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。虽然体育疟原虫的传播通常达到19-21 ° C 13，P的传输约氏疟原虫和体育chabaudi进行例行在23-25 ​​° C 14。 Ookinetes的P.疟原虫未能发展成卵囊，在温度低于16 ° C或更高的超过24 ° C 15 。除了温度，接种量的大小可以影响血液中疟原虫的繁殖率。虽然一个大的接种量（5 × 10 6 iRBC或以上）的注射，将产生更高原虫感染的早期阶段，它并不一定会导致更大的数字配子体或更高的传染性蚊子。加兹比和其他人（2009年）16表明，P. chabaudi阿达米配子体是目前从3天到第20天（13天峰）在1 × 10 6 iRBC血液后感染，但感染后第6天唯一一天后，蚊子被感染。此外，管理一个大的接种量的快速增长和致命的寄生虫，如克隆N67（nigeriensis），17XL和ym P.约氏疟原虫， 克隆体育安卡株疟原虫 ，P. chabaudi阿达米克隆局副局长，结果往往是严重的贫血和小鼠的病理，从而导致的小鼠体温明显下降。因此，小鼠成为感染蚊子少（未发表意见）。不过已进行了一个标准的接种量（10 6 iRBC），啮齿类动物疟疾寄生虫传播。人们已经发现，P. 疟原虫和P.约氏疟原虫蚊子是传播至5天3阶段诱导小鼠17的血后感染，而P. chabaudi chabaudi和P. chabaudi阿达米只传染给蚊子阶段引起的血液感染小鼠16，18后第6天。 重组后代生产的影响因素，包括两个父母在小鼠品系配子体的比例。从理论上讲，重组后代的最大生产发生时父母克隆男女配子体在同等数量和自体受精和交叉受精发生在相同的频率。在这种情况下，所产生的受精卵的一半将是混合动力车，其余的将包括比例相等的两个亲本的克隆线。重组子代克隆的生产将大大减少时，配子体的比例是偏向一个家长的克隆，导致不相称的自体受精。此外，寄生虫往往有不同的增长率，并可能有不同的能力，在产生配子体16， 19。因此，它是必要的，以优化在父母的寄生虫的蚊子喂养的老鼠注射的混合比例。 克隆蚊子喂养后的血液寄生虫的时间也是一个重要的考虑因素。最好是到原虫之间的0.1-1.0％（血液中的太多的复制周期之前），以避免克隆复制克隆时，克隆后代的遗传交叉。快速或缓慢增长的寄生虫可能会大量在一定时间内，与具有相同表型的克隆和克隆快速或缓慢增长的寄生虫的高峰将结束。 总之，执行使用灭鼠疟疾寄生虫的基因十字架是相对容易，比执行的人类疟疾寄生虫 P.遗传交叉便宜得多恶性疟原虫 ，这需要一个非人灵长类动物感染。重组的遗传杂交后代的遗传连锁图谱的建设和疟疾的重要特征，包括寄生虫的发展，耐药性和毒力的映射是非常有用的工具。

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

Nanyang Technological University

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

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

蚊子养育的议定书“（冈比亚按蚊）

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

科研

生物学

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

遗传学

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

免疫与感染

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the &#966;C31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces &#966;C31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the &#966;C31 integrase. We report two representative results of &#966;C31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. &#966;C31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the &#966;C31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the &#966;C31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

位点导向 的φC31介导的疟疾 按蚊 载体中的整合和盒式交换

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the ...

Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control strategies. CRISPR-based homing gene drive systems have made transgenic technologies more accessible within the scientific community. Evaluation of transgenic mosquito performance and comparisons with wild-type counterparts in small laboratory cage trials provide valuable data for the design of subsequent field cage experiments and experimental assessments to refine the strategies for disease prevention. Here, we present three different protocols used in laboratory settings to evaluate transgene spread in anopheline mosquito vectors of malaria. These include inundative releases (no gene-drive system), and gene-drive overlapping and non-overlapping generation trials. The three trials vary in a number of parameters and can be adapted to desired experimental settings. Moreover, insectary studies in small cages are part of the progressive transition of engineered insects from the laboratory to open field releases. Therefore, the protocols described here represent invaluable tools to provide empirical values that will ultimately aid field implementation of new technologies for malaria elimination.

The protocols reported here illustrate three alternative ways to assess the performance of genetically-engineered mosquitoes destined for vector control in laboratory-contained small cage trials. Each protocol is tailored to the specific modification the mosquito strain bears (gene drive or non-gene drive) and the types of parameters measured.

基因工程按蚊的小笼实验室试验

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control ...

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. ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

生成转基因 伯氏疟原 虫孢子

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

一种多检测试验为疟疾蚊子发射

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

<meta charset="utf8"></head><body>鉴定疟疾寄生虫中的激酶重新布线:一种化学遗传学方法

Understanding the Development of Compensatory Pathways in a Mutant Malaria Parasite Harbouring Hypomorphic Allele of Plant-Like Kinases

One of the mechanisms for subverting the effect of drugs by&#160;the malaria parasite is through rewiring of its transcriptome. The effect is more pronounced for target genes belonging to the multigene family. Plasmodium falciparum protein kinases belonging to the CDPK family are essential for blood stage development. As such, CDPKs are considered good targets for the development of anti-malarial compounds. The chemical genetics approach has been historically used to elucidate the function of protein kinases in higher eukaryotes. It requires the substitution of gatekeeper residue for another amino acid with a different side chain through genetic manipulation. Amino acid substitution at the gatekeeper position modulates the activity of a protein kinase and changes its susceptibility to a specific class of compounds known as bumped kinase inhibitors (BKIs) that help in the functional identification of the target gene. Here, we have exploited the chemical genetics approach to understand compensatory mechanisms evolved by a mutant parasite harboring a hypomorphic allele of cdpk1. Overall, our approach helps in identifying compensatory pathways that may be simultaneously targeted to prevent the development of drug resistance against individual kinases.

<meta charset="utf8"></head><body>作者聚焦:鉴定含有必需蛋白激酶亚型等位基因的疟疾寄生虫中的代偿途径

Chemical genetics involves the substitution of a gatekeeper residue with an amino acid containing a different side chain at the target locus. Here, we have generated a mutant parasite containing a hypomorphic allele of cdpk1 and identified compensatory pathways adopted by the parasite in the mutant background.

生产的灭鼠疟疾寄生虫的遗传交叉议定书"

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此视频中的章节

蚊子养育的议定书“（冈比亚按蚊）

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫

位点导向的φC31介导的疟疾按蚊载体中的整合和盒式交换

基因工程按蚊的小笼实验室试验

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

生成转基因伯氏疟原虫孢子

一种多检测试验为疟疾蚊子发射

了解携带植物样激酶亚型等位基因的突变疟疾寄生虫中补偿途径的发展

了解携带植物样激酶亚型等位基因的突变疟疾寄生虫中补偿途径的发展

生产的灭鼠疟疾寄生虫的遗传交叉议定书"

摘要

探索更多视频

此视频中的章节

蚊子养育的议定书“（冈比亚按蚊）

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫

位点导向 的φC31介导的疟疾 按蚊 载体中的整合和盒式交换

基因工程按蚊的小笼实验室试验

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

生成转基因 伯氏疟原 虫孢子

一种多检测试验为疟疾蚊子发射

了解携带植物样激酶亚型等位基因的突变疟疾寄生虫中补偿途径的发展

了解携带植物样激酶亚型等位基因的突变疟疾寄生虫中补偿途径的发展

位点导向的φC31介导的疟疾按蚊载体中的整合和盒式交换

生成转基因伯氏疟原虫孢子