SD was supported by a Sir Henry Wellcome Fellowship [092201/Z/10/Z]. This work was funded by Wellcome Trust grants [WT066839MA][104627/Z/14/Z][108445/Z/15/Z] to Professor Keith Gull. We would like to thank Professor Keith Gull for helpful conversations and insights.

1，96孔龙引物PCR <ol><li>预冷冻96孔PCR冷却器在-80℃冷冻机中。 </li><li>在一个10毫升管，准备预混一个:2.100微升的DDH 2 O，105微升PCR级DMSO，105微升10毫米的dNTP，105微升PPOT模板（25毫微克/微升）9，每盘2415微升总体积。 </li><li>等分试样23微升到96孔PCR板的每个孔中。 </li><li>加入2微升100μM汇集引到每个加载以及使用P20 12通道多道移液器的。用膜，代替板密封在预冷的PCR冷却器并允许冻结在-80℃至少20分钟。 </li><li>在10ml的管中，制备主混合物A:2048微升DDH 2 O的，525微升10×缓冲液，52.5每板2626微升的总体积微升聚合酶。 </li><li>从-80℃冰箱中取出板和PCR凉爽。 </li><li>随着板块仍然在PCR冷却器，在冻结的顶部加入25微升主混合物BN主控一个混合的50微升反应总体积。这是时间关键的，并且应完成之前混合物A可以解冻。封板与薄膜。 </li><li>设定热循环如下:94℃10分钟，然后94℃30个循环持续15秒，65℃，30秒，72℃2分钟，然后72℃7分钟的最后延伸期间。 </li><li>加载PCR板的块已到达94℃后。 </li></ol> 2. 96-龙引物的PCR验证<ol><li>投大1％（重量/体积）在Tris醋酸EDTA（TAE）琼脂糖凝胶用4×28孔的梳子运行缓冲液，得到一种凝胶具有4行孔中。对准一12信道多道移液器的尖端的梳子的交替齿。仔细淹没在TAE凝胶电泳缓冲液的车道加载后。 </li><li>负载1 kb的DNA梯到1 次和28 次良好的每一行。 </li><li>使用P20 12通道多道移液器，转移2微升PCR产物迪尔ectly到凝胶（ 图1A）的各泳道。为此迅速减少污染扩增子的可能性。 <ol><li>装载从PCR板的行A中的PCR产物到凝胶的第1行的奇数泳道（A1 - 泳道3，A 2 - 泳道5 ...... A11 - 车道23，A12 - 泳道25）和加载PCR产物从PCR板的列B到凝胶的1行的偶数道（B1 -泳道4，A 2 -泳道6 ...... B11 -车道24，B12 -泳道26）。 </li><li>如上所述，与C行到奇数通道及D行到偶数泳道加载来自使用相同的图案的PCR板的行C和D的PCR产物。直到PCR板的每个孔中装载到凝胶继续这个装载模式。 DNA的加样缓冲液不需要加载此凝胶。 </li></ol></li><li>将凝胶进入运行罐和TAE加满油箱运行缓冲液直至凝胶被淹没。在100V进行30分钟运行，并可视化使用UV透照。参见图1B的例子凝胶。 注意:PCR产物可被储存在-20℃下几个星期转染前</li></ol> 3. 96孔转<ol><li>使用T.布氏 procyclic形式的细胞系SMOXP9 10此过程和生长在含有10％FCS 10的SDM-79的介质。 </li><li>使用1×10 7个细胞每转上的C末端标记时（总共1.1×10 9个细胞）的蛋白质的N末端 ​​和每转染2×10 7个细胞标记时（总共2.2×10 9个细胞）的蛋白质。保持在对数中期（1.2×10 6 -1×10 7细胞/ ml）的细胞在转染前几天和收获转染的细胞以5-8×10 6细胞/ ml的密度。 </li><li>允许冷冻PCR产物进行转染，在室温下解冻。 </li><li>使用血红素细胞计数ocytometer或自动细胞计数。 </li><li>在800 XG 10分钟多50ml试管颗粒所需数量的细胞。 </li><li>离心后弃上清，重悬在10毫升的细胞改性cytomix（0.8毫EGTA，24毫米氯化钾，0.15毫氯化钙 ，10mM磷酸钾缓冲液pH7.6的，25毫米的HEPES-KOH pH7.6的，2.6毫MgCl 2的， 0.5％（重量/体积）葡萄糖，每管100微克/毫升BSA，1mM的次黄嘌呤，144毫蔗糖）。传输所有细胞溶液到单个管中并在800 xg离心再次旋转10分钟。 </li><li>离心后，弃去上清，重悬23毫升改性cytomix的细胞的5×10 7个细胞/ ml的终浓度。 </li><li>而细胞被纺纱，加入1 ml的SDM-79的介质，以4×24孔组织培养板的各孔中。