Name: protocol for production of a genetic cross of the rodent malaria parasites
Uploaded: 2011-01-03T17:30:00
Description: Watch this Scientific Journal Video about Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites at JoVE.com

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?...

<ol>
	<li>Hayton, K., Ranford-Cartwright, L. C., Walliker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfadoxine-pyrimethamine+resistance+in+the+rodent+malaria+parasite+Plasmodium+chabaudi.">Sulfadoxine-pyrimethamine resistance in the rodent malaria parasite Plasmodium chabaudi.</a> Antimicrob Agents Chemother. 46, 2482-2489 (2002).</li><li>Cravo, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+mefloquine+resistance+in+the+rodent+malaria+parasite+Plasmodium+chabaudi.">Genetics of mefloquine resistance in the rodent malaria parasite Plasmodium chabaudi.</a> Antimicrob Agents Chemother. 47, 709-718 (2003).</li><li>Hunt, P., Cravo, P. V., Donleavy, P., Carlton, J. M., Walliker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroquine+resistance+in+Plasmodium+chabaudi:+are+chloroquine-resistance+transporter+(crt)+and+multi-drug+resistance+(mdr1)+orthologues+involved?.">Chloroquine resistance in Plasmodium chabaudi: are chloroquine-resistance transporter (crt) and multi-drug resistance (mdr1) orthologues involved?.</a> Mol Biochem Parasitol. 133, 27-35 (2004).</li><li>Yuan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mapping+of+targets+mediating+differential+chemical+phenotypes+in+Plasmodium+falciparum.">Genetic mapping of targets mediating differential chemical phenotypes in Plasmodium falciparum.</a> Nat Chem Biol. 5, 765-771 (2009).</li><li>Su, X., Kirkman, L. A., Fujioka, H., Wellems, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+polymorphisms+in+an+approximately+330+kDa+protein+are+linked+to+chloroquine-resistant+P.+falciparum+in+Southeast+Asia+and+Africa.">Complex polymorphisms in an approximately 330 kDa protein are linked to chloroquine-resistant P. falciparum in Southeast Asia and Africa.</a> Cell. 91, 593-603 (1997).</li><li>Hayton, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte+binding+protein+PfRH5+polymorphisms+determine+species-specific+pathways+of+Plasmodium+falciparum+invasion.">Erythrocyte binding protein PfRH5 polymorphisms determine species-specific pathways of Plasmodium falciparum invasion.</a> Cell Host Microbe. 4, 40-51 (2008).</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=Congenicity+and+genetic+polymorphism+in+cloned+lines+derived+from+a+single+isolate+of+a+rodent+malaria+parasite.">Congenicity and genetic polymorphism in cloned lines derived from a single isolate of a rodent malaria parasite.</a> Mol Biochem Parasitol. 157, 244-247 (2008).</li><li>Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hundreds+of+microsatellites+for+genotyping+Plasmodium+yoelii+parasites.">Hundreds of microsatellites for genotyping Plasmodium yoelii parasites.</a> Mol Biochem Parasitol. 166, 153-158 (2009).</li><li>Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Typing+Plasmodium+yoelii+microsatellites+using+a+simple+and+affordable+fluorescent+labeling+method.">Typing Plasmodium yoelii microsatellites using a simple and affordable fluorescent labeling method.</a> Mol Biochem Parasitol. 155, 94-102 (2007).</li><li>Walliker, D., Carter, R., Morgan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+recombination+in+malaria+parasites.">Genetic recombination in malaria parasites.</a> Nature. 232, 561-562 (1971).</li><li>Sinden, R. E., Hartley, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+meiotic+division+of+malarial+parasites.">Identification of the meiotic division of malarial parasites.</a> J Protozool. 32, 742-744 (1985).</li><li>Ozaki, L. S., Gwadz, R. W., Godson, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+centrifugation+method+for+rapid+separation+of+sporozoites+from+mosquitoes.">Simple centrifugation method for rapid separation of sporozoites from mosquitoes.</a> J Parasitol. 70, 831-833 (1984).</li><li>Yoeli, M., Most, H., Bone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+berghei:+cyclical+transmission+by+experimentally+infected+Anopheles+quadrimachulatus.">Plasmodium berghei: cyclical transmission by experimentally infected Anopheles quadrimachulatus.</a> Science. 144, 1580-1581 (1964).</li><li>Landau, I., Boulard, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Life cycles and Morphology. , (1978).</li><li>Vanderbergh, J. P. Y., M, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+temperature+on+sporogonic+development+of+Plasmodium+berghei.">Effects of temperature on sporogonic development of Plasmodium berghei.</a> J Parasitol. 52, 559-564 (1966).</li><li>Gadsby, N., Lawrence, R., 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=A+study+on+pathogenicity+and+mosquito+transmission+success+in+the+rodent+malaria+parasite+Plasmodium+chabaudi+adami.">A study on pathogenicity and mosquito transmission success in the rodent malaria parasite Plasmodium chabaudi adami.</a> Int J Parasitol. 39, 347-354 (2009).</li><li>Pattaradilokrat, S., Culleton, R. L., Cheesman, S. J., Carter, R. 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>

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 ...

Research

JoVE Journal

Immunology and Infection

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

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

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

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 ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

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

Genetic Manipulation in ÃƒÅ½Ã¢â‚¬Âku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

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

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

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

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

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

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

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

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 ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcÃƒÅ½Ã‚Â³R Cross-linking

巨噬细胞 ros 产生的流式细胞仪测量对 fcγr 交联的响应

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...