我们是鹰二胁田和延Bukh JFH1和J6CF株，分别表示感谢。此外，我们感谢查尔斯·赖斯HuH-7.5细胞和9E10的抗体，史蒂芬Foung为E2特定的抗体CBH-23，实验病毒学，有益的建议和讨论Twincore部的所有成员。

用PEG细胞融合 1。细胞培养<ol><li>咦，7.5 HeLa和Hep56.1D的天真或包装细胞株15厘米的完整的DMEM培养液（DMEM培养液cplt）中型细胞培养皿12文化，培养液补充2毫米L-谷氨酰胺，1个非必需氨基酸，100 U / L mL青霉素，100μg/ mL链霉素，10％胎牛血清。运用适当的选择如表1所示的稳定细胞线。 </li></ol> 2。丙型肝炎病毒RNA的转染<ol><li>洗两次了7.5细胞（1合流15厘米盘子）用磷酸盐缓冲液（PBS），trypsinize，在1.5×10 7细胞/ ml在Cytomix浓度（120毫米氯化钾， 氯化钙 0.15毫米，10毫米ķ悬浮2 HPO 4 pH值7.6 - 25毫米的HEPES，EGTA 2毫米，5毫米MgCl 2的 ，调整到7.6 pH值与氢氧化钾和过滤消毒），其中包含2毫米ATP和5 mM的谷胱甘肽。 </li><li>转染通过electroporat离子400μL细胞悬液，10微克，丙型肝炎病毒RNA（pFKi389Luc-EI/NS3-3“_JFH1_dg 13）使用一个0.4厘米的缝隙宽度和一个基因脉冲Xcell系统（Biorad公司）设置为975试管（6X10 6个细胞） μF和270 V的描述以前14。 </li><li>立即转了7.5细胞转移至10毫升培养液cplt介质和种子成一个直径10厘米的单培养皿整个悬挂。为培养24 h的细胞在37°C间和5％的CO 2。 </li></ol> 3。经PEG诱导融合<ol><li> 2.1收获细胞悬浮在培养液cplt中等和稀释到5×10 5细胞/ ml。 </li><li>生成咦-7.5的单细胞悬液，HeLa细胞，并包装细胞Hep56.1D，并稀释至5×10 5细胞/ mL，2×10 5细胞/毫升，2×10 5细胞/ ml，分别。需要注意的是包装细胞异位两个明确的丙型肝炎病毒蛋白的核心，素E1，E2，P7，NS2的后转依赖慢病毒载体转导的C-E1和E2-P7，NS2的表达盒，分别为13，15。 </li><li>准备用2-3每口井的无菌玻璃盖的融合效率的进一步分析单6孔培养板。 </li><li>结合1毫升转了1毫升的包装细胞，种子7.5窝藏亚基因组复制子细胞到6孔板，在37°C和5％的CO 2孵育24小时。 </li><li>在这个时间点，细胞密度应介于60-80％汇合诱导细胞融合前，以便在靠近细胞膜的融合细胞。 </li><li>吸共培养细胞的培养基，用1 mL PBS洗一次，然后小心加入500μL预热的40％PEG-1500或PBS作为控制和孵化，在37°C诱导异核体的形成，为5分钟。 </li><li>吸聚乙二醇，并仔细清洗3-5次为1分钟（分钟）2毫升PBS，每洗，以去除多余的PEG和最后加2毫升培养液cplt中每口井，并于37°C和5％的CO 2。 </li><li>四十八小时后融合，收集各上清通过0.45微米的过滤器的过滤器。 </li><li>测量生产在融合heterokaryons的传染性颗粒天真了7.5细胞（8x10的4个细胞/孔接种在12孔板，24小时前接种）接种500μL细胞培养液和随后测定荧光素酶活动14。 </li></ol> 4。