标签板AD，绘制出周围环以及A1在永久性标记每块板，以帮助板方向。 </li><li>板处理程序连接到电穿孔。在electropora器单元设置的电压为1500伏，脉冲长度为100微秒和脉冲12的数目和脉冲的时间间隔为500毫秒。上盘处理，设定脉冲计数为1。这确保了电穿孔将应用于单个脉冲到各个电穿孔板的12列。 </li><li>使用P200的12信道多道移液器，从PCR板转移PCR反应，以96孔一次性电穿孔板，4毫米间隙。 </li><li>当细胞已被纺丝并重新悬浮于5×10 7个细胞/ ml（步骤3.7）的最终浓度，转移到试剂容器。吸移管200微升的细胞溶液进入使用P200的12信道多道移液器电穿孔板的各孔中，通过移液混合用的PCR产物。 </li><li>采用组织从板块以避免短路的顶部移除任何液滴。 </li><li>在板包装提供的密封膜适用于电板，POS机的顶部itioning它以便留下在每一列的顶部和底部露出的电极上的孔。避免覆盖用于指导电影提出的问题。 </li><li>加载电板进入平板处理器，并盖上盖子。按电穿孔单位"脉冲"。 </li><li>电穿孔后，迅速从96孔电穿孔板的4×24孔组织培养板转移的细胞。为了转移细胞中，使用P200 12通道多道移液器与所有其他尖端缺失，使得剩余的6个技巧排队与24孔培养板的6行。转移在96孔电穿孔板中的电穿孔细胞在预先定义的模式的24孔组织培养板中（ 图2A）。 </li><li>准备P200枪头 - 每个96孔转染准备96提示2盒拆下隔列。 <ol><li>传递孔A1 / A3 / A5 / A7 / A9 / A11的96孔电穿孔板板24孔培养板的排A，B1 / B3 / B5 / B7 / B9 / B11成排B，C1 / C3 / C5 / C7 / C9 / C11到C行和D1 / D3 / D5 / D7 / D9 / D11进行D.每组6个孔从96孔板的24孔平板的转移后，在96孔板的孔中，然后用培养基洗涤从相应的孔中的24-孔板和洗涤，然后转移回至孔在24孔板中。 </li><li>传递孔的E1 / E3 / E5 / E7 / E9 / E11上的96孔电穿孔板24孔盘B行A，F1 / F3 / F5 / F7 / F9 / F11成列B，G1 / G3 / G5 / G7 / G9 / G11成在96 C行和H1 / H3 / H5 / H7 / H9 / H11到行D.从96孔板每组6个孔的转移到24孔板后，将孔-Well板然后用培养基洗涤从相应的孔在24孔板中，然后在洗涤被传回至孔在24孔板中。 </li><li>转让井A2 / A4 / A6 / A8 / A10 / A12的96孔电穿孔板24孔板C列A，B2 / B4 / B6 / B8 / B10 / B12成排B，C2 / C4 / C6 / C8 / C10 / C12到C行和D2 / D4 / D6 / D8 / D10 / D12成排D.从96每组6口井的转移后-Well板至24孔板，在96孔板的孔中，然后用培养基洗涤从相应的孔在24孔板中，然后在洗涤被传回至孔在24孔板中。 </li><li>转让井E2 / E4 / E6 / E8 / E10 / E12的96孔电穿孔板24孔板d行A，F2 / F4 / F6 / F8 / F10 / F12到B行，G2 / G4 / G6 / G8 / G10 / G12的进行C和H 2 / H4 / H6 / H8 / H10 / H12到行D.从96孔板每组6个孔的转移到24孔板后，在96孔-Well板然后用培养基洗涤从相应的孔在24孔板中，然后在洗涤被传回至孔在24孔板中。 </li></ol></li><li>使用倒置显微镜，研究1井从每列检查细胞。会有活的和死的细胞用的混合物大量的细胞碎片。活细胞可以从死细胞区分开来，因为他们将在相差显微镜明亮，会动。 </li><li>放置在一个干净的，塑料盒24孔板与形成紧密密封的盖。把盒子在28℃培养箱。 </li><li> 6-8之间小时后，加入1 ml SDM-79的含2倍选择性抗生素，每孔10％FCS。根据我们的经验，其中该基因的标签上的N末端细胞系是对较高浓度选择药物的抗性。因此，在使用杀稻瘟素作为一个例子，标记上的N末端要求20微克的终浓度/ ml的杀稻瘟素和标记在C末端需要10微克/ ml的杀稻瘟素的最终浓度。 注:转染后的转基因细胞系将成为可见的9-10天。 </li><li>在新鲜的药物和媒体至少两次分析前通道。 图2B示出从一个典型的96孔transfe结果ction上的N末端标记96个不同的蛋白质。评估每孔用相差显微镜，以确定它是否包含健康，药物抗性细胞。健康以及主要包含（&lt;95％）高活性的同相合同显微镜明亮细胞。通过荧光显微镜或免疫印迹随后的分析是必需的，以确定该蛋白已成功标记。 </li></ol>

