This work was supported by The MRC DPFS grant (MR/M015696/1) and Ministry of Sciences and Technology of China (2013DFG32080).

1.目标RNA和Cas9构造和维修供体载体的制备<ol><li> gRNAs的克隆<ol><li>针对设计痘苗病毒按照先前25所列的原则N1L基因的引导-RNA靶序列。对准对牛痘病毒基因组中的导向RNA靶序列（在此情况下，痘苗病毒的利斯特株），以排除任何脱靶gRNA靶序列。 </li><li>合成和克隆gRNA寡成gRNA克隆载体如前面所述25。确认每个单独gRNA在所得载体用Sanger测序33的序列。命名产生gRNA建设作为gRNA N2 33。 注:gRNA目标站点是N1L基因内，并且是在左臂和右臂之间的区域中，该修供体载体盖（ 见图1）。 </li></ol></li><li>克隆Cas9基因的核定位信号（NLS）缺少进pST1374矢量并指定构建物PST-Cas9 33。 注意:任何哺乳动物表达克隆载体可用于Cas9表达载体的构建。 </li><li>人力资源修复捐助载体的构建<ol><li>使用VV的N1L基因作为目标区域进行修改33。确保左右臂的长度各自是约500-600碱基对，和侧翼目标区域。 注:左手臂/右手臂最多可以有与目标区域50个基点重叠（ 见图1）。 </li><li>克隆N1L区域修复供体载体如先前33,34描述和指定维修供体载体为PT-N1L。 注:请参阅图1的修为供体载体和它的目标区域。维维的其它区域可使用区域（基因）特异性gRNA（S）和特定区域的修复供体载体进行定位。任何克隆载体可用于修复供体载体的构建。 </li> &lt;/ OL&gt; </li><li>质粒的拓展<ol><li>变换质粒入化学感受态根据制造商的说明大肠杆菌 。纯化使用质粒提取试剂盒指的是由制造商提供的协议的质粒。 </li></ol></li></ol> 2.种子细胞（1天） <ol><li>保持CV-1细胞（猴肾成纤维细胞）在Dulbecco氏改良Eagle氏培养基（DMEM），补充有5％胎牛血清（FBS），100U / mL青霉素和100微克/毫升链霉素，在37℃用5％的CO 2。 </li><li> Trypsinize CV-1细胞是80-90％汇合<ol><li>除去从含有CV-1细胞在T-175烧瓶中的细胞培养基。冲洗用5mL无菌PBS的单层，以除去残留的血清和吸出PBS中。加2.5毫升1×胰蛋白酶-EDTA溶液，以覆盖细胞，并放置在烧瓶中37℃培养箱中约23分钟，以帮助细胞剥离。 </li><li>加10毫升细胞培养基（参见步骤2.1），以暂停通过温和移液动作的细胞。算使用血球细胞数。 </li><li>种子细胞在6孔板<ol><li>种子2×10 5个 CV-1细胞在2ml细胞培养基的成6孔板的每个孔中的转染的前一天。 注意:一个附加的离心步骤以除去胰蛋白酶是不需要的。 </li></ol></li></ol></li></ol> 3. Cas9质粒和gRNA质粒转染（2日） <ol><li>转染CV-1细胞用0.5微克PST-Cas9质粒和使用根据制造商的说明转染试剂0.5微克gRNA N 2质粒（在步骤2.2.3.1制备）。 </li><li>孵育24小时的细胞在培养箱（37℃，5％的CO 2），然后替换用2毫升的新鲜细胞培养基（在步骤2.1中所述）的介质。 </li></ol> 4.感染CV-1细胞及穿梭供体的V埃克特转染HR（3日） <ol><li>稀骨干VVL15病毒用DMEM细胞培养基以2×10 5 PFU / mL的浓度。 Cas9和gRNA质粒的24小时后转染，添加100微升稀释的病毒进入转染CV-1细胞的每个孔中。 </li><li> 2小时后的病毒感染，转染1.0μg的穿梭供体载体成使用根据制造商的说明转染试剂VVL15感染细胞的人力资源。放置转染的细胞在37℃培养箱，5％CO 2下24小时。 </li></ol> 5.收获重组病毒（4日） <ol><li>在步骤4.2的HR穿梭供体载体转染的24小时后，分离使用细胞刮转染CV-1细胞。收集细胞悬液到一个离心管，并在-80℃以供将来使用存储。 注:立即擦去消毒剂任何细胞培养液溢出，然后用70％乙再次擦拭醇。 </li></ol> 6.纯化改性VV（第一轮） <ol><li>在第1天，接种15 6孔板用3×10 5个 CV-1细胞在每孔中。 <ol><li>第2天，解冻来自步骤5.1的水浴中的冷冻的细胞悬浮液，在37℃，3分钟，并大力旋涡振荡冷冻管30秒，以获得细胞裂解物不去除细胞碎片。 </li></ol></li><li>感染CV-1细胞中的一个6孔板的，稀释细胞裂解物的1微升用3mL的DMEM，并添加0.5毫升稀释的细胞裂解物进6孔板的每个孔中，对媒体顶部已经当下。感染所有在步骤6.1中制备的6孔板中。 注:不需要的细胞碎片的去除。 </li><li>感染后两天（ 即第4天），发现RFP阳性斑块使用10X物镜在荧光显微镜下，并贴上标签斑块用记号笔由盘旋斑块位置板下方。 注:请参阅<st荣&gt;图3为代表的RFP阳性牌匾。 <ol><li>制备含有200微升无血清的DMEM细胞培养基中拾起六个不同的RFP阳性噬斑6标记的冷冻管。吸从井用标记斑块（S）（步骤6.3）的细胞培养基。附加200微升尖端P200的吸管，将音量设置为30μL，并采取〜从一个冷冻管10微升的细胞培养基。 </li><li>握住吸管按钮而不释放介质，并使用前端划伤一种标记斑块的面积。松开吸管按钮抬起脱落的细胞，并将其转移到含有无血清DMEM 190微升冷冻管。 </li><li>乘坐从与划伤细胞（从最后一步）小瓶另外10μL中，重复拿起相同的RFP阳性斑块三次，以收集尽可能多的划伤细胞尽可能的过程。传输所有细胞进入同一小瓶中，将小瓶储存于-80℃。 </ol></li></ol> 7.纯化改性VV（第二轮） <ol><li>种子3×10 5个 CV-1细胞到每个孔六6孔板并培养所述细胞24小时。 </li><li>解冻在37℃水浴冷冻冷冻管（从步骤6.3.3）3分钟，然后涡冷冻管剧烈30秒，以获得细胞裂解物。 <ol><li>添加混合细胞裂解物的0.5微升至6孔板的一个孔中。感染每个斑块一个6孔板中。用5％的CO 2 2天（第二轮噬斑纯化）孵育VV感染6孔板在37℃。然后重复第6.3节。 注:六点多RFP阳性斑块可以捡到;两个或更多个6孔板可用于各斑块纯化。 </li></ol></li></ol> 8.斑块普瑞更多轮fication <ol><li>进行进一步的三到五轮纯化的以下步骤7中所述，直到从一个阳性噬菌斑形成的所有的噬菌斑表现为RFP阳性显微镜下相同的协议。 注意:然后，这个斑块被认为是纯粹的。 </li><li>一旦斑块是纯粹的（ 图4，表达RFP所有被感染的细胞），如6.3节所述收获了积极的牌匾进入混和，存于-80℃。验证以下步骤9和10中描述的协议的斑块。 </li></ol> 9.展开纯化的斑块（S） <ol><li>种子3×10 5个 CV-1细胞到病毒感染前的6孔板一天每孔中。 </li><li>解冻从8.2冷冻冷冻管在37℃水浴中3分钟以获得细胞裂解物。添加所有溶胞产物（不除去细胞碎片）到CV-1细胞的6孔平板的一个孔中（在步骤9.1接种）。孵育感染CV-1细胞在37℃，5％CO 2的最多3天。 <ol><li>展开所有纯化的斑（至少三个噬斑）。 注:感染时间从1-3天，取决于病毒的裂解物的量而变化。 </li></ol></li><li>收获使用细胞刮刀VV感染的细胞时，50％以上的细胞显示在显微镜下观察细胞病变效应（细胞被四舍五入，容易脱落和RFP阳性）。用细胞培养基到15毫升管收集脱离的细胞一起。 <ol><li>离心以300g沉淀细胞3分钟。去除上清，并保持细胞沉淀在-80℃直至进一步使用或立即使用细胞沉淀为步骤10。 </li></ol></li></ol> 10.突变VV的利用PCR改性的验证<ol><li>提取用DNA提取试剂盒根据制造商的协议，在步骤9.3.1制备的细胞沉淀VV的DNA。洗脱用的双蒸水200微升的DNA。 注意:使用粒料的体积的一半以提取的DNA，并保持其余为病毒产生，如果克隆被验证为含有正确修饰/插入。 </li><li>所述N1L基因的缺失和RFP基因的插入N1L区域的验证。 <ol><li>扩增跨越N1L基因（L025），并使用设计针对N1L基因的引物的L026基因的DNA片段。使用放大RFP特异性引物RFP基因。扩增使用特定A52R-引物通过PCR跨越A52R一个对照DNA片段。 注:请参阅材料清单对特异性引物。 <ol><li>使用PCR主混合试剂盒进行PCR。设置25使用DNA模板的2微升PCR反应100μL，变性在94℃，PCR反应进行2分钟，然后运行下列步骤的PCR 30个循环:在94℃使DNA变性15秒，然后退火引物的DNA模板在52℃15秒延伸72℃30秒的PCR反应。 </li></ol></li></ol></li><li>解决使用1％琼脂糖凝胶33的PCR产物。捕获使用UV胶文档的凝胶图像。如果N1L PCR是阴性和A52R和RFP的PCR是阳性，那么斑块中正确的N1L区域进行修改。 </li></ol>

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Authors have no competing financial interests.