免疫测定融合效率<ol><li>后收集细胞培养上清液（3.8节），洗井含玻璃盖单，一次1分钟1毫升的PBS。在室温（RT），应执行以下所有步骤和应用1 mL PBS洗涤每洗1分钟。 </li><li>修复细胞600μL3％多聚甲醛（PFA）的PBS 15分钟，然后用PBS洗3倍。仔细转让盖玻片到24孔板使用镊子。 </li><li>通透细胞500μL0.5％Triton的PBS中X-100的5分钟，随后用PBS洗3倍。 </li><li>准备辅以5％山羊血清抗体在PBS溶液。在浓度为6.4毫微克/毫升的浓度在0.5微克/毫升和人类单克隆抗E2抗体（CBH-23）使用反病毒NS5A抗体（9E10）。适用于250μL，每口井，并在室温下孵育45分钟。 </li><li>用PBS洗3倍和250μL，中学的羊抗鼠或羊抗人IgG特异性抗体结合Alexa的氟546或网站，福陆公司488约束的主要抗体检测浓度2微克/毫升的PBS，辅以5％山羊血清。在室温30分钟，在黑暗中培养。 </li><li>然后，用PBS洗1X和随后用250微升的DAPI（4&#39;，6&#39;-diamidino-2-苯基二胺）在PBS稀释1:3,000，在室温下为1分钟，在黑暗中孵育染色细胞核。 </li><li>洗净用PBS和1X 4X与H 2 </sUB&gt;澳，然后装入盖玻片，在显微镜幻灯片Fluoromount下降到倒挂，让在黑暗和评估的样品干燥的间接免疫荧光显微镜。 </li></ol>瞬时转染的原型泡沫病毒（PFV）糖蛋白的融合 5。病毒接种的制备<ol><li> HuH-7.5细胞在体外转录丙型肝炎病毒RNA转染和收获后48 h和72 h细胞培养上清中所述先前14产生高滴度病毒的股票。限制稀释法（TCID 50）13，确定病毒滴度。 </li></ol> 6。细胞培养，细胞融合的膜融合PFV糖蛋白的转染<ol><li>文化Lunet氮，HeLa细胞，并Hep56.1D hCD81细胞根据表1说​​明，在培养液cplt介质中包含适当的选择。 </li><li>分离细胞，如2.1中所述，准备在培养液cplt米的单细胞悬液edium，并共同培养Lunet列印与HeLa或Hep56.1D hCD81细胞株的细胞产生的总细胞密度1.5×10 5细胞每在适当的比例（如HeLa细胞Hep56.1D hCD81或1:1 1:2） 12孵育24小时在37°C和5％的CO 2。 </li><li>第二天，瞬时转染用脂质体2000根据制造商的指示的高度膜融合的PFV包膜蛋白（pczHFVenvEM066 16）的细胞。 </li></ol> 7。感染试验<ol><li>接种heterokaryons的病毒库存为12Ĥ一夜之间350μL（内政部〜2.3）的转染30小时后，用PBS洗一次，添加1毫升培养液cplt每口井的媒介。 48小时在37°C和5％的CO 2。 </li><li>收获上清异核文化，通过0.45微米的过滤器和接种了7.5细胞，接种于96孔板（1×10 4细胞/孔）的前一天，在限制稀释法，过滤器<sup&gt; 13。 </li><li> 72小时后，感染了7.5细胞染色与丙型肝炎病毒特异性抗体（NS5A的; 9E10）量化传染性后代颗粒生产。 </li></ol> 8。细胞融合的可视化<ol><li>可视化的融合事件，准备在DMEM培养基的5至10μM的解决方案CellTracker染料无添加剂和前完成共培养6小时以下的步骤（6.2和6.3节中所述）。 </li><li>一次用5毫升的PBS洗涤贴壁细胞，然后孵育细胞4毫升45分钟，每10厘米的菜37染色解决方案°C和5％CO 2（染色Lunetñ与CellTracker橙CMTMR HeLa或Hep56.1D的细胞hCD81细胞CellTracker绿色CMFDA，分别）。 </li><li> 1分钟，5毫升的PBS洗涤细胞一次和添加培养液cplt介质，为6小时37°C和5％CO 2孵育。 </li><li>如上所述，在节6.2，然而，加盖玻片的准备播种染细胞和转染播种前的细胞培养皿。 </li><li>转染后30小时，洗涤细胞1毫升的PBS精心为1分钟，然后用250微升3％的煤灰修复细胞在室温为15分钟。用1 mL PBS洗涤细胞1分钟后，用DAPI染色1分钟，如4.6节所述，1分钟1毫升PBS洗一次，重复清洗与H 2 O和安装盖玻片，载玻片上（见4.7节）。用荧光显微镜分析细胞。 </li></ol><table border="1"><tbody><tr><td> 种类 </td><td> 细胞株 </td><td> 起源 </td><td> 生长介质与选择 </td></tr><tr><td>人的</td><td> 呵呵7.5 </td><td>亚克隆的HuH-7肝癌细胞株17 </td><td> DMEM培养液cplt </td></tr><tr><td></td><td> 呵呵-7.5 [CE1的] [E2p7NS2] </td><td>稳定表达病毒蛋白的核心，E1，E2，P7，NS2的，转由慢病毒基因T转让（BOT）的两个独立的基因盒</td><td> DMEM培养液cplt +稻瘟5微克/毫升</td></tr><tr><td></td><td> HuH-7 Lunet列印 </td><td>亚克隆的HuH-7肝癌细胞株9 </td><td> DMEM培养液cplt </td></tr><tr><td></td><td> HeLa细胞 </td><td>宫颈腺癌细胞株（ATCC编号：CCL-2） </td><td> DMEM培养液cplt </td></tr><tr><td></td><td> HeLa细胞[CE1的[E2p7NS2] </td><td>稳定表达病毒蛋白的核心，E1，E2，P7，NS2的，转由两个独立的基因盒的慢病毒基因转移</td><td> DMEM培养液cplt + G418的750微克/毫升;稻瘟5微克/毫升</td></tr><tr><td>鼠标</td><td> Hep56.1D </td><td>原发性肝癌（研究恩克样的礼物） 成年C57BL/6J小鼠</td><td> DMEM培养液cplt </td></tr><tr><td></td><td> Hep56.1D [CE1的[E2p7NS2] </td><td>稳定前按病毒核心蛋白，E1，E2，P7，NS2的，转由两个独立的基因盒的慢病毒基因转移</td><td> DMEM培养液cplt + G418的750微克/毫升;稻瘟5微克/毫升</td></tr><tr><td></td><td> Hep56.1D hCD81 </td><td>稳定表达人CD81转慢病毒基因转移</td><td> DMEM培养液cplt +稻瘟5微克/毫升</td></tr></tbody></table>完成后，G418的：，H geneticin： 人类起源和文化用细胞系Cplt条件表1规格。 9。代表结果在这项研究中，我们采用了两种体细胞融合，使我们专门调查heterokaryons的丙型肝炎病毒复制周期完成的制约因素的影响，不同的方法。