<ol><li>Kelly, S., Reed, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+genomics+in+Trypanosoma+brucei%3A+a+collection+of+vectors+for+the+expression+of+tagged+proteins+from+endogenous+and+ectopic+gene+loci%5BTitle%5D)+AND+%22Mol+Biochem+Parasitol%22%5BJournal%5D)">Functional genomics in Trypanosoma brucei: a collection of vectors for the expression of tagged proteins from endogenous and ectopic gene loci.</a> Mol Biochem Parasitol. 154 (1), 103-109 (2007).</li>
<li>Wirtz, L. E., Leal, S., Ochatt, C., Cross, G. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+tightly+regulated+inducible+expression+system+for+conditional+gene+knock-outs+and+dominant-negative+genetics+in+Trypanosoma+brucei%5BTitle%5D)+AND+%22Mol+Biochem+Parasitol%22%5BJournal%5D)">A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei.</a> Mol Biochem Parasitol. 99 (1), 89-101 (1999).</li>
<li>Shi, H., Djikeng, A., Mark, T., Wirtz, L. E., Tschudi, C., Ullu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+interference+in+Trypanosoma+brucei+by+heritable+and+inducible+double-stranded+RNA%5BTitle%5D)+AND+%22RNA%22%5BJournal%5D)">Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA.</a> RNA. 6 (7), New York, NY. 1069-1076 (2000).</li>
<li>Alsford, S., Turner, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+phenotyping+using+parallel+sequencing+of+RNA+interference+targets+in+the+African+trypanosome%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome.</a> Genome Res. 21 (6), 915-924 (2011).</li>
<li>Alsford, S., Eckert, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+decoding+of+antitrypanosomal+drug+efficacy+and+resistance%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">High-throughput decoding of antitrypanosomal drug efficacy and resistance.</a> Nature. 482 (7384), 232-236 (2012).</li>
<li>Mony, B. M., MacGregor, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+dissection+of+the+quorum+sensing+signalling+pathway+in+Trypanosoma+brucei%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Genome-wide dissection of the quorum sensing signalling pathway in Trypanosoma brucei.</a> Nature. 505 (7485), 681-685 (2014).</li>
<li>Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., Séraphin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration+%3A+Abstract+Nature+Biotechnology%5BTitle%5D)+AND+%22Nature+Biotechnol%22%5BJournal%5D)">A generic protein purification method for protein complex characterization and proteome exploration : Abstract Nature Biotechnology.</a> Nature Biotechnol. 17 (10), 1030-1032 (1999).</li>
<li>Los, G. V., Encell, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HaloTag%3A+A+Novel+Protein+Labeling+Technology+for+Cell+Imaging+and+Protein+Analysis%5BTitle%5D)+AND+%22ACS+Chem+Biol%22%5BJournal%5D)">HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis.</a> ACS Chem Biol. 3 (6), 373-382 (2008).</li>
<li>Dean, S., Sunter, J. D., Wheeler, R. J., Hodkinson, I., Gluenz, E., Gull, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+toolkit+enabling+efficient%2C+scalable+and+reproducible+gene+tagging+in+trypanosomatids%5BTitle%5D)+AND+%22Open+biol%22%5BJournal%5D)">A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids.</a> Open biol. 5 (1), 140197(2015).</li>
<li>Poon, S. K., Peacock, L., Gibson, W., Gull, K., Kelly, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modular+and+optimized+single+marker+system+for+generating+Trypanosoma+brucei+cell+lines+expressing+T7+RNA+polymerase+and+the+tetracycline+repressor%5BTitle%5D)+AND+%22Open+biol%22%5BJournal%5D)">A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor.</a> Open biol. 2 (2), 110037(2012).</li>
<li>Lam, S. S., Martell, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Directed+evolution+of+APEX2+for+electron+microscopy+and+proximity+labeling%5BTitle%5D)+AND+%22Nature+meth%22%5BJournal%5D)">Directed evolution of APEX2 for electron microscopy and proximity labeling.</a> Nature meth. 12 (1), 51-54 (2014).</li>
<li>Shu, X., Lev-Ram, V., Deerinck, T. J., Qi, Y., Ramko, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genetically+encoded+tag+for+correlated+light+and+electron+microscopy+of+intact+cells%2C+tissues%2C+and+organisms%5BTitle%5D)+AND+%22PLoS+biology%22%5BJournal%5D)">A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms.</a> PLoS biology. , (2011).</li>
<li>Schimanski, B., Nguyen, T. N., Günzl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+efficient+tandem+affinity+purification+of+trypanosome+protein+complexes+based+on+a+novel+epitope+combination%5BTitle%5D)+AND+%22Eukaryotic+Cell%22%5BJournal%5D)">Highly efficient tandem affinity purification of trypanosome protein complexes based on a novel epitope combination.</a> Eukaryotic Cell. , (2005).</li>
<li>Burkard, G., Fragoso, C. M., Roditi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+efficient+stable+transformation+of+bloodstream+forms+of+Trypanosoma+brucei%5BTitle%5D)+AND+%22Mol+Biochem+Parasitol%22%5BJournal%5D)">Highly efficient stable transformation of bloodstream forms of Trypanosoma brucei.</a> Mol Biochem Parasitol. 153 (2), 220-223 (2007).</li>
</ol>

