Vanessa Garcia is funded by a Queen Elizabeth II Ontario Graduate Scholarship in Science and Technology and Cory Batenchuk by a Natural Sciences and Engineering Research Council fellowship.

1，样品制备<ol><li>得到含有荧光素酶表达的病毒（本文VSVΔ51-Fluc为例）为滴定并转移至96孔板的样品。交替地，执行在96孔组织培养板直接与使用上清液感染。 </li><li>留下两列未处理列入标准曲线。用于实验的实验（40小时后的感染）或存储在-80℃和滴度的末端在96孔板中，滴度直接做在稍后的日期。 </li></ol> 2，准备允许细胞进行病毒滴定<ol><li> 24小时前滴定，制备Vero细胞的悬浮液以2.5×10 5个细胞/在Dulbecco改良的Eagle培养基毫升（DMEM）中含有10％胎牛血清（FBS），30mM的HEPES的浓度，和1％青霉素/链霉素。注:尽管本协议使用Vero细胞，任何适当的宽容细胞系的VSV感染可以bê使用。 </li><li>在使用微板的分配器96孔白色固体的平底板种子2.5×10 4个细胞（100微升）。 <ol><li>制备12毫升细胞悬浮液，每片加5毫升涂刷。 </li><li>干净微分配器盒通过冲洗管路用50ml无菌水。 </li><li>填充微分配器线路用细胞悬浮液，并让5毫升细胞悬浮液的流过。 </li><li>选择一个程序，将分配100μl的在白壁96孔板的每个孔中。分配，并重复所必需的附加板。还种子几孔中96孔透明平底板如果不透明底白墙板用于验证的细胞的健康和密度。 </li><li>完成后，冲洗细胞放回原包装内。通过运行50毫升70％乙醇中的随后加入50毫升的温无菌水通过管道清洁盒。 </li><li>确保磁带和油管是AP使用之间propriately清洗。 </li></ol></li><li>培养在湿润的5％CO 2培养箱中的细胞24小时，在37℃。 </li></ol> 3，病毒的制备标准曲线<ol><li>制备VSVΔ51-Fluc在无血清DMEM中的标准曲线，使得噬斑形成单位（pfu）每毫升转印后的最终浓度到Vero细胞上是如下:10 8 PFU / ml的10 7 PFU / ml的， 10 6 PFU / ml的10 5 PFU / ml的10 4 PFU / ml的，10 3 PFU /毫升，10 2 PFU / ml，并将10 1 PFU / ml的。准备50微升每盘Vero细胞上加上一个额外的10％，每个浓度。 请注意:在荧光素酶的病毒原液的效价将需要在一个经典方式10为了产生标准曲线，将允许为绝对定量评估。否则，相对定量可以在不准确的效价信息通过任意设定病毒滴度达到基础在稀释步骤。例如在标准曲线的第一稀释度可以被设置为10 8病毒单元和以下10稀释到10 7等。在这种情况下，应该表示的值作为倍数变化相比预先确定的样本作为绝对定量将不准确。 </li></ol> 4，样品转移上清到允许细胞<ol><li>检查Vero细胞接种在透明底的96孔板，用光学显微镜来确认单层是至少95％汇合。 </li><li>转账25微升样品上清液到Vero细胞接种于白墙板。不转移上清液到指定的标准曲线2列。注意:此可同时对所有的孔上使用96通道的液体处理的单个板来完成。 </li><li>每种稀释液的标准曲线的制备在步骤3到Vero细胞中的使用8位或12通道多通道移液器，加25微升2指定列。 </li><li>离心平板5分钟，在430 XG在RT。 </li><li>在潮湿的5％CO 2的培养箱中孵育5小时，在37℃。 </li></ol>细胞活性的5评价<ol><li>请注意:当从在清晰的96孔板进行感染实验中获得的上清液开始，样品活力之前可以量化与细胞存活率指示剂染料诸如刃天青评价。 </li><li>的量等于该体积的10％，在96孔板中含有病毒和细胞样品的每个孔中加入刃天青。包括细胞只控制以及媒体只控制分别确定为100％和0％生存力的值。 </li><li>后2-4小时（温育时间将取决于细胞类型和对染料的市售的或再造的粉末的浓度变化），读取并使用荧光板读数器（530-560激发，590发射）记录该信号。报告细胞viabili×100％根据下式的相对代谢活性= - （ - 负控制信号）/（细胞只能控制信号的负控制信号）（试验样品信号）TY为样品</li></ol> 6，制备荧光素底物<ol><li> 30分钟5小时孵育结束前，制备荧光素，以获得2毫克/毫升的溶液在无菌磷酸盐缓冲盐水（PBS）中。准备每盘2.5毫升加上一个额外的2毫升避光的解决方案。注:荧光素，也可以在当天早些时候制备并保存于4℃直至使用。 </li></ol> 7，读生物发光<ol><li> 5小时培养结束后，在白色的固体平板添加25μl的荧光素对Vero细胞的每个孔中。手动或使用自动分配器集成在光度计中添加荧光素。注意:只有单和终点使用的仪器读取可能需要添加荧光素酶裂解兼容的BUFFER之前加入萤光素底物，以改善一致性。 </li><li>阅读以下参数的板: <ol><li>摇动5秒。 </li><li>等待30秒。 </li><li>阅读发光在适当的固定感光度/曝光值。如果该选项可用在仪器上，使用多点读取（ 例如，3×3矩阵），以提高准确性。 </li></ol></li><li>记录并保存定量生物发光强度。 </li><li>如果一个自动化分配器用于添加荧光素清除萤光素和素用温水无菌水的线条。 </li></ol> 8，数据分析<ol><li>为准确的结果，解决了非线性回归，以产生从标准曲线希尔方程。套用这个公式的滴定样品来计算病毒表达单位。某些病毒或情况可能会产生的标准曲线的线性关系;在这种情况下求解线性方程。 </li></ol>