值得注意的是，两个不同类型的细胞融合后的反式互补的条件设计，确保了（方法2）只有在人类肝细胞和非允许细胞株，病毒大会（方法1）或病毒进入和充分的丙型肝炎病毒复制周期之间的真正heterokaryons的完成。 在第一种方法，咦-7.5，高度宽松的丙型肝炎病毒，细胞转染与亚基因组复制子表达支持亚基丙型肝炎病毒RNA的复制（咦-7.5复制子细胞）的荧光素酶基因和丙型肝炎病毒非结构蛋白。这些细胞共同培养和融合细胞系中表达丙型肝炎病毒核心结构蛋白，E1，E2以及配件蛋白质P7和NS2。 Figure1A显示了实验过程的概述。重要的是，粒子装配所需的所有蛋白质的PEG融合后，应该在形成异核体，可以通过对个别病毒蛋白的免疫证实。图1B描绘了一个模范的融合事件，在双方的NS5A的信号在HuH-7.5复制子细胞和E2表示在Huh-7.5 [CE1的表达[E2p7NS2]包装细胞检测PEG诱导异核体形成后，在相同的细胞质。当共同培养的细胞，而不是用PBS治疗，诱导无融合，因此，只有单阳性细胞和无信号的共定位观察。此外，为了控制转基因表达，我们进行了酶联免疫吸附具体的核心和E2（以前15详细报道）。咦7.5除了控制包装细胞系[CE1的[E2p7NS2，我们产生的HeLa和人类非肝小鼠肝细胞的代表，分别Hep56.1D包装细胞。在这些后者细胞丙型肝炎病毒RNA的复制效率低和丙型肝炎病毒的组装和释放并没有被证明。图1C所示的融合实验的结果。收集上清共培养48小时后融合感应和用于随后的荧光素酶检测天真了7.5细胞接种。值得注意的是，当了7.5复制子细胞融合，要么天真的细胞株或用PBS作为对照治疗，没有传染性的培养液中检测到。然而，经PEG诱导之间咦-7.5复制子和包装细胞系的细胞融合时，复制子和结构蛋白组成型表达之间的跨互补救出病毒造成heterokaryons的生产和传染性病毒颗粒释放到培养液。因此，我们得出的结论是病毒生产所需的细胞融合，并在包装​​细胞株的病毒蛋白的表达。引人注目的是，不仅咦-7.5肝癌细胞的heterokaryons的，但也与人类非肝（CE1的HeLa细胞[] [E2p7NS2]）和小鼠肝（Hep56.1D [CE1的[E2p7NS2）细胞允许病毒释放heterokaryons的，表明，组装和释放不显性抑制由这些细胞类型中的制约因素。在弗伦岑在包装细胞系和地道入口进入靶细胞的结构蛋白的表达<em&gt;等15。 利用细胞融合的一个替代方法，以分析可能影响进入细胞，RNA翻译和RNA复制的复制周期阶段的限制，并排除高转咦7.5复制子细胞中的病毒的负担会妨碍饱和制约因素检测。为此，我们开发了一个基于HCVcc和检测新病毒在这些细胞生产heterokaryons的接种独立检测。正如图2A中所述，诱导体细胞融合后的细胞共培养膜融合糖蛋白的表达。这种方法被选中，因为敏感性增加可能是由于提高融合效率。 heterokaryons的是由不同的细胞系与前共同培育的CellTracker染料染色的可视化。一个异核体与细胞质内的染料均匀分布表明，两种类型的细胞融合作为表明D图2B。只有在的Lunet列印HeLa细胞或Hep56.1D hCD81细胞与细胞heterokaryons的所有丙型肝炎病毒进入的因素，从而选择性地表达渲染heterokaryons的易患丙型肝炎病毒进入细胞。与HCVcc的挑战，这是显而易见的， 从头生产的约10倍以上的检测（图2C）的背景病毒感染后代heterokaryons的完整的丙型肝炎病毒的复制。作为对照，Lunet N细胞Lunet N细胞融合。