图1总结了以下协议的插图。 安全注意事项：所有材料，在接触病毒颗粒组装需要用卫康消毒剂溶液或其他合适的消毒剂消毒。 1。制备的质粒DNA股（1〜2天） <ol><li>以下质粒： <ul><li> pRV1 - 含AAV2 Rep和Cap序列</li><li> pH21 - 含AAV1 Rep和Cap序列</li><li> pFdelta6 - 腺病毒辅助质粒</li><li>腺相关病毒质粒含有重组表达盒两侧AAV2包装信号（倒终端重复，ITRS） </li></ul></li><li>虽然pFdelta6应在Stbl2主管细胞生长，防止部分缺失，pRV1和pH21可以生长在DH5alpha主管细胞。腺相关病毒质粒可以种植Stbl2细胞，如果DH5alpha细胞出现部分缺失。 </li><li>质粒DNA应该是高品质和RNA的污染物。质粒进行筛选，使用下面的摘要完整性： <ul><li> pRV1 - 消化用XbaI 7.5 kb和3.8 kb的带</li><li> pH21 - 用EcoRI消化4.5 kb的条带，2.8 kb和0.2 kb的</li><li> pFdelta6 - 消化与HindIII位给予5.5 kb的条带，3 KB，3 KB，2.3 kb和1.5 kb的</li></ul></li></ol> 2。制备人胚肾293（HEK293）细胞转染（2 - 3天） <ol><li>板两个80％融合150厘米2到5个直径15厘米的NUNC组织培养皿HEK293细胞培养瓶。细胞应在70 - 80％，转染前（约48小时后电镀）汇合。培养细胞与低血糖在标准贝科的改良Eagle培养基（DMEM），含10％小牛血清和100 U / ml青霉素/ 100微克/ ml链霉素。 </li><li>转染前3小时删除DMEM和更换与Iscove“小号修改贝科的培养基（IMDM）含5％胎牛血清。 </li></ol> 3。病毒质粒转染（〜1小时转，3天孵化） <ol><li>准备一个批处理病毒（5 × 15厘米培养板）以下内容： <ul><li> 62.5微克腺相关病毒质粒</li><li> 125微克pFdelta6 </li><li> 31.25微克pRV1 </li><li> 31.25微克pH21 </li><li> 1650微升2.5米氯化钙2 </li><li> 12毫升DH 2 O </li></ul></li><li>在2类组织文化引擎盖无菌过滤到50毫升管转染混合物。 </li><li>虽然震荡的解决方案，迅速加入13毫升2 × HEPES缓冲液（pH值7.05）。更换50毫升管的盖子，并继续为15秒涡。离开站为1分45秒，罚款应形成白色沉淀。 </li><li>轻轻地转解决方案，以每15厘米的组织培养皿中加入5毫升。旋流板组合，并返回到孵化器。 </li><li>转染后16小时删除IMDM培养液，用DMEM更换。 </li></ol> 4。裂解细胞和收获的rAAVs（2小时） <ol><li>转染后72小时，取出细胞培养板的媒体和丢弃。所有废弃物应被视为与卫康消毒剂溶液或其他合适的消毒剂。 </li><li>轻轻洗细胞，在温暖的1X磷酸缓冲液（PBS，pH值7.4）。 </li><li>加入25毫升温PBS将每块板，轻轻地清除细胞与细胞刮刀。收集悬浮在50毫升管。 </li><li>颗粒细胞在10分钟的800 XG。 </li><li>倒掉上清，重悬沉淀在150 mM氯化钠，20毫米的Tris pH值8.0，利用组织培养板每10毫升。拆分成两个50毫升管。 </li><li>准备一个10％的脱氧胆酸钠的新鲜解决方案，在DH 2 O终浓度为0.5％，每管加入1.25毫升。添加benzonase核酸到终浓度为每毫升50个单位。彻底混合管。 </li><l我在37 ° C孵育1小时。 </li><li> 3000 XG离心15分钟，取出细胞碎片。转移到新鲜管50毫升，确保所有细胞碎片已被删除，以防止肝素列阻塞（见第5步）。在这个阶段，样品可存放于-20 ° C，然后再继续。我们已储存在-20 ° C的几个星期没有在减少传染性样本。 </li></ol> 5。 rAAVs肝素柱纯化（2 - 3小时） <ol><li>安装HiTrap肝素列列在1毫升每分钟5.2至5.4的步骤，使解决方案流通过使用蠕动泵。重要的是要确保无气泡进入肝素列介绍。 </li><li>平衡10毫升150毫米，20毫米的Tris，pH值8.0氯化钠列。 </li><li> 50毫升的病毒解决方案，应用列，并允许流过。 </li><li> 20毫升100毫米，20毫米的Tris，pH值8.0氯化钠洗净列。 </li><li>使用5毫升注射器，继续洗柱瓦特第i个1毫升200毫米氯化钠，20毫米的Tris，pH值8.0，1毫升300毫米，20毫米的Tris，pH值8.0氯化钠。丢弃的流量通过。 </li><li>从列由申请使用5毫升的注射器和柔和的压力洗脱病毒： <ul><li> 1.5毫升400毫米，20毫米的Tris，pH值8.0氯化钠</li><li> 3.0毫升450 mM氯化钠，20毫米的Tris，pH值8.0 </li><li> 1.5毫升500毫米，20毫米的Tris，pH值8.0氯化钠</li><li>在15 mL离心管中收集这些。 </li></ul></li></ol> 6。浓度和rAAVs无菌过滤（1小时） <ol><li>使用了10万分子量截止Amicon超4离心式过滤器，浓缩载体。在2000年为2分钟（室温）XG装入浓缩和离心列洗脱液4毫升。流穿，用剩余的病毒解决方案，并重复离心和重载选矿厂丢弃。集中量应约250μL。如果集中体积比这更显著，丢弃流量吨hrough和继续离心机在一分钟内的步骤，直到体积约250μL。 </li><li>最终体积为500μL加入250μL的PBS病毒删除选矿厂。 </li><li>通过13毫米直径为0.2微米的注射器过滤器的过滤器载体。媒介应分装和保存于-80℃直至所需。 </li></ol> 7。病毒性股票的滴定（3天） 这需要一个推动者，是活跃在HEK293细胞驾驶报告基因或免疫细胞化学检测基因产物。当产生一个向量是不兼容的HEK293细胞的其他细胞系或初级细胞培养可用于的。 <ol><li> 18聚- L -赖氨酸涂层玻片NUNC玻片或18井准备播种HEK293细胞，使他们有40 - 50％融合。对于创建依赖rAAVs滴定使用HEK293细胞中稳定表达Cre重组酶2。 </li><li>感染每口井与串行Dilutions的AAV载体（图2A）。使用3口井，每稀释。我们经常直接添加病毒每口井。 </li><li> 72小时后加入等体积的4％多聚甲醛（PFA）（给予终浓度为2％煤灰）在室温下10分钟至中等HEK293细胞修复。 </li><li>的PBS洗涤细胞3次，冲洗卫生署2 O和盖玻片。 </li><li>计数三口井的最高稀释倍数，细胞的数量，但仍然含有受感染的细胞，发现这些平均乘以稀释倍数，给每微升传染性单位（图2B）。 </li></ol> 8。预期结果 HEK293细胞转导增强型绿色荧光蛋白（EGFP）如图2b所示的病毒编码。我们一般实现一致的滴度约为6 × 10 6％微升传染性颗粒。我们经常使用立体仁济这些载体ction到成年的啮齿动物的大脑。这提供了一个强大的技术，为特定区域的基因表达 2 。选择性基因表达的细胞类型，我们注入的Cre转基因小鼠的目标2地区的依赖Cre重组rAAVs要结合本地区的特殊性。图2C - E显示立体定向注射编码成不同的大脑区域的成人白蛋白- Cre转基因小鼠3 EGFP的依赖CRE - rAAVs的例子。 <img alt="图1" src="/files/ftp_upload/3348/3348fig1.jpg" /> 图1。示意图的rAAV生产协议。 1）HEK293细胞镀成长为70 - 80％汇合。 2）腺相关病毒和辅助质粒转染HEK293细胞。 3）中变回DMEM培养16小时后转染HEK293细胞培养56小时，。 4。）HEK293细胞收获和裂解。从细胞碎片中分离出来，由C的rAAV载体entrifugation。 5）。纯化病毒颗粒通过肝素浓度和消毒前列。 6）。HEK293细胞是生长在24孔板和感染AAV载体的系列稀释，以确定传染病的单位数量。 <img alt="图2" src="/files/ftp_upload/3348/3348fig2.jpg" /> 图2。滴定和在体内rAAV1 / 2的应用。 （一）设置的HEK293细胞病毒滴度测量。 HEK293细胞转rAAVs系列稀释。 C，未受感染的控制，N，1μL未稀释的病毒股票。 （二）感染HEK293细胞表达EGFP rAAVs。 （CE）病毒EGFP的表达仅限于表达CRE -白蛋白- Cre转基因小鼠Cre重组酶的激活rAAVs立体定向注射后不同的目标区域中的神经元。示例图像显示GFP免疫反应，（三）网状丘脑和苍白球（四）齿状回和（E）海马CA1区。狄刚，报ntate回; RT，网状丘脑核，GP，苍白球。比例尺：B，500微米，C，100微米，D，100微米，E，500微米。

