> 6。用有限稀释克隆后代<ol><li>孢子注射后73个小时，收集从两个捐助者与薄血涂片，姬姆萨染色后镜检和红血细胞密度的测量不同的疟原虫株（见补充材料的详细程序）感染小鼠尾血。 </li><li>选择一个鼠标，显示0.1至1％，原虫，其受感染的血红细胞中含有克隆过程的捐助者多为单发的寄生虫。重复步骤6.1接下来的日子里，如果老鼠微观负​​。 14天之内，如果动物不感染，重复步骤2至5，确定的孢子在步骤5.11）。 </li><li>收获的尾巴血液稀释1:1小牛血清和哺乳动物的林格氏液（氯化钾2.7毫米，1.8毫米氯化钙，154毫米氯化钠）在寒冷的解决方案，从而达到一个反面0.6对中，每100μL1感染的红血细胞，并储存在冰接种。在这种方式下，个别鼠标应该会收到一个或零寄生虫。 </li><li>静脉注射注入100μL稀释50至100只老鼠，每年感染者的血液。 </li><li>五到七天后，屏幕上的小鼠研究姬姆萨染色薄血涂片血阶段寄生虫。在一个成功的实验中，有20-50％的老鼠受到感染，将显示在感染后第8天原虫0.1-5.0％。 </li></ol> 7。利用遗传标记后代的表征<ol><li>收集在1.5毫升的离心管中，含有0.2毫升冷冻的生理柠檬酸盐溶液，旋涡简要，并在3000 rpm到颗粒的血细胞在室温下3分钟离心的20％到40μL鼠尾血。立即提取DNA，或保持在-80 ° C。颗粒</li><li>准备使用任何标准的DNA提取方法的寄生虫的DNA。迅速隔离的DNA，使用一个高的纯PCR模板制备试剂盒（罗氏应用生物科学）。 </li><li>重组后代确定后，利用微卫星（MS）或不同的染色体上，可以区分两个父母的遗传交叉菌株的单核苷酸多态性（SNP）基因分型。一个简化的方法，使用荧光标记的引物的质谱基因分型的体育单可以使用约氏疟原虫 （见文献8,9）。 </li></ol> 8。血液阶段的疟疾寄生虫的冷冻保存<ol><li>收集从5-20％5-10毫升原虫麻醉小鼠心脏穿刺的感染者的血液0.5-1毫升生理盐水溶液冷冻。 </li><li>在室温下在2000 RPM离心5分钟。弃上清。 </li><li>添加到血颗粒落明智的Glycerolyte 57解决方案的2倍量（Fenwal实验室），拌匀。 </li><li>将暂停cryotubes（0.2-0.5毫升每管）。 </li><li>商店，在-80℃（长达1个月）或液氮冻存的血液。 </li></ol> 9。代表性的成果： 在一个成功的实验，克隆线的5-10％将独立重组的后代。 <img src="/files/ftp_upload/2365/2365fig1.jpg" alt="图1" /> 图1。一个生命周期，在一个遗传交叉基因重组事件的示意图。在发展早期阶段的蚊子进行基因重组事件。继“单倍体”的男性和女性不同的遗传背景，在蚊子的中肠配子受精，“二倍体”受精卵的形成。受精卵构成寄生虫的生命周期只有二倍体阶段;减数分裂如下受精12小时内，蚊子和哺乳动物中的所有后续阶段仍然单倍体。产生的受精卵发育成ookinetes和卵囊。每个卵囊的生长和有丝分裂产生成千上万的孢子，这是蚊子“hemoceal发布。唾液腺迁移到这些孢子是传染给哺乳动物的主机，肝脏和红细胞阶段的发展发生的位置。一些寄生虫的红血细胞周期的过程中，可以分化成成熟的男性和女性配子体。寄生虫的生命周期完成时，这些配子体是由另一蚊子，延续传输。 <img src="/files/ftp_upload/2365/2365fig2.jpg" alt="图2" /> 图2。疟疾寄生虫和生产重组后代的核基因的继承。可以通过染色体重组或交叉事件产生的基因重组。生产相同的父母克隆1或2（A和D），分别配子之间自交纯合后代。二）由两个不同的家长的克隆，重组细胞核（蓝色椭圆形）仅由染色体重组形成的配子之间的相互交流产生的杂合后代。三）从两个不同父克隆，重组核同源染色体的非姐妹染色单体之间的交叉形成红色椭圆形配子之间的相互交流产生的杂合子后代。 <img src="/files/ftp_upload/2365/2365fig3.jpg" alt="图3" /> 图3。啮齿动物疟原虫约氏疟原虫的形态。成熟的雄性配子体一）环滋养体阶段，二），C），裂殖体，D）的裂殖子，E）不成熟的雄性配子体，F）的，G）的未成熟的雌性配子体，高）的成熟女性配子体，我）第10天，后肠卵囊喂养（用箭头表示的成熟卵囊; 200X的放大倍率），J）的蚊子的唾液腺（100倍的放大倍率），K）的孢子（400倍的放大倍率）。