The authors have nothing to disclose.

在蛋白质组学和转录方法在过去的5 - 10年的灵敏度显着改善提供了数以千计的基因及其产物的有价值的数据。然而，这些工具来解决这些蛋白质的功能没有跟上。 标记的蛋白质有利于无数次的实验，以确定其功能。例如，蛋白质可以在各种不同的颜色，以促进定位和共定位研究来融合于荧光蛋白。标签电子显微镜，如APEX2或miniSOG 11,12开发，使标签蛋白的超微结构定位。标签为生物化学，例如在TAP标记，并ProtC-TEV-PROTA（PTP）标记7,13允许与蛋白质的结合伙伴的身份或体外生物化学测定相关联配合物的纯化。 那些对protoc的成功至关重要的具体步骤醇有:二甲基亚砜掺入PCR主混合物1之前，在加入主混合物2中，在主混合物2中使用的商业聚合酶和cytomix电穿孔缓冲液的修饰的冻结PCR主要混合液1。在我们的经验，这是必要的，以便达到阳性孔的相似的比例使用细胞的双数为C末端标记的转染，作为N末端标记的转染。因此，如所描述，应执行的所有步骤。 这种技术只可能转染虫procyclic形式锥虫时获得成功。血液形式具有较低的转染效率14，而且他们很可能是由于这是无关的选择药物的密度依赖毒性选择过程中死亡。因此，我们以前的协议代表了血液形式锥虫9标记当前最先进的技术。它也可能是反式染效率会变化取决于特定的锥虫分离物。其他菌株可能需要额外的优化 - 这个协议使用927 SMOX procyclic形式进行了优化。可能增加成功的概率的措施包括:增加PCR扩增子的量，增加包括在转染细胞的数目。 我们提出了数百蛋白质可以并行标记的方法。这将有利于本地化和互动的大规模研究，补充现有大型数据集，并提供宝贵的信息社会。底漆模板可根据要求和质粒模板列表可以从:http://www.sdeanresearch.com/