重要的是，只有非常低的水平感染丙型肝炎病毒接近检出限进行了观察。当Lunet N细胞与天真Hep56.1D细胞的融合获得了类似的结果。在这两种情况下，人类CD81的缺席，使HCV富有成效感染heterokaryons的，。这后者在这两个案件中发现的传染性是非常低的可能由于Lunet N细胞和/或残留的接种传染性病毒输入的低水平感染水平低。在对比HeLa细胞和他p56.1D hCD81细胞表达人CD81，从而补充缺乏的细胞中的异核项因素，并允许丙型肝炎病毒进入细胞。因此，增加10倍的水平感染丙型肝炎病毒，在涉及这两个细胞系heterokaryons的媒介检测可能反映从头 heterokaryons的传染性颗粒的生产。因此，我们认为，丙型肝炎病毒复制周期的完成，并不限于不含主导HCV制约因素，在这些细胞株中的表达。 <img alt="图1" src="/files/ftp_upload/4029/4029fig1.jpg" /> 图1。 跨互补丙型肝炎病毒在heterokaryons的组装和释放。（一）PEG介导的细胞融合的实验过程纲要。天真了7.5细胞转染荧光素酶的JFH1记者复制子吕克-NS3蛋白-5B电第二天，细胞分离和共培养幼稚细胞或包装<html>细胞表达HCV核心，E1和E2，P7和NS2。 24小时后共同培育，异核体形成的诱导治疗，40％的PEG作为阴性对照PBS。 48小时后收获，无细胞上清液接种天真了7.5细胞。测定荧光素酶活性的传染性。 （二）检测的异核细胞，免疫组化染色对E2和NS5A单克隆抗体。同时表达这两种蛋白在细胞内，细胞融合。 （三）荧光素酶的测量进行量化病毒传染性从heterokaryons的产生。三个独立的实验和手段的标准偏差的平均值。单杠代表背景的RLU在未受感染了7.5细胞决定的。 <a href="http://www.jove.com/files/ftp_upload/4029/4029fig1large.jpg" target="_blank">点击这里查看大图</a> 。 9/4029fig2.jpg“/&gt; 图2。完成后丙型肝炎病毒复制周期接种的heterokaryons的。（一）实验过程中的总体示意图。 HuH-7 Lunet的N细胞缺乏CD81的指示细胞株缺乏或表达人CD81共同培养。重要的是，这些后者细胞缺乏至少有一个丙型肝炎病毒进入的因素，因此不能进行富有成效的感染。第二天，发起的膜融合PFV病毒包膜蛋白转染融合。 30小时后，细胞与感染丙型肝炎病毒颗粒的挑战。来衡量进入细胞，RNA复制及病毒生产由这些heterokaryons的持续，释放在细胞培养液的传染性被确定接种后48小时。为此，天真了7.5细胞作为靶细胞在限制稀释法（TCID 50）。 （乙）检测的异核细胞染色，与CellTracker绿色或6小时CellTracker橙合作，cultiv前ATION和转染。 30小时后，细胞被固定，并用DAPI染色。 （三）Lunet N细胞进行融合，HeLa细胞，的天真Hep56.1D或Hep56.1D细胞表达人CD81。这些文化与HCVcc（2.3 MOI），接种。 48 h后收集培养液和新从heterokaryons的粒子释放TCID 50测定，用天真了7.5靶细胞。 3次独立实验的平均值。单杠检测的检测限。 <a href="http://www.jove.com/files/ftp_upload/4029/4029fig2large.jpg" target="_blank">点击这里查看大图</a> 。

