我们感谢团队Bioccelerator的病毒生产和开发单位，特别是马库斯·米勒，西尔维娅·明斯特曼，芭芭拉Liebetrau，曼迪罗雪尔。这项研究已部分由联邦教育和研究部（BMBF）和亥姆霍兹联合会在德国Krebsforschungszentrum /杜大CancéropôleEST在应用肿瘤病毒学联合计划的框架内部赠款支持。

请注意，与生物安全2级实验室重组细小病毒的（recPV）生产的需要。 该协议是在两个主要部分细分。第一部分通过转）（recPV生产需要作为接种服务的协议的第二部分（通过感染recPVs生产）生产最小recPVs的金额。一旦产生少量的recPVs，该协议的第一部分可以省略，只能通过提供感染细小病毒衣壳蛋白通过腺病毒辅助（见下文）的编码基因扩增和重组细小病毒。 1。生产，通过转染recPVs 1.1病毒DNA转染它可以使用任何既定的DNA转染的方法，在此节上阳离子脂质或磷酸钙为基础的协议。我们通常使用FugeneĤð转染试剂。为了控制转染效率，我们建议转染与表达质粒增强型绿色荧光蛋白（ 如染pEGFP-N1，Clontech公司）的额外板。染是考虑病毒的生产效率和最佳的结果转染EGFP阳性后24小时内至少50％的细胞群。 <ol><li> 1天前转，种子2万HEK293T细胞，在一个10厘米的板，获得50％的细胞转染当天汇合。增长在10毫升杜尔贝科的改良Eagle培养基（DMEM培养基），另外10％胎牛血清，2毫米L-谷氨酰胺，100 U，每毫升青霉素和100μg/ mL链霉素的细胞。 </li><li>准备在1.5毫升管病毒的混合物（摩尔比为1:2:1）: <ol class="lalpha"><li> 3微克recPV载体窝藏感兴趣的转基因（EI PHH-1-GFP 6）。 </li><li> 6微克载体pCMV /副总裁5窝藏细小病毒衣壳基因 ​​组</li><li> 8微克Øf派生腺病毒辅助质粒pXX6 7。 </li></ol></li><li>用无血清培养基850μL（20 ng /μl的最终浓度）稀释的质粒组合。 </li><li>加入42.5μLFu​​gene HD（2.5:1μL的Fugene:微克DNA）。加入Fugene到中期时，请勿触摸管侧。 </li><li>涡，轻轻地为5-10秒。 </li><li>在室温下孵育30分钟的混合物。 </li><li>添加下拉明智的细胞转染混合物。轻轻地摇动板均匀地分布整个混合物。 </li></ol> 1.2生产病毒<ol><li>在37°C，湿度92％和5％CO 2，收获前72小时的孵育板。在此期间，后代细小颗粒形成细小病毒质粒。 </li></ol> 1.3病毒的收获<ol><li>在组织文化的引擎盖上，刮细胞内的介质，吸和转让暂停从板成15毫升的塑料管。 </li><li>在250 XG离心管在室温下5分钟。 </li><li>取出上清的95％。 </li><li>涡管轻轻将剩余的上清液（约0.5毫升）悬浮颗粒。 </li><li>冻结管在-80°C。这是可能的，在这个阶段停止生产或继续使用该协议。 </li><li>除霜暂停，并把它在室温下。 </li><li>在液氮浴和温水解冻冻结悬挂在37°C。涡管大力为15秒，重复的冻融循环两次。 </li><li>离心管，在15分钟的16000 XG 4°C。 </li><li>上清收集到一个新的管。在这个阶段的管包含原油病毒提取的准备。 </li><li>进行病毒滴定（见下文）。为recPV携带绿色荧光蛋白基因，一个典型的生产应给予1-3×10 7病毒传染性单位约1 -3×10 10病毒基因组含有颗粒。 </li></ol> 1.4病毒的存储<ol><li>对于长期储存，冻结管含有原油病毒提取物在-80°C。它可以使用在协议的第二部分作为接种病毒粗提取物。 </li></ol> 2。生产，通过感染recPVs 通过的感染recPV生产开始之前，生产和纯化腺病毒携带细小病毒基因副总裁（AD-VP帮手，描述5）根据标准协议8。它也需要有少量的账面权益的转基因（ 例如 ，如上所述，通过转染生产）recPVs。 下面，我们描述了在175厘米2瓶（T175）格式recPV生产。以这种方式产生的病毒库存的进一步放大，可以进行10多层CellSTACK的文化室（康宁）提出的协议的轻微调整。 2.1感染<ol><li> 1天前感染，板材10万NB324K在T175烧瓶获得60-80％细胞汇合，在一天的感染细胞。准备第二瓶含有相同数量作为控制细胞（非感染的细胞）。每个T175应该有作为最终的体积，辅以5％小牛血清，2毫米L-谷氨酰胺，每毫升含青霉素100U和100μg/ mL链霉素，20毫升的最低基本培养基（MEM）的。 </li><li>第二天早上，准备在2毫升的塑料管组成的病毒混合: <ol class="lalpha"><li> AD-VP辅助在感染复数（MOI，传染性单位/细胞）5 = 10（1×10 8传染性单位）; </li><li> recPVs在MOI = 0.5（对应5×10 6传染性单位）。完成1.5毫升的MEM。 </li></ol></li><li>适用于T175烧瓶整个病毒的混合物。 </li><li>孵育瓶2小时在37°C，湿度92％，5％CO 2轻轻晃动，每15分钟。 </li><li>进一步为20-24小时，在37℃，湿度92％，5％的CO 2。 </li><li>更换新鲜培养基培养基。 </li></ol> 2.2生产病毒在37°C孵育48小时内92％的湿度和5％的CO 2瓶。高效的病毒生产的指示，明确迹象的细胞毒性，应观察瓶含有病毒感染的细胞，开始感染后36-48小时。 2.3病毒的收获<ol><li>在组织文化的吸油烟机，吸转移到50 mL离心管上清液从烧瓶。 </li><li> 1.5毫升PBS洗细胞两次。 </li><li> 1.5毫升0.25％Trysin-EDTA Trypsinize细胞。加入细胞悬液，以移除介质。 </li><li>在室温下5分钟离心在5000 XG细胞和上清的温升。 </li><li>弃上清。 </li><li> TE缓冲液加入1毫升（1毫米的Tris-HCl，0.1 mM的EDTA，pH值8.7）的细胞沉淀，轻轻涡管悬浮细胞。 </li><li>在液氮浴和温水解冻冻结的细胞悬液，在37°C。涡管大力为15秒和冻融循环重复三次。 </li><li> 50 U / ml的Benzonase核酸为30分钟，在37°C间治疗的细胞悬液，以消化细胞基因组和非包装病毒的DNA。 </li><li> 20分钟离心管在5000 XG在4°C。 </li><li>上清收集到一个新的管。在这个阶段的上清含有原油的病毒提取物制剂。典型的产量的recPV-GFP的生产后，这个协议应该在1-10×10 8个病毒感染单位的范围。 </li></ol> 2.4病毒的存储<ol><li>适用于短期存储（最多2个月），商店病毒在4°C </li><li>为LON的Ğ长期存储（超过2个月），存储病毒在-80°C。 </li></ol> 3。 RecPV纯化<ol><li>对于从原油裂解recPVs净化，我们建议运行碘克沙醇间断梯度，根据Zolotukhin 等。9。我们建议至少使用5毫升高效的病毒纯化病毒粗提取物（五T175烧瓶从生产中获得）。 </li></ol> 4。重组细小病毒滴定<ol><li> EL-Andaloussi 等执行recPV滴定5。 </li></ol> 5。质量控制<ol><li>使用EL-Andaloussi 等5所述检查不受欢迎复制主管账面指标在NB324K细胞细小斑块检测出的病毒（RCV）的存在的协议。 </li></ol> 6。代表结果一个的recPVs生产的例子ñ通过转染腺病毒基因组元素的存在或没有在图3所示。细胞转染PCMV / VP（执行副总裁细小病毒衣壳蛋白编码基因的质粒），连同PHH-1-GFP（A recPV窝藏绿色荧光蛋白基因）或PHH-1-荧光素酶（A recPV窝藏萤火虫荧光素酶基因） （+ pXX6）或无（pXX6）的pXX6质粒（携带E2A，E4（ORF6）和VA RNA基因的腺病毒）。等量的细胞粗提取物被应用到细胞，绿色荧光蛋白转导或荧光素酶检测NB324K执行在厄尔尼诺-Andaloussi 等报道。5。一个得到明显增加在recPVs生产中存在的pXX6生产增加0.3绿色荧光蛋白转导单位（TU）的/细胞获得约5 TU /细胞得到我们的方法（ 图3A，B）的常规协议。有显着增加（约24倍）的recPV producti上也有人在PHH-1-荧光素酶（ 图3C）。这些结果表明，在pXX6所载的遗传物质是能提高细小的生产。 图4显示了一个典型的例子是通过感染recPVs生产。 NB324K细胞共感染各种recPVs（如下图所示）和Ad-VP辅助（窝藏光伏副总裁衣壳蛋白编码基因）。进一步加强广告的副总裁助手recPV产量可达70叶锡恩每种子细胞（ 图4A）在不增加发生不良复制主管病毒颗粒（ 图4B）。 <img alt="图1" src="/files/ftp_upload/3518/3518fig1.jpg" /> 图1。基于自主细小病毒的载体。 （A）上方 :转基因替换副总裁编码基因的一部分，是根据病毒启动子P38的控制。 NS1蛋白/ 2的基因重新tained和他们的表达是由病毒启动子P4的控制。载体基因两侧的细小病毒的国际电信规则"，其中含有重组基因的复制和包装所需的顺式作用元素。底部:下一个异种（ 如巨细胞病毒），或自体（ 如 P38的）子（PX）的VP基因质粒携带，提供重组细小病毒生产过程中的反以弥补结构基因的破坏重组基因组中。艾提科信，倒末端重复。适应从图4（二）用于生产recPVs古典协议示意图。 HEK293T细胞瞬时转染病毒DNA（载体和辅助质粒），三天后，收集细胞和病毒收获。 <img alt="图2" src="/files/ftp_upload/3518/3518fig2.jpg" /> 图2。生产与recPVs的帮助广告副总裁。 （一）原理图recPV和AD-VP基因组。在recPV含有外源基因取代的VP地区的一部分。 AD-VP怀有副总裁细小病毒基因。（二）在本手稿中描述的协议的示意图。 NB324K细胞与recPV和AD-VP病毒共同感染。三天后细胞收获和recPV颗粒从细胞裂解液中回收。 <img alt="图3" src="/files/ftp_upload/3518/3518fig3.jpg" /> 图3。刺激recPV生产由基于腺病毒载体pXX6。 PCMV / VP PHH-1-GFP（A和B）或PHH-1-荧光素酶（三）转染HEK293T细胞，接种在10厘米的菜，生产recPVs结合。同时，细胞共转染与派生的腺病毒辅助质粒pXX6或不表示。三天后转染，细胞收获和裂解三个冻融循环。等量的CRUDé病毒提取物应用于NBK指标细胞，进行了转导实验。（一）（二）代表显微镜显示GFP阳性细胞内融合NB324K单层。每种子细胞在转导单位（TU）表示量化的GFP转导检测（C）荧光素酶活性的定量表达的相对荧光素酶单位（RLU）。荧光素酶检测执行在厄尔尼诺-Andaloussi 等描述。5。列代表从三个平均值与标准差酒吧复制。人数顶部+ pXX6列，（B和C），表示在recPV病毒滴度倍，在存在的pXX6获得与无。 <img alt="图4" src="/files/ftp_upload/3518/3518fig4.jpg" /> 图4。通过重组Ad-VP辅助病毒recPV生产的刺激。 （一）NB324K感染细胞，泰德与纯化的AD-10内政部副总裁辅助病毒（AD-X的单位/细胞，腺-X的快速滴度套件滴定），然后与重组细小病毒智HH-1-EGFP的11（0.1以下任superinfected恩/单元）或H-1-GFP（0.5叶锡恩/细胞）5。感染后一天被改变，中，两天后，收集细胞，并通过三个冷冻和冻融裂解。细胞粗提取物被用来确定传导检测病毒滴度，根据EL-Andaloussi 等人。5。杜叶锡恩，转导单位（B）在AD-VP存在的情况下产生的病毒分批进行了分析其复制能力的病毒颗粒（垃圾车）NB324K指标细胞斑块检测的内容。