布氏锥虫是原生动物寄生虫引起牛非洲人类trypanosomasis和那加那T。布氏是蛋白质功能的分析的理想生物体由于高品质的基因组，众多蛋白质组学和转录数据集和发达分子工具1-3的组合。蛋白质组学和测序的进步导致了突出潜在的有趣的基因4-6大型数据集;然而，许多基因都与他们现有的数据库相关联的最少信息。因此，有需要一种高通量方法，以帮助蛋白质功能表征。 一个标签蛋白的表达能给见解的多样性成蛋白质的功能。例如，标记有荧光蛋白或表位的蛋白质可以通过荧光显微镜，其中给出了关于其中PROT信息进行本地化EIN可能会发挥其生物效应。另外，贴上了TAP 7的蛋白质，HaloTag 8或他的标签可以用于生化分析及其合作伙伴的互动鉴定纯化。 我们最近开发出一种强大的标注方法为T.布氏 9。这在过去很长的引物PCR生成转染DNA，并允许数十种蛋白质并行的标记 - 一个重大改进现有的协议。现在，我们已经通过了一个数量级提高该协议在procyclic形式的可扩展性。这里，我们提出我们的方法，其中，我们执行PCR和转染到96-孔板，以恢复和选择在24孔板中发生。数百蛋白质现在可以在并行进行标记该方法提供用于标记整个锥虫基因组中的具有成本效益的，可行的方法。

<table><tbody><tr><td>Oligonucleotide</td><td>Life technologies</td><td>na</td><td>desalt only (do not use additional purification)</td></tr><tr><td>DMSO</td><td>Roche</td><td>Included with the Expand HiFi pack</td><td>Must be PCR grade</td></tr><tr><td>dNTP</td><td>any reputable</td><td></td><td></td></tr><tr><td>Expand HiFi polymerase</td><td>Roche</td><td>11759078001</td><td></td></tr><tr><td>BTX ECM830</td><td>Harvard Apparatus</td><td></td><td>Electroporator</td></tr><tr><td>HT-200 plate handler</td><td>Harvard Apparatus</td><td>HT-200</td><td>Handles the 96-well eletroplates</td></tr><tr><td>MOS 96 ELECTROPLATE 4 mm</td><td>Harvard Apparatus</td><td>45-0452</td><td>Electroplate for the 96-well electroporation</td></tr><tr><td>Blasticidin S Hydrochloride</td><td>Melford</td><td>3513-03-9</td><td></td></tr><tr><td>Hygromycin b Gold</td><td>Invivogen</td><td>ant-hg-1</td><td></td></tr><tr><td>Phleomycin</td><td>Melford</td><td>P0187</td><td></td></tr><tr><td>225 cm TC Flask, canted neck, phenolic cap</td><td>Appletonwoods</td><td>BC006</td><td></td></tr><tr><td>24 well culture plates</td><td>Appletonwoods</td><td>BC017</td><td></td></tr><tr><td>Eppendorf&amp;reg; PCR Cooler, iceless cold storage system for 96 well plates and PCR tubes</td><td>Sigma Aldrich</td><td>Z606634-1EA</td><td></td></tr><tr><td>96-well Multiply&amp;reg; PCR plate with lateral skirt</td><td>Sarstedt</td><td>72.1979.203</td><td></td></tr></tbody></table>

high-throughput gene tagging in trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