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	<li>Grigorov, B., Rabilloud, J., Lawrence, P., Gerlier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+titration+of+measles+and+other+viruses:+optimization+with+determination+of+replication+cycle+length.">Rapid titration of measles and other viruses: optimization with determination of replication cycle length.</a> PLoS One. 6 (e24135), (2011).</li><li>Lizee, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+reverse+transcriptase-polymerase+chain+reaction+as+a+method+for+determining+lentiviral+vector+titers+and+measuring+transgene+expression.">Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.</a> Hum Gene Ther. 14, 497-507 (2003).</li><li>McSharry, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uses+of+flow+cytometry+in+virology.">Uses of flow cytometry in virology.</a> Clin Microbiol Rev. 7, 576-604 (1994).</li><li>Watcharatanyatip, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispecies+detection+of+antibodies+to+influenza+A+viruses+by+a+double-antigen+sandwich+ELISA.">Multispecies detection of antibodies to influenza A viruses by a double-antigen sandwich ELISA.</a> J Virol Methods. 163, 238-243 (2010).</li><li>Dawson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+Direct+Quantification+of+Viruses+in+Solution+Using+the+ViroCyt+Virus+Counter.">Rapid, Direct Quantification of Viruses in Solution Using the ViroCyt Virus Counter.</a> Journal of Biomolecular Techniques. 23 (S10), (2012).</li><li>Snyder, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV+and+RT-PCR:+true+or+false.">AAV and RT-PCR: true or false.</a> Mol Ther. 1, 389-390 (2000).</li><li>Diallo, J. S., Roy, D., Abdelbary, H., De Silva, N., Bell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+infection+of+live+tissue+with+oncolytic+viruses.">Ex vivo infection of live tissue with oncolytic viruses.</a> J Vis Exp. 52, (2011).</li><li>Gaush, C. R., Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+and+plaque+assay+of+influenza+virus+in+an+established+line+of+canine+kidney+cells.">Replication and plaque assay of influenza virus in an established line of canine kidney cells.</a> Appl Microbiol. 16, 588-594 (1968).</li><li>Green, M., Loewenstein, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adenoviruses:+propagation,+purification,+quantification,+and+storage.">Human adenoviruses: propagation, purification, quantification, and storage.</a> Curr Protoc Microbiol. 14 (14C 11), (2006).</li><li>Diallo, J. S., Vaha-Koskela, M., Le Boeuf, F., Bell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation,+purification,+and+in+vivo+testing+of+oncolytic+vesicular+stomatitis+virus+strains.">Propagation, purification, and in vivo testing of oncolytic vesicular stomatitis virus strains.</a> Methods Mol Biol. 797, 127-140 (2012).</li><li>Hoenen, T., Groseth, A., Callison, J., Takada, A., Feldmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Ebola+virus+expressing+luciferase+allows+for+rapid+and+quantitative+testing+of+antivirals.">A novel Ebola virus expressing luciferase allows for rapid and quantitative testing of antivirals.</a> Antiviral Res. 99, 207-213 (2013).</li><li>Diallo, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+pharmacoviral+approach+identifies+novel+oncolytic+virus+sensitizers.">A high-throughput pharmacoviral approach identifies novel oncolytic virus sensitizers.</a> Mol Ther. 18, 1123-1129 (2010).</li></ol>

The authors have nothing to disclose.