<ol>
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总磷收到Biotest咨询费。

我们提出了两种方法在培养细胞诱导异核分析显性负的限制，排除丙肝病毒的复制形成。使用这些程序时，我们排除了存在的一个组成明示或病毒引起的各种人类非肝小鼠肝细胞株的主导因素。第一法主要分析制约因素，防止传染性后代的丙型肝炎病毒的组装和释放。因为在这种情况下包装细胞融合丙型肝炎病毒复制子细胞，包装细胞中表达的因素可能限制只获取的细胞，已经有一个高负荷的病?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司，卡尔斯鲁厄，德国</td><td> 41965-039 </td></tr><tr><td> L-谷氨酰胺</td><td> Invitrogen公司，卡尔斯鲁厄，德国</td><td> 25030-024 </td></tr><tr><td>非必需氨基酸</td><td> Invitrogen公司，卡尔斯鲁厄，德国</td><td> 11140-035 </td></tr><tr><td>青霉素/ </td><td> Invitrogen公司，卡尔斯鲁厄，德国</td><td> 15140-122 </td></tr><tr><td>胎牛血清</td><td>临时机场管理局，科尔贝，德国</td><td> A15151 </td></tr><tr><td> α-E2（CBH23的） </td><td>慷慨地提供由史蒂芬Foung 10 </td><td></td></tr><tr><td>三磷酸腺苷</td><td> Steinheim，德国西格玛， </td><td> A2833-106 </td></tr><tr><td>谷胱甘肽</td><td> Steinheim，德国西格玛， </td><td&gt; G4251-1G </td></tr><tr><td>稻瘟</td><td> invivo根，圣地亚哥，美国</td><td> ANT-BL-1 </td></tr><tr><td> G418（geneticin） </td><td> Invitrogen公司，卡尔斯鲁厄，德国</td><td> 11811-064 </td></tr><tr><td>聚乙二醇-1500 </td><td>罗氏，德国曼海姆</td><td> 10783641001 </td></tr><tr><td>多聚甲醛</td><td>罗斯，卡尔斯鲁厄，德国</td><td> 0335.3 </td></tr><tr><td>的Triton X-100 </td><td>罗斯，卡尔斯鲁厄，德国</td><td> 3051.2 </td></tr><tr><td>山羊血清</td><td> Steinheim，德国西格玛， </td><td> G9023-5ML </td></tr><tr><td> α-NS5A的（9E10） </td><td>请提供由查尔斯·赖斯7 </td><td></td></tr><tr><td>的DAPI（4&#39;，6&#39;-diamidino-2-苯基盐酸盐） </td><td> Invitrogen公司</td><td> D1306 </td></tr><tr><td> Alexa的氟546 - 羊抗人IgG </td><td> Invitrogen公司，卡尔斯鲁厄，德国</td><td> A21089 </tD&gt; </tr><tr><td> Alexa的氟488 - 羊抗鼠IgG </td><td> Invitrogen公司，卡尔斯鲁厄，德国</td><td> A10680 </td></tr><tr><td>脂质体2000 </td><td> Invitrogen公司，卡尔斯鲁厄，德国</td><td> 11668-019 </td></tr><tr><td> CellTracker CMTMR </td><td> Invitrogen公司，卡尔斯鲁厄，德国</td><td> C2927 </td></tr><tr><td> CellTracker CMFDA </td><td> Invitrogen公司，卡尔斯鲁厄，德国</td><td> C2925 </td></tr><tr><td> fluoromount </td><td> Steinheim，德国西格玛， </td><td> F4680-25ML </td></tr><tr><td>所有其他化学品</td><td>罗斯，卡尔斯鲁厄，德国</td><td></td></tr><tr><td>细胞培养材料</td><td>萨尔斯塔特，Nümbrecht，德国</td><td></td></tr></tbody></table>