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没有利益冲突的声明。

重组AAV载体在动物体内研究越来越有价值。在这里，我们生产rAAVs一个简单而廉价的的协议，可能有助于避免病毒的生产成本高昂的外包公司的广泛应用这项有益的载体。协议（基于参考文献1）描述生产嵌合rAAV1 / 2，其中包含从4等于比率在双方父母的血清型衣壳蛋白的载体。通过肝素列结合的rAAV载体净化依靠AAV2衣壳蛋白的表达，但可以用比AAV1这里列出的血清型相结合。此外，AAV6还报道肝素结合，虽然减少的亲和力，并可能与使用肝素这个过程 5,6列纯化。但是应该指出，其他血清型会要求不同浓度的NaCl，肝素柱洗脱5,7 。包装的rAAV基因组显示在包装效率大幅减少到8 KB 5.2 4.1和4.9 kb的之间最佳。这种包装限制，可以考虑的rAAV系统的最大缺点，因为细胞类型特异性的转基因表达的调节通常是由大顺行事，不能住小腺相关病毒颗粒内的元素实现。为了克服低滴度批次，如那些试图包密切AAV的包装限制，我们结合加入PBS 6.3步，而不是单独的病毒批次，集中到一个首批500μL到250μL的基因是导致。相结合，创建驱动小鼠和有条件的rAAV盒（见下文），以避免拉伸包装限制，可以实现可靠的细胞类型的具体传导。 值得注意的是，虽然此协议试图提供易于遵循的指示，生产高T国际热核实验堆的rAAV，它要求肝素亲和层析的纯化AAV2为核衣壳蛋白基团的存在。使用替代程序9血清型比AAV2必须净化。肝素柱纯化的一个好处是，它被认为是产生有传染性率大于生产的使用氯化铯梯度10的AAV载体。然而，这种方法也存在着一些弊端。例如，结合肝素的其他蛋白质也可能是目前在纯化病毒的股票。虽然可以通过载体滴度10，我们的方法，具体目标无论是20兴奋性或抑制性神经元（图2）的影响肝素纯化载体嗜采用诱导Cre重组AAV介导的转基因表达的激活和滴度独立。肝素柱纯化的另一种方法是使用碘克沙醇密度梯度，从而产生较高的病毒滴度比使用素净准备氯化铯梯度10。这种方法也可以结合肝素柱纯化，以产生一种载体，是大于99％的纯 11 。因此，需要认真考虑采取个别团体病毒提纯方法最适合其特定的下游应用。 与此协议相关的最常见的问题是低的病毒滴度。这在我们的经验，通常可以追溯到转染效率低，或病毒从肝素列洗脱。转染前，所有转材料，应在室温下进行测试转染应在每个实验室中找到最佳的条件。其他转染方法，脂质体等，是一种高效的，但可以非常昂贵。此外，肝素柱洗脱解决方案的浓度和pH值是完整的病毒粒子洗脱witho肝素列的关键UT损害。另外很重要的一点是，包装细胞，必须坚持以组织培养皿的表面。如果在某个阶段的细胞分离，在手术过程中，建议终止实验和除霜一个新的细胞小瓶。 在啮齿类动物中的转基因表达的时空控制很容易实现准确的解剖定位和发育阶段的动物。高效的腺相关病毒介导的基因转移已被证明在子宫内12,13。在成人大脑，立体定向注射后的rAAV颗粒感染的地区，可以从非常小的有针对性的地区，不仅在病毒滴度的变化，但也到更大的领域，通过改变注入参数进行调整。这些措施包括注射量和速度，列入多元醇与病毒悬液14甘露醇。甘露醇也可用于增强全身的rAAV注射15后的各种组织感染 &lt;/ SUP&gt;。 rAAV的包装容量（约4.7 KB）可能是在可以从这些病毒载体基因表达方面，最重要的限制因素。然而，最近的研究表明，这可以部分克服分裂更大的基因或表达盒的rAAV载体为单独的和引进架桥序列，启动基因表达的病毒粒子感染同一个细胞 16时。的rAAV载体携带的基因表达水平，也可以大大提高使用自免费的rAAV 载体 17 。立体定向注射rAAV的载体诱导在一个特定区域的的方式的基因表达提供了一种快速，廉价和功能强大的方法。 18 rAAVs结合第二代RNA干扰策略也可以用于特定区域的基因击倒。 rAAVs可以结合的Cre转基因小鼠或细胞类型特异性启动子，以实现电路和单元式特定基因的表达，允许高空间分辨率2,19-21的遗传操作，而装修rAAVs如TET系统增加了对病毒介导的基因表达22,23的时间控制。这些例子说明这些向量组合巨大潜力，预测在今后几年中的rAAV为基础的研究的快速增长。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Amicon超离心式过滤器</td><td> Millipore公司</td><td> UFC810024 </td><td> 10万截留分子量</td></tr><tr><td> Benzonase核酸</td><td> Sigma - Aldrich公司</td><td> E1014 </td><td></td></tr><tr><td> Dulbeco的改良Eagle培养基（DMEM） </td><td> Invitrogen公司</td><td> 10567014 </td><td>补充10％胎牛血清和10单位/ ml penicillin/100μg/ mL链霉素</td></tr><tr><td> HEK293细胞</td><td>美国式文化的收集</td><td> CRL - 1573 </td><td>在通过使用小于30 </td></tr><tr><td> HEPES缓冲液</td><td> Sigma - Aldrich公司</td><td> 51558 </td><td></td></tr><tr><td> HiTrap肝素墨盒</td><td>西格玛- Aldrich公司</td><td> 54836 </td><td></td></tr><tr><td> Iscove的修改贝科的中期</td><td> Invitrogen公司</td><td> 12440-053 </td><td>补充5％FCS </td></tr><tr><td>脱氧胆酸钠</td><td> Sigma - Aldrich公司</td><td> D5670 </td><td>每个批次的新鲜解决方案</td></tr><tr><td> 15厘米的组织培养皿</td><td> Fisher Scientific则</td><td> 157150 </td><td></td></tr><tr><td> Stbl2主管细胞</td><td> Invitrogen公司</td><td> 10268-019 </td><td></td></tr><tr><td>卫康消毒剂的解决方案</td><td> Fisher Scientific则</td><td> NC9821357 </td><td>消毒之前丢弃在一夜之间流失许可</td></tr></tbody></table>协议要求的特定试剂见表1 。