这里描述的荧光素酶为基础的方法提供了许多优于其它现有的方法，包括其方便，快速，最小的设备的需要，并且成本相对较低。一个关键因素，这是避免连续稀释步骤。尽管如此，这个协议的系列稀释基衍生物是肯定可行的，最近被用于评估在高通量抗病毒药屏幕11荧光素酶表达埃博拉滴度。而本质上更耗时且较昂贵，这样的修改可以针对病毒的定量提供更大的动态范围，必要时。除了是特别适合用于评估病毒滴度在高通量筛选的上下文中，我们的一步法荧光素酶基的病毒定量方法产生在复制病毒的情况下，感染性病毒粒子的准确估计。此外，发光以及来自同一实验的细胞毒性数据读数得到更的靶细胞上的实验条件下的效果完整图像，这是在溶瘤病毒和药物屏幕的背景下尤其有用。 这里所示的例子中使用了复制的负单链RNA病毒;然而，该协议可以适应许多复制型或非复制型病毒有一些小的协议的调整。这包括DNA病毒，如牛痘，单纯疱疹病毒，和腺相关病毒（参见图5）。感染细胞内的病毒，如牛痘病毒，例如样品，需要事先向量化（ 例如 ，至少一个冷冻-解冻周期）的病毒的释放步骤。当应用该技术对其他的病毒，它是必要的，以从病毒上清液或裂解物由光度计转移到所述板的读取优化的温育时间。该参数将主要取决于病毒在允许细胞系和复制周期p的强度romoter驾驶荧光素酶的表达。这最好通过使用完整的标准曲线，在优化步骤中完成的。要做到这一点，必须有感染的编制标准曲线的各种重复的适当宽松细胞系和阅读每个重复在不同的时间点后转移。理想情况下，孵育时间点被选择，导致LOG（RLU）和LOG（效价）之间的线性关系跨越预期样品的滴度范围。为VSVΔ51-Fluc，这通常是从10 4 PFU / ml的-10 7 PFU / ml的为5小时的温育时间。如果更低或更高的滴度从样品预期，可以简单地增加或减少培养时间分别。或者，样品可以稀释到下降多达通常用于ELISA进行的范围内。 如上面所提到的，这种方法非常适合于执行使用药物库的大多数步骤可以是自动化的高通量药物屏幕。细胞可以被镀èfficiently使用自动化微板的分配器，所述药物库可使用96通道的液体处理器加入，病毒可以使用微分配器被添加和平板阅读使用自动化的光度计。从理论上讲，这也适用于384孔或更小的格式;但是，不限于该端是多个小区可以被镀的，给予较少的细胞导致更窄的范围中的LOG（RLU）的线性度，以LOG（效价）的关系。最后，使用刃天青或其他代谢染料细胞活力评估可以很容易地在工作流中引入，从而允许细胞毒性化合物的抗病毒帘或鉴定的化合物，导致协同杀伤与病毒12组合判断。然而，这种方法的局限性包括:荧光素酶转基因表达的病毒，这并不总是可能的要求，并具有足够允许细胞系的可用性。然而，这很可能能够适应该方法与其它报告基因的使用（ 例如 ，GFP）提供记者定量方法具有适当的线性度和信噪比。总体而言，所描述的高通量的方法可以进行修改，以适应许多不同的病毒，并适用于多种应用。