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我们什么都没有透露。

我们已经表明，recPV生产，可以增强腺病毒基因组元素的存在。我们已增加超过10倍（从0.3到5 TU /细胞），通过转染腺病毒基因组元素提供超过100倍的合作感染与recPV结合的细胞与AD-VP辅助recPV产量比较传统的协议。这里所描述的协议可以进一步优化，确定交付的腺病毒基因组元素和被感染的AD-VP帮手的最佳浓度为最适当的时机。 较大的转基因（超过1600个碱基）RecPVs是生产效率较低。这可能是由于插入外源基因，光伏适当的病毒DNA包装必要的顺式作用元件被删除。最佳的转基因可分为光伏基因组插入，而不影响其生产规模是700基地第6条 。插入的基因排序，也可能会影响生产recPV（ 如细胞毒蛋白编码基因插入可能会导致在较低的recPV产量）。 考虑recPVs生产过程中的另一个重要方面是复制能力的病毒可能发生（RCV），可以自发地形成之间的重组细小病毒VP含质粒通过同源重组。本协议不加强重组的风险。然而RCV的质量控制，应常规进行生产（ 如斑块检测NB324K指标细胞5）为了保证病毒的股票只包含一个微不足道的垃圾车。 协议描述克服以前在选择使用的包装细胞线的限制，因为它使用的细胞系，很难转染（呈现可能因此不适合上染的协议），但良好的PV生产商。 如 。 NB324K细胞10，在我们的手中更高效的生产recPVs比HEK293T细胞（数据未显示）。不同的细胞系的筛选，现在可以申请新的协议，并可以更有效的细胞确定为recPV生产。这也铺平了大规模重组细小病毒，如生产系统的进一步勘探的方式。在悬浮培养细胞生物反应器。