在这个代表转染的引物使用TagIt perl脚本9设计和商业合成。在96孔PCR进行和验证所描述（ 图1A）;在这个例子中，95/96的PCR成功（ 图1B）。在我们的经验，重复或者与原始引物或重新合成的引物失败的反应不会导致成功的PCR。 扩增子被转移到96孔电穿孔板电穿孔（ 图2A），然后转移到用于恢复和选择的24孔培养板中。后有足够的时间以允许发生的选择，各孔打进之前进一步分析存活。在这个例子中，九十六分之八十八井（92％）为15天之后选择阳性。 <i毫克ALT ="图1"SRC ="/文件/ ftp_upload / 54342 / 54342fig1.jpg"/&gt; 图1:长引物的PCR（A）的图案的验证 PCR产物转移从96孔PCR平板的琼脂糖凝胶进行PCR验证。 （B）为在N末端 ​​标记95的基因的代表性96孔PCR验证凝胶，和不含有5&#39;靶向同源充当在转染的阴性对照1扩增子。样品装载到1％（W / V）琼脂糖凝胶，并在100伏解决30分钟。 <a href="https://www.jove.com/files/ftp_upload/54342/54342fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54342/54342fig2.jpg" /> 图2:96孔电穿孔和选择 （A）模式的电穿孔细胞从96孔电开发平台转移。E要24孔培养板。 （B）示意图，显示在后24孔板选择15天一个典型的活/死得分。绿色代表着成功转染，灰色代表也只有死细胞。在这个例子中，井九十六分之八十八（92％）含有成功转染寄生虫。 <a href="https://www.jove.com/files/ftp_upload/54342/54342fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about High-throughput Gene Tagging in Trypanosoma brucei at JoVE.com

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

高通量基因定位在<em&gt;布氏锥虫</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

high-throughput-gene-tagging-in-trypanosoma-brucei

Research

JoVE Journal

Immunology and Infection

High-throughput Gene Tagging in Trypanosoma brucei

7.9K 次观看.  University of Oxford. Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

视频: High-throughput Gene Tagging in Trypanosoma brucei

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

审核端粒G -悬结构锥虫布氏</em

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

科研

免疫与感染

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Here, we describe a protocol to introduce a gene knockout into the extracellular amastigote of Trypanosoma cruzi, using the CRISPR/Cas9 system. The growth phenotype can be followed up either by cell counting of axenic amastigote culture or by proliferation of intracellular amastigotes after host cell invasion.

使用CRISPR/Cas9系统将基因挖空直接引入锥虫瘤的肌瘤阶段

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target ...

遗传学

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).

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 ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

利用荧光蛋白监视非洲锥虫Glycosome动态

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing &#946;-galactosidase

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many non-endemic countries due to the increase in migration. This disease affects nearly 8 million people, with new cases estimated at 50,000 per year. In the 1960s and 70s, two drugs for ChD treatment were introduced: nifurtimox and benznidazole (BZN). Both are effective in newborns and during the acute phase of the disease but not in the chronic phase, and their use is associated with important side effects. These facts underscore the urgent need to intensify the search for new drugs against T. cruzi.
T. cruzi is transmitted through hematophagous insect vectors of the Reduviidae and Hemiptera families. Once in the mammalian host, it multiplies intracellularly as the non-flagellated amastigote form and differentiates into the trypomastigote, the bloodstream non-replicative infective form. Inside the insect vector, trypomastigotes transform into the epimastigote stage and multiply through binary fission.
This paper describes an assay based on measuring the activity of the cytoplasmic &#946;-galactosidase released into the culture due to parasites lysis by using the substrate, chlorophenol red &#946;-D-galactopyranoside (CPRG). For this, the T. cruzi Dm28c strain was transfected with a &#946;-galactosidase-overexpressing plasmid and used for in vitro pharmacological screening in epimastigote, trypomastigote, and amastigote stages. This paper also describes how to measure the enzymatic activity in cultured epimastigotes, infected Vero cells with amastigotes, and trypomastigotes released from the cultured cells using the reference drug, benznidazole, as an example. This colorimetric assay is easily performed and can be scaled to a high-throughput format and applied to other T. cruzi strains.

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing &#946;-galactosidase

We describe a high-throughput colorimetric assay measuring &#946;-galactosidase activity in three life cycle stages of Trypanosoma cruzi, the causative agent of Chagas disease. This assay can be used to identify trypanocidal compounds in an easy, fast, and reproducible manner.

体外 使用表达β半乳糖苷酶的寄生虫针对 克氏锥虫 的所有生命周期阶段进行药物筛选

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many ...