Classical viral plaque assays continue to be a mainstay in virus research even though they can be notoriously time-consuming and constitute a significant bottleneck to obtaining results from experiments. More rapid indirect virus quantification methods have emerged including quantitative polymerase chain reaction (qPCR), ELISA, and flow cytometry1-4. Recent innovations such as the Virocyt virus counter can directly count viruses using advanced flow sorting technology and through a combination of protein and DNA/RNA dyes5. While all of these methods have undoubtedly quickened the pace of virus research, each method has its advantages and drawbacks. For example, qPCR can allow for quantification of specific viral genome sequence but cannot effectively discriminate infectious from defective virions6. ELISAs can be very specific however require a suitable antibody against the desired target viral protein and can be very expensive. While flow cytometry technology offers many advantages and has improved significantly, throughput and accessibility to highly specialized equipment nonetheless remains a hurdle. Importantly, all of these techniques are not ideally suited for high-throughput screening, for which the ease and time requirement of the virus quantification step is of critical importance.
Here we describe a high-throughput and easily automatable technique to titer viruses that express a Firefly luciferase (Fluc) transgene. This method generates approximate viral titers in a test sample based on luminescence signal reads through the parallel use of a standard curve of known amounts of virus. Samples containing unknown quantities of luciferase-expressing virus are transferred on to a permissive &#8220;plaquing&#8221; cell line in parallel with the standard virus dilution curve and virus-associated luminescence is read after a few hours incubation time. This allows for rapid, quantitative, often same-day generation of results, unlike classic plaque assay protocols which typically require several days of incubation in order to manually count visible plaques7-9. 
The protocol outlines the steps of our titration method using oncolytic Vesicular Stomatitis Virus encoding a Fluc transgene (VSV&#8710;51-Fluc) as an example and provides an overview of 1. Sample preparation 2. The plating of a permissive cell line for virus titration using an automated dispenser 3. The preparation of the viral standard curve 4. The transfer of the sample supernatants onto the permissive cell line using a 96-well manual pipettor 5. The assessment of sample cytotoxicity using a cell viability reagent 6. The preparation of the luciferin substrate 7. Reading of bioluminescence and 8. Data analysis.

<table><tbody><tr><td>Dulbeccos&#039; Modified Eagle&#039;s Medium (DMEM)</td><td>Corning</td><td>SH30243.01</td><td></td></tr><tr><td>Fetal bovine serum</td><td>NorthBio Inc.</td><td>NBSF-701</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-1</td><td>Prepare a 1 mM solution, pH 7.3</td></tr><tr><td>alamarBlue&amp;nbsp;</td><td>AbD Serotec</td><td>BUF012B</td><td></td></tr><tr><td>D-Luciferin potassium salt</td><td>Biotium</td><td>10101-2</td><td></td></tr><tr><td>96-well solid white flat bottom polystyrene TC-treated microplates</td><td>Corning</td><td>3917</td><td>384-well plates can also be used for higher throughput</td></tr><tr><td>Synergy Mx</td><td>BioTek</td><td>SMTBL</td><td>Monochromator microplate reader</td></tr><tr><td>Liquidator96</td><td>Mettler Toledo</td><td>LIQ-96-200</td><td>96 tip manual pipetting system</td></tr><tr><td>Liquidator96 LTS Tips sterilized with filters</td><td>Mettler Toledo</td><td>LQR-200F</td><td>Any sterile filtered tips compatible with pipettors of choice are appropriate</td></tr><tr><td>Microflo</td><td>BioTek</td><td>111-206-21</td><td>Used to plate cells in a 96-well plate</td></tr><tr><td>Fluoroskan Ascent FL</td><td>Thermo Scientific</td><td>5210450</td><td>Microplate fluorometer</td></tr></tbody></table>

high-throughput titration of luciferase-expressing recombinant viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