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G. e. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=encoding+erythrocyte+binding+ligand+linked+to+blood+stage+multiplication+rate+phenotype+in+Plasmodium+yoelii+yoelii.">encoding erythrocyte binding ligand linked to blood stage multiplication rate phenotype in Plasmodium yoelii yoelii.</a> Proc Natl Acad Sci U S A. 106, 7161-7166 (2009).</li><li>Pattaradilokrat, S., Cheesman, S. J., Carter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linkage+group+selection:+towards+identifying+genes+controlling+strain+specific+protective+immunity+in+malaria.">Linkage group selection: towards identifying genes controlling strain specific protective immunity in malaria.</a> PLoS One. 2, e857-e857 (2007).</li><li>Mackinnon, M. J., Read, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Relationships+between+Parasite+Virulence+and+Transmission+in+the+Rodent+Malaria+Plasmodium+chabaudi.">Genetic Relationships between Parasite Virulence and Transmission in the Rodent Malaria Plasmodium chabaudi.</a> Evolution. 53, 689-703 (1999).</li></ol>

因为作者是政府雇员，这是一个政府工作，工作是在美国的公共领域。尽管有任何其他的协议，美国国立卫生研究院提供的工作PubMedCentral，展示和使用公共有权，并PubMedCentral可能标记或修改其习惯做法的工作保持一致。您可以建立的美国主题之外的政府使用许可权。

我们展示的技术，生产的灭鼠疟疾氏疟原虫，这也适用于其他啮齿类动物malarias的遗传杂交生产中的遗传交叉。单身父母克隆小鼠的感染通常以确定父母寄生虫的成功传输，以确保父母在生产前进行跨功能的配子主管。 成功传输通过蚊子是由多种因素，包括温度insectary，用于灭鼠疟疾寄生虫的种类，和血阶段寄生虫的剂量​​（接种量）的影响。虽然体育疟原虫...

补充材料： <ul><li>实验小鼠的维修</li><li>维护实验室蚊子</li><li>薄血涂片染色镜检，用吉姆萨染色</li><li>红血细胞密度的测量</li></ul>实验小鼠的维修近交系的实验室小鼠品系的BALB / c，年龄在5至8周龄，雌性在研究中使用。小鼠被安置在一个标准的固底聚碳酸酯笼与电线杆盖配备馈线和一瓶水，。小鼠保持在一个恒定的温度（25 ± 1 ° C）12:12小时光照：黑暗周期。允许小鼠饲料2018S哈伦Teklad全球19％的蛋白质挤压灭鼠饮食（消毒;从哈伦Teklad），并提供酸化饮水自由采食。动物实验的动物护理和使用，根据协议LMVR11E（，马里兰州贝塞斯达国家卫生研究院）在美国国立过敏和传染病研究所委员会规定的准则和法规的规定执行。 维护实验室蚊子蚊子是从按蚊实验室饲养的殖民地。成年人都保持在尼龙笼存放在温度和湿度控制的房间（23日至25 ° C 氏疟原虫和恶性疟原虫chabaudi，疟原虫 19日至21 ° C，湿度80％至95％;上12:12小时光照：黑暗的周期）。美联储与10％葡萄糖和2.00％对氨基苯甲酸（PABA）补充水液成蚊。要获得高品质的成人，500幼虫生长在低密度条件在1000 厘米 3开放盘提供约1毫克碳酸氢钠在1 L蒸馏水。孵化后，幼虫tetramin粉（PETCO），直到他们发展成蛹阶段，并转移到为新兴的成蚊笼。 薄血涂片染色镜检，用吉姆萨染色使用干净的剪刀剪断了感染小鼠的尾巴尖（1.0毫米）。广场尾血一滴到一个干净的标本幻灯片（0.5〜1.0μL）。鼠标将在1-2分钟停止出血。滴血上放置一个干净的吊具幻灯片，维持在45度角相对的标本幻灯片，让血液吸附到吊具的整个宽度。保持试样的幻灯片和推进吊具幻灯片迅速和顺利地生产薄涂片。让血片干燥，然后沉浸在绝对甲醇的幻灯片。允许的幻灯片，在空气中晾干后方可再次覆盖姬姆萨染色（10％姬姆萨染色，蒸馏水）。孵化薄血片在室温为10-15分钟后，小心地用自来水冲洗的幻灯片，并让它空气干燥。检查感染的红血细胞的数量（iRBC;受感染的红细胞形态见图3），与1000倍的放大倍率（100X物镜）并计算原虫（红细胞计数的iRBC每100）浸泡在油光显微镜下。不同的疟原虫株不同的增长速度和致病性。可进行24小时监测血阶段parasitaemias注射后，根据剂量的疟疾寄生虫的血液阶段。例如，小鼠注射10 7感染红细胞腹腔或10 6静脉注射感染红细胞时，将24小时显微镜积极。 红血细胞密度的测量像parasitaemias，感染小鼠的红细胞（RBC）的密度水平变化在整个感染过程。红细胞密度应测前1-2小时内的单一和混合克隆感染的启动和克隆实验。红细胞密度的测量方法有两种：手动计数，使用纽鲍尔血球自动计数使用一个Cellometer（Nexcelom生命科学）。在这两种方法，撤回1μL鼠尾使用玻璃毛细管（VWR）血液稀释在10毫升的PBS，拌匀。要使用纽鲍尔血球，加载到hemacytometer 20μL中止。将与10倍物镜，光镜下的hemacytometer。 hemacytometer包含网格划分成9个大方格，并在角落里的4大广场，进一步划分成16小方格。计数细胞总数在16小方块四角方块。为了避免计数的偏见或计数细胞重叠的网格线，被视为“在”一个单元格，如果重叠的顶部或右行和“出”，如果它重叠的上下或左右线。估计每一个小广场上的细胞数，除以0.00625（一个小广场上的体积是6.25 NL）。这就产生了每微升的细胞数（＆亩，L）。从这个数据，计算出最终的红血细胞密度乘以10,000（稀释倍数）。冲洗盖玻片，用蒸馏水和70％的乙醇和计数室，空气干燥。此外，加载到Cellometer计数室幻灯片20μL中止。幻灯片中插入一个Cellometer幻灯片室（读者）。启动Cellometer软件，选择“红细胞”选项，并输入10000的稀释倍数。记录红细胞密度。