two methods of heterokaryon formation to discover hcv restriction factors

丙型肝炎病毒（HCV）是一种嗜肝病毒与宿主范围只限于人类和黑猩猩。虽然已在人类非肝和小鼠细胞株观察丙型肝炎病毒RNA复制，效率非常低，需要长期的选拔程序，用丙型肝炎病毒复制子构建表达占主导地位的抗生素选择标记1-5。因此，丙型肝炎病毒在体外的研究仅限于人类肝癌细胞株病毒进入和完成病毒生命周期的宽容。由于医疗券计划狭窄的物种的取向，有没有免疫的小动物模型，维持完整的丙型肝炎病毒复制周期6-8。低效的丙型肝炎病毒复制非人类细胞如鼠标起源的可能是由于缺乏主机依赖基本因素和/或制约因素表达的遗传不相容。 我们调查是否抑制丙型肝炎病毒的传播是由占主导地位的限制，发无论是从非肝组织或在小鼠肝细胞株人类细胞系的构造函数中。为此，我们制定了两个独立的条件反式互补的方法，依靠体细胞融合。在这两种情况下，完成病毒的复制周期是唯一可能在heterokaryons的。因此，成功的反式互补，这是通过测量从头传染性病毒后代的生产决定，表示没有占主导地位的限制。 具体来说，亚基携带丙型肝炎病毒复制子的荧光素酶基因转染非常宽容的人肝癌细胞（Huh-7.5细胞）。随后，这些细胞共培养和融合各种人类和小鼠细胞表达丙型肝炎病毒结构蛋白的核心，信封1和2（素E1，E2）和辅助蛋白P7和NS2。提供治疗与聚乙二醇（PEG）发起，细胞融合，文化释放病毒PA工艺品的感染，其中幼稚细胞在受体依赖的方式。 评估完整的病毒生命周期的主导限制，包括进入细胞，RNA的翻译，复制和病毒装配的影响，我们采取了一个人的肝细胞株（HuH-7 Lunet N细胞）缺乏CD81的内源性表达的优势，丙型肝炎病毒的基本项因素。在异位表达CD81的情况下，这些细胞基本上是难治性丙型肝炎病毒感染10。重要的是，当共同培养和融合细胞表达人CD81，但缺乏起码的另一个关键进入细胞因子（即SR-BI的CLDN1，OCLN），只有heterokaryons的显示一整套进入丙型肝炎病毒感染的必要因素。因此，分析优势制约因素，如果抑制丙型肝炎病毒复制周期完成，我们融合Lunet N细胞与人类和小鼠的起源的各种细胞履行上述暴ERIA。当共同培养的细胞转染高度膜融合病毒包膜蛋白突变体的原型泡沫病毒（PFV 11），并随后与感染丙型肝炎病毒颗粒（HCVcc）的挑战， 从头传染性病毒的生产进行了观察。这表明，丙型肝炎病毒成功地完成其复制周期，从而排除了在这些细胞株的主导限制因子的表达heterokaryons的。这些新的条件反式互补的方法将是有益的筛选丙型肝炎病毒的特定优势制约因素表达的细胞株和原代细胞的大面板。

Watch this Scientific Journal Video about Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors at JoVE.com

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

我们描述有条件的两种方法<em&gt;反</em&gt;丙型肝炎病毒（HCV）的组装和完成的全病毒的生命周期，这对异核体的形成依靠的互补。这些技术是合适的细胞系表达占主导地位的制约因素，其中排除感染丙型肝炎病毒的后代生产的屏幕。

两种异核体的形成方法，发现丙型肝炎病毒的制约因素

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

two-methods-heterokaryon-formation-to-discover-hcv-restriction

Research

JoVE Journal

Immunology and Infection

11.5K 次观看.  Twincore, Centre for Experimental and Clinical Infection Research. 我们描述有条件的两种方法反丙型肝炎病毒（HCV）的组装和完成的全病毒的生命周期，这对异核体的形成依靠的互补。这些技术是合适的细胞系表达占主导地位的制约因素，其中排除感染丙型肝炎病毒的后代生产的屏幕。

视频: Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

科研

免疫与感染

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

限制性内切酶克隆方法，以评估<em&gt;在体外</em&gt; HIV-1 C亚型的Gag-MJ4嵌合病毒的复制能力

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-&#947;, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-&#947;, TNF-&#945;, and MIP-1&#945; were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest.

Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure.

方法评估细胞毒性的可燃烟草产品的准备和免疫抑制

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

方法来提高高分辨率的敏感性疟疾熔单核苷酸多态性基因分型

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

方法来抑制细菌Pyomelanin生产和确定敏感性氧化应激的影响相应增加

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

破译和成像发病机制与盘带<em&gt;脓肿分枝杆菌</em&gt;斑马鱼胚胎

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

方法调查小RNA和核糖体占用的调控作用<em&gt;恶性疟原虫</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...