production and titering of recombinant adeno-associated viral vectors

近年来的重组腺相关病毒载体（AAV）已成为越来越有价值的， 在动物体内研究，而目前也正在测试在人体临床试验。野生型腺相关病毒是一个非致病的细小病毒家族成员固有的复制缺陷。广泛传导，低免疫反应，以及与这些载体实现的强大和持久的转基因表达，使他们为流行和通用的工具 ， 在体外和体内基因传递。 rAAVs可以轻松，廉价地在实验室中产生的，基于其良好的安全性，一般都是低安全分类。在这里，我们描述了一个含有AAV1和AAV2衣壳蛋白嵌合rAAVs的生产和滴定的方法。使用这些所谓的嵌合载体，结合双方父母的血清型，如高滴度的股票（AAV1）和净化的好处通过亲和层析（AAV2）。这些腺相关病毒血清型是最好的腺相关病毒血清型研究，及个别有广泛的传染性格局。这里所描述的嵌合载体应该有AAV1和AAV2传染病属性，因此可以预计大范围的组织，包括神经细胞，骨骼肌，胰腺，肾等等的感染。这里介绍的方法使用肝素柱纯化，方法认为给予较高的病毒滴度和病毒编制比其他净化方法，如通过氯化铯梯度离心，清洁。此外，我们描述了如何将这些载体可以快速，轻松地滴度，以提供准确的读数产生的传染性颗粒数量。

Watch this Scientific Journal Video about Production and Titering of Recombinant Adeno-associated Viral Vectors at JoVE.com

Production and Titering of Recombinant Adeno-associated Viral Vectors

重组腺相关病毒（rAAVs）载体正变得越来越有价值的<em&gt;在体内</em在动物身上的实验。我们描述了如何rAAVs可以在实验室中产生，并如何将这些载体可以被滴定给一个准确的读数产生的传染性颗粒数量。

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 ...

production-and-titering-of-recombinant-adeno-associated-viral-vectors

Research

JoVE Journal

Immunology and Infection

61.3K 次观看.  University of Aberdeen. 重组腺相关病毒（rAAVs）载体正变得越来越有价值的在体内

视频: Production and Titering of Recombinant Adeno-associated Viral Vectors

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 ...

科研

免疫与感染

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

从核衣壳的病毒DNA的制备

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

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 ...

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties1. These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors2. A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol1,2. Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling3, i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically &gt;80% for most AAV serotypes), or peptide display4,5, i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular AAV evolution.

Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.

工程和演变的合成腺相关病毒（AAV），基因治疗载体，通过DNA家族改组

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique ...

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 1010 infectious particles per ml and can be directly administrated in vivo.

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro&#160;and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

生产的非复制型犬2型腺病毒衍生载体与纯化

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

细胞因子产生的病毒逃避解剖先天免疫信号

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...