protocol for production of a genetic cross of the rodent malaria parasites

在抗疟疾药物，疟疾寄生虫的致病性变异是生物和医疗的重要性。连锁图谱已经导致成功的基本在1-3啮齿类动物和人类4-6疟疾寄生虫的各种性状的基因或位点的识别。 氏疟原虫疟疾寄生虫是许多疟疾从非洲野生啮齿类动物中分离的物种之一，已经适应了在实验室中生长。该物种繁殖许多人类疟疾寄生虫的生物学特性，遗传标记，如微和扩增片段长度多态性（AFLP）标记也已开发的寄生虫 7-9 。因此，在啮齿类动物疟疾寄生虫的基因研究可以进行补充对恶性疟原虫的研究。在这里，我们展示的技术， 生产 P.遗传交叉约氏疟原虫 ，第一个率先由Drs。大卫Walliker，理查德卡特和他的同事在10爱丁堡大学。 在体育的遗传杂交约氏疟原虫和其他啮齿类动物疟疾寄生虫是由一个包含了两个基因不同的克隆，在利益的表型，使蚊子感染小鼠感染后第4天的饲料不同配子体接种感染小鼠的小家鼠。小鼠血液中的男性和女性配子体的存在是微观喂奶前确认。喂奶后48小时内，在蚊子的中肠，单倍体的配子体分化为雌雄配子，施肥，并形成一个二倍体的合子（图1）。在发展成一个动合子受精卵，减数分裂发生11。如果通过基因的两个不同的寄生虫病，遗传交流（染色体重组和跨接一对同源染色体的非姐妹染色单体之间;图2）配子之间的相互交流中衍生的受精卵是可能发生的，在重组同源基因位点的遗传物质。每个受精卵经过连续两次核部门，导致4个单倍体核。一个动合子，进一步发展成一个卵囊。一旦卵囊的成熟，成千上万的孢子（跨后代）形成和释放到蚊子hemoceal。孢子是收获的唾液腺发生前红细胞和红细胞阶段发展到一个新的小鼠主机，的注入。红细胞形式的克隆和分类方面区分亲本遗传连锁映射之前的字符。个别家长克隆控制感染是在相同的方式作为一种遗传性的交叉的生产。

Watch this Scientific Journal Video about Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites at JoVE.com

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

啮齿类动物疟疾寄生虫的基因杂交进行喂养蚊子的两个基因的不同的寄生虫。克隆小鼠血液，让蚊虫叮咬感染小鼠后，重组的后代。本视频演示了如何产生遗传杂交<em&gt;约氏疟原虫</em&gt;，并适用于其他啮齿类动物疟疾寄生虫。

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

protocol-for-production-genetic-cross-rodent-malaria

Research

JoVE Journal

Immunology and Infection

15.1K 次观看.  National Institutes of Health. 啮齿类动物疟疾寄生虫的基因杂交进行喂养蚊子的两个基因的不同的寄生虫。克隆小鼠血液，让蚊虫叮咬感染小鼠后，重组的后代。本视频演示了如何产生遗传杂交约氏疟原虫，并适用于其他啮齿类动物疟疾寄生虫。

视频: Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

从黄热病的蚊子的血细胞的收集和染色的议定书埃及伊蚊</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

遗传操纵<em&gt;Δku80</em&gt;株功能基因组分析<em&gt;弓形虫</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

一个简单的红细胞溶解法对B淋巴母细胞系的建立

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

巨噬细胞内细胞内生长的定量化是评价利什曼原虫寄生虫毒力的快速可靠方法

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...