<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>西格玛</td><td> D5796 </td></tr><tr><td>的MEM </td><td>西格玛</td><td> M4655 </td></tr><tr><td>胎儿牛血清</td><td>临时机场管理局</td><td> A15-101 </td></tr><tr><td> L-谷氨酰胺</td><td> Gibco公司</td><td> 25030-024 </td></tr><tr><td> fugene </td><td>罗氏公司</td><td> 047097050001 </td></tr><tr><td>胰蛋白酶EDTA </td><td> Invitrogen公司</td><td> 25300062 </td></tr><tr><td> benzonase核酸</td><td>西格玛</td><td> E8263 </td></tr><tr><td>腺病毒-X的快速滴度套件</td><td> Clontech公司</td><td> 632250 </td></tr></tbody></table>

efficient recombinant parvovirus production with the help of adenovirus-derived systems

如鼠的H-1PV和MVM的啮齿动物细小病毒（PV），是小二十面体，单链DNA病毒。其基因组包含两个启动子P4和P38调节的非结构性（NS1和NS2）和衣壳蛋白（VP1和VP2），分别为1的表达。他们作为抗癌药物的溶瘤和oncosuppressive能力，而人类2非致病性高利率吸引。 NS1蛋白是该病毒的细胞毒性3的主要效应。为了进一步提高他们的天然抗癌活动，从这些载体的衍生工具已产生取代衣壳蛋白编码基因与治疗基因（ 如细胞毒性多肽，细胞因子，趋化因子，抑癌基因等）4。 （recPVs）重组细小病毒载体保留的NS1 / 2编码序列和光伏病毒DNA扩增和包装所必需的基因组端粒。生产recPVs只发生在生产者细胞（一般染HEK293T），合作的第二个表达载体为VP蛋白编码基因（ 图1）4（PCMV-VP）转染细胞。以这种方式产生的recPV载体是复制缺陷。 ，虽然recPVs证明拥有与尊重父母，他们已产生病毒增强oncotoxic活动，其生产仍然是一项重大的挑战和强烈阻碍了这些药物的使用在抗癌的临床应用。 我们发现，引进含有HEK293T细胞，E2A，E4（ORF6）和VA RNA基因（ 如 pXX6，质粒）提高超过10倍相比，在使用中的其他协议的recPVs生产AD-5派生载体。基于这一发现，我们已经构建一种新型的广告副总裁的助手包含腺病毒基因组元素以及必要加强recPVs生产parvovir我们VP基因单位5。 AD-VP辅助使用，允许生产REC-PVS使用协议，完全依赖于病毒感染步骤（如反对质粒转染），使人们有可能使用难以转染的细胞株（ 如 NB324K）（ 图2）。我们提出了一个方法，大大提高了重组病毒的产生量，降低生产时间和成本，不影响最终产品的质量5。此外，大规模生产的recPV（悬浮细胞和生物反应器）现在是可以想象的。

Watch this Scientific Journal Video about Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems at JoVE.com

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

在这里，我们描述只对细胞的感染，从而提高重组细小的生产效率超过100倍相比，在使用中的其他协议为基础的协议。此协议依赖于使用一种新型腺病毒含有的细小副总裁转录单位（AD-VP）5，基于辅助。

重组细小病毒与腺病毒派生系统的帮助下高效生产

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

efficient-recombinant-parvovirus-production-with-help-adenovirus

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.9K 次观看.  German Cancer Research Center (DKFZ). 在这里，我们描述只对细胞的感染，从而提高重组细小的生产效率超过100倍相比，在使用中的其他协议为基础的协议。此协议依赖于使用一种新型腺病毒含有的细小副总裁转录单位（AD-VP）5，基于辅助。

视频: Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

估计在水生系统的病毒产率

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

科研

免疫与感染

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

重组逆转录病毒的生产和B细胞的感染

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

高效转染树突状细胞的无细胞的成熟优化的协议

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

重组腺相关病毒载体的生产和滴定

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

在FDA批准的Vero细胞疫苗开发产生重组沙粒

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

有效生成荧光标记疫苗病毒蛋白的方法

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

简单而高效的生产和鼠标髓鞘少突胶质糖蛋白的纯化的实验性自身免疫性脑脊髓炎研究

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

重组原型泡沫病毒整合纯化过程中核酸污染的检测与去除

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...