描述了高通量方法的工作流程的概要示于图1，图2示出了一个典型的标准曲线ofVSVΔ51-Fluc的结果。四参数非线性回归分析所产生的积山从未知输入PFU（病毒滴度的估计），可以插。这些估计滴度被称为病毒表达单位（VEU）。 图3A示VEUs和滴度通过执行具有相同的样品10标准的空斑测定法得到的。样品源自其中的各种化学品使用，以增强复制和786-0细胞扩散VSVΔ51-Fluc的一个实验。 VEUs从标准曲线内插必须由这是基于样品的上清液的稀释因子乘以被转移到Vero细胞中（在这种情况下，稀释因子为5）。滴度和VEU之间的线性相关性， 如图3B所示</strong&gt;与R 2的0.8083和0.899（P &lt;0.0001）皮尔森的Ř得分。在图4中 ，从代谢法得到的典型的细胞的细胞毒性数据显示为与化学品在感染前用VSVΔ51-Fluc处理786-0细胞。最后， 图5示出了具有各种病毒获得的典型的标准曲线（单纯疱疹病毒（HSV），牛痘病毒，和腺相关病毒（AAV），所有表达萤火虫萤光素酶），并描述的孵育时间和冻融循环，根据需要。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig1highres.jpg" width="500" /> 图1中的工作流的高通量病毒滴定和细胞毒性测定用VSVΔ51-Fluc。 （一）种子2.5×10 4 Vero细胞每孔（100微升）细胞培养在37℃（B）24小时后转移25μl的标准曲线的对向Vero细胞（每盘2列），（C）转移25μl的样品进行滴定到剩余的Vero细胞。离心机板，并在37℃（D）在相等的量在体积的10％的孵化均匀，加入刃天青试剂含有原始样品板。培养板在37℃（E）的 3小时后，读取并记录荧光和评估细胞毒性（F）第5小时后，加入25微升2毫克/毫升的溶液的荧光素对Vero细胞的每个孔中。阅读发光并计算病毒表达单位。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig2highres.jpg" width="500" /> 图2。预期的VSVΔ51-Fluc的标准曲线。荧光素酶的表达是我asured内使用发光井和生物发光是用平均相对光单位（RLU）的五个不同点5小时后上清液转移。平均RLU作图对已知输入PFU / ml的解决非线性回归和生成希尔方程。两个复制曲线和标准误差线的平均值示相关（r 2 = 0.9993）。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig3highres.jpg" width="500" /> 图的标准空斑测定滴定度与通过高通量方法3得到的比较。 （A）在PFU / ml的通过标准空斑测定在Vero细胞上获得与使用的高通量荧光素酶测定法滴定相同的样品计算的病毒滴度（VEU /毫升）进行比较（B）线性VEU /毫升之间关系的病毒滴度并通过标准纵获得滴度laque检测。线性回归曲线和决心（R2）的系数显示。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig4highres.jpg" width="500" /> 图4样品的可行性。样品活力之前，上清液转移到Vero细胞上通过评估使用市售的刃天青溶液细胞代谢活性测定。原始荧光值标准化为未处理，未感染的孔中。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig5highres.jpg" width="500" /> 图5。预计单纯疱疹病毒，牛痘，和AAV病毒的标准曲线。荧光素酶的表达是衡量五个不同点内使用发光良好，生物发光是用平均RELA略去光单位（RLU）:（A）HSV标准曲线，加入到Vero细胞接种24小时前在每孔中（100微升）和萤光素酶测定为2.5×10 4个细胞的密度被制成17小时后supernantant转移（R 2 = 0.9489，n = 1时）。甲希尔方程，通过求解非线性回归产生的（B）牛痘标准曲线，加入到Vero细胞上镀每孔（100微升）2.5×10 4个细胞的24小时更早的密度和在37℃下温育2.5小时C，之后被带到荧光素酶测定（R2 = 0.9892）。的线性方程，通过求解线性回归产生的（C）的AAV标准曲线，加入到人肺癌细胞（A549细胞）接种24小时前在每孔中（100微升）和萤光素酶测定为2.5×10 4个细胞的密度有人24小时后感染。甲希尔方程，通过求解非线性回归产生的。平均五个复制曲线和标准误差条示（R 2 = 0.9926）。

Watch this Scientific Journal Video about High-throughput Titration of Luciferase-expressing Recombinant Viruses at JoVE.com

High-throughput Titration of Luciferase-expressing Recombinant Viruses

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

high-throughput-titration-of-luciferase-expressing-recombinant-viruses

Nanyang Technological University

Research

JoVE Journal

Biology

12.8K 次观看.  Ottawa Hospital Research Institute. This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

视频: High-throughput Titration of Luciferase-expressing Recombinant Viruses

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

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 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

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 ...

科研

免疫与感染

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

癌症研究

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 ...

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

慢病毒生产

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

生物学

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

在生物安全2级环境中制备假型颗粒研究高致病性冠状病毒

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

体外转录 rna 基荧光素酶记者法研究类病毒感染细胞的翻译规律

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

用于病毒感染活成像和定量的路西酶-荧光报告流感病毒

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

评估假型病毒对 SARS-CoV-2 的血清中和

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

作者聚焦：研究宿主-病毒与假型病毒的相互作用 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

假型病毒作为通过中和测定监测针对 SARS-CoV-2 的体液免疫反应的分子工具

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...

萤光素酶表达的重组病毒的高通量滴定

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此视频中的章节

重组细小病毒与腺病毒派生系统的帮助下高效生产

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

在FDA批准的Vero细胞疫苗开发产生重组沙粒

慢病毒生产

在生物安全2级环境中制备假型颗粒研究高致病性冠状病毒

体外转录 rna 基荧光素酶记者法研究类病毒感染细胞的翻译规律

用于病毒感染活成像和定量的路西酶-荧光报告流感病毒

使用自制双辉光萤光素酶检测的高通量筛选功能

假型病毒作为通过中和测定监测针对 SARS-CoV-2 的体液免疫反应的分子工具

假型病毒作为通过中和测定监测针对 SARS-CoV-2 的体液免疫反应的分子工具