Name: two methods of heterokaryon formation to discover hcv restriction factors
Uploaded: 2012-07-16T12:00:00
Description: Watch this Scientific Journal Video about Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors at JoVE.com

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?...

<ol>
	<li>Zhu, Q., Guo, J. T., Seeger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+hepatitis+C+virus+subgenomes+in+nonhepatic+epithelial+and+mouse+hepatoma+cells.">Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells.</a> J. Virol. 77, 9204-9210 (2003).</li><li>Kato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonhepatic+cell+lines+HeLa+and+293+support+efficient+replication+of+the+hepatitis+C+virus+genotype+2a+subgenomic+replicon.">Nonhepatic cell lines HeLa and 293 support efficient replication of the hepatitis C virus genotype 2a subgenomic replicon.</a> J. Virol. 79, 592-596 (2005).</li><li>Ali, S., Pellerin, C., Lamarre, D., Kukolj, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+subgenomic+replicons+in+the+human+embryonic+kidney+293+cell+line.">Hepatitis C virus subgenomic replicons in the human embryonic kidney 293 cell line.</a> J. Virol. 78, 491-501 (2004).</li><li>Date, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+2a+hepatitis+C+virus+subgenomic+replicon+can+replicate+in+HepG2+and+IMY-N9+cells.">Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells.</a> J. Biol. Chem. 279, 22371-22376 (2004).</li><li>Chang, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+hepatitis+C+virus+(HCV)+RNA+in+mouse+embryonic+fibroblasts:+protein+kinase+R+(PKR)-dependent+and+PKR-independent+mechanisms+for+controlling+HCV+RNA+replication+and+mediating+interferon+activities.">Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities.</a> J. Virol. 80, 7364-7374 (2006).</li><li>Zhong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+hepatitis+C+virus+infection+in+vitro.">Robust hepatitis C virus infection in vitro.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 9294-9299 (2005).</li><li>Lindenbach, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+replication+of+hepatitis+C+virus+in+cell+culture.">Complete replication of hepatitis C virus in cell culture.</a> Science. 309, 623-626 (2005).</li><li>Wakita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+infectious+hepatitis+C+virus+in+tissue+culture+from+a+cloned+viral+genome.">Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.</a> Nat. Med. 11, 791-796 (2005).</li><li>Witteveldt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD81+is+dispensable+for+hepatitis+C+virus+cell-to-cell+transmission+in+hepatoma+cells.">CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells.</a> J. Gen. Virol. 90, 48-58 (2009).</li><li>Bitzegeio, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+hepatitis+C+virus+to+mouse+CD81+permits+infection+of+mouse+cells+in+the+absence+of+human+entry+factors.">Adaptation of hepatitis C virus to mouse CD81 permits infection of mouse cells in the absence of human entry factors.</a> PLoS Pathog. 6, e1000978 (2010).</li><li>Lindemann, D., Goepfert, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+foamy+virus+envelope+glycoproteins.">The foamy virus envelope glycoproteins.</a> Curr Top Microbiol Immunol. 277, 111-129 (2003).</li><li>Brohm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+determinants+important+for+hepatitis+C+virus+p7+function+in+morphogenesis+by+using+trans-complementation.">Characterization of determinants important for hepatitis C virus p7 function in morphogenesis by using trans-complementation.</a> J. Virol. 83, 11682-11693 (2009).</li><li>Steinmann, E., Brohm, C., Kallis, S., Bartenschlager, R., Pietschmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+trans-encapsidation+of+hepatitis+C+virus+RNAs+into+infectious+virus-like+particles.">Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like particles.</a> J. Virol. 82, 7034-7046 (2008).</li><li>Koutsoudakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+early+steps+of+hepatitis+C+virus+infection+by+using+luciferase+reporter+viruses.">Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses.</a> J. Virol. 80, 5308-5320 (2006).</li><li>Frentzen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Completion+of+hepatitis+C+virus+replication+cycle+in+heterokaryons+excludes+dominant+restrictions+in+human+non-liver+and+mouse+liver+cell+lines.">Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines.</a> PLoS Pathog. 7, e1002029 (2011).</li><li>Lindemann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+particle-associated+glycoprotein+signal+peptide+essential+for+virus+maturation+and+infectivity.">A particle-associated glycoprotein signal peptide essential for virus maturation and infectivity.</a> J. Virol. 75, 5762-5771 (2001).</li><li>Blight, K. J., McKeating, J. A., Rice, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+permissive+cell+lines+for+subgenomic+and+genomic+hepatitis+C+virus+RNA+replication.">Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication.</a> J. Virol. 76, 13001-13014 (2002).</li></ol>

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 ...

Research

JoVE Journal

Immunology and Infection

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

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

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

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

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

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

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

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

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

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

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

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus 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

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

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 ...