生物化学

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

生物发光成像检测到非洲锥虫病的晚期感染

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

检测<em&gt;布氏锥虫</em&gt;变体表面糖蛋白通过磁性细胞分选和流式细胞仪开关

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations.
HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity.
Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal.
These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns

This method of trypanosome separation from blood depends on their surface charge being less negative than mammalian blood cells. Infected blood is placed and treated on an anion-exchanger column. This method, the most fitting diagnostic for African trypanosomiasis, provides purified parasites for immunological, biological, biochemical, pharmaceutical and molecular biology investigations.

阴离子交换剂 (二乙基氨基乙基纤维素柱) 纯化包括非洲在内的细胞外锥虫体

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is ...

基于 pHluorin2 的布氏锥虫糖体 pH 传感器的开发

Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. Little is known about the cellular responses generated when environmental glucose levels change. In both bloodstream and procyclic form (insect stage) parasites, glycosomes house most of glycolysis. These organelles are rapidly acidified in response to glucose deprivation, which likely results in the allosteric regulation of glycolytic enzymes such as hexokinase. In previous work, localizing the chemical probe used to make pH measurements was challenging, limiting its utility in other applications.
This paper describes the development and use of parasites that express glycosomally localized pHluorin2, a heritable protein pH biosensor. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid)-dependent decrease in excitation at 395 nm while simultaneously yielding an increase in excitation at 475 nm. Transgenic parasites were generated by cloning the pHluorin2 open reading frame into the trypanosome expression vector pLEW100v5, enabling inducible protein expression in either lifecycle stage. Immunofluorescence was used to confirm the glycosomal localization of the pHluorin2 biosensor, comparing the localization of the biosensor to the glycosomal resident protein aldolase. The sensor responsiveness was calibrated at differing pH levels by incubating cells in a series of buffers that ranged in pH from 4 to 8, an approach we have previously used to calibrate a fluorescein-based pH sensor. We then measured pHluorin2 fluorescence at 405 nm and 488 nm using flow cytometry to determine glycosomal pH. We validated the performance of the live transgenic pHluorin2-expressing parasites, monitoring pH over time in response to glucose deprivation, a known trigger of glycosomal acidification in PF parasites. This tool has a range of potential applications, including potentially being used in high-throughput drug screening. Beyond glycosomal pH, the sensor could be adapted to other organelles or used in other trypanosomatids to understand pH dynamics&#160;in the live cell setting.

作者聚焦：使用 pHluorin2 生物传感器监测 布氏锥 虫糖体 pH 值的进展

We describe a method to study how pH responds to environmental cues in the glycosomes of the bloodstream form of African trypanosomes. This approach involves a pH-sensitive heritable protein sensor in combination with flow cytometry to measure pH dynamics, both as a time-course assay and in a high-throughput screen format.

测量布氏活锥虫的动态糖体 pH 值变化

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. ...

高通量基因定位在

摘要

探索更多视频

审核端粒G -悬结构锥虫布氏

使用CRISPR/Cas9系统将基因挖空直接引入锥虫瘤的肌瘤阶段

遗传操纵

利用荧光蛋白监视非洲锥虫Glycosome动态

体外使用表达β半乳糖苷酶的寄生虫针对克氏锥虫的所有生命周期阶段进行药物筛选

生物发光成像检测到非洲锥虫病的晚期感染

检测

阴离子交换剂 (二乙基氨基乙基纤维素柱) 纯化包括非洲在内的细胞外锥虫体

测量布氏活锥虫的动态糖体 pH 值变化

测量布氏活锥虫的动态糖体 pH 值变化

高通量基因定位在

摘要

探索更多视频

审核端粒G -悬结构锥虫布氏

使用CRISPR/Cas9系统将基因挖空直接引入锥虫瘤的肌瘤阶段

遗传操纵

利用荧光蛋白监视非洲锥虫Glycosome动态

体外 使用表达β半乳糖苷酶的寄生虫针对 克氏锥虫 的所有生命周期阶段进行药物筛选

生物发光成像检测到非洲锥虫病的晚期感染

检测

阴离子交换剂 (二乙基氨基乙基纤维素柱) 纯化包括非洲在内的细胞外锥虫体

测量布氏活锥虫的动态糖体 pH 值变化

测量布氏活锥虫的动态糖体 pH 值变化

体外使用表达β半乳糖苷酶的寄生虫针对克氏锥虫的所有生命周期阶段进行药物筛选