有关于用于治疗目的VV的修改两个主要目标。之一是要删除一个特定的基因，如胸苷激酶（TK）基因，以衰减病毒抗肿瘤的治疗用途。另一种是手臂VV与期望的治疗基因（如GM-CSF）或标记基因（如荧光素酶基因）。实现任一这些涉及的目标区域/基因的缺失。一个直接的DNA连接方法35和体外包装方法36先前已被用来产生的TK基因突变的修改痘苗病毒。然而，这些方法都对基因组中的其他区域的突变应用由于缺乏在整个基因组的独特限制酶位点的限制。当前的方法来创建突变的VV涉及用选择标记（RFP或GFP）到CV-1细胞感染VV后两小时的穿梭（供体）载体的转染以诱导同源重组incorporati纳克选择标记到目标区域。标记阳性噬斑的选择，随后可以通过纯化后24小时转染。斑块净化过程可能需要长达10个回合，最后3-4周，并常常导致不希望重组病毒插入在非目标区域选择标记。 DNA双链断裂能有效诱导同源重组在哺乳动物细胞37，以及通过利用这种机制，产生突变的VV的效率可以大大提高。所述gRNA引导Cas9系统已在基因组中的编辑被成功地并增强同源重组的效率在真核生物22,25。最近，在宿主细胞中我单纯疱疹病毒是由gRNA引导Cas9编辑腺病毒和类型的基因组系统24。最近已经证明，CRISPR Cas9系统是用于有效地编辑VV基因组，构建一个VV载体用于表达的有力工具特定基因。 既N1L gRNA制导Cas9和传统的同源重组（HR）方法导致在N1L的相同靶位点成功的人力资源的事件。然而有趣的是gRNA引导Cas9引起HR在更高水平的效率比传统的人力资源的方法。因此，使用gRNA制导Cas9的无需纯化许多斑块以获得突变的VV。在这项工作中，我们显著提高用于产生使用gRNA引导Cas9系统33突变的VV的效率和时限。值得注意的是，用于获得同源重组的一种高效率的一个关键步骤是在执行传统的同源重组之前转染CV-1细胞用表达Cas9和gRNA质粒24小时。在24小时的时间间隔允许Cas9和gRNA至在合理的水平来表示。此外，靶向特定基因的gRNA序列可能需要被优化的，因为我们发现，不同gRNA序列吨argeting N1L基因的效率33，34变化。 在编辑VV为CRISPR / Cas9的限制是在第一轮重组RFP阳性斑块的低速率。以获得足够数量的含有重组病毒的RFP阳性噬斑，通常至少十个，需要6孔板，这使得第一轮筛选乏味。因此，仍有需要优化来克服这种限制的协议。 RFP阳性噬斑的小尺寸是另一个潜在的问题，这是由于用于感染6孔板高容量裂解物。为了克服这个，裂解物的一个较小的体积，应用于感染6孔板，以获得更大的尺寸的RFP阳性噬菌斑。使用裂解物的一个更小的体积也将使从RFP-消极的RFP阳性噬斑的更好的分离。因此较少轮噬斑纯化的需要获得纯的RFP阳性噬菌斑。 <p类="jove_content"&gt;关靶效应总是在基因的编辑不需要的问题。 CRISPR-Cas9表明脱靶效应。然而，随着gRNA的精心设计，脱靶可以编辑VV基因组的33，34时避免了影响。这是使用gRNA引导Cas9系统用于编辑VV的基因组的特定益处。由于VV的承诺，使用CRISPR / Cas9系统将加快生物医学研究和转化医学突变些VV的发展。此外，这样的系统也将加速在细胞生物学的发现，例如用于其基于肌动蛋白的蠕动使用VV的信号通路的清扫。

痘苗病毒（VV）是属于痘病毒家族包膜DNA病毒，并且在消灭天花的药物最伟大的成就之一，起到了至关重要的作用。在消灭了天花后时代，维维已经发展成为通过插入由各种病原体基因导入VV载体基因交付防治艾滋病毒和其他传染性疾病的疫苗1-7的向量。 VV也被广泛地用作载体的癌症免疫治疗8-14特别是对于肿瘤靶向复制溶瘤痘苗病毒的发展。为了创建与癌细胞改进选择性的有效疫苗载体，病毒基因组内的修改是必要的，包括基因缺失或导入治疗基因。 用DNA技术的发展和更好地理解分子生物学和病毒学，外源DNA的插入到VV最初实现d。使用同源重组（HR）20世纪80年代15。这种方法仍然是广泛用于构建VV载体。遗传修饰的导入是通过使用了HR的穿梭载体，它与在预感染的细胞的基因组中的VV重组实现。然而，这种方法已被证明是非常低效的（小于1％的同源重组效率16）和通常导致选择标记的随机插入非目标区域和/或在病毒扩张所述标记的损失。 DNA的同源重组在基因组位点插入的外源DNA的效率可以在双链断裂（DNA双链断裂）17的存在而显着提高。因此，可以在目标位点DNA双链断裂诱导技术适用于VV基因组工程的巨大潜力。 最近开发的CRISPR-Cas9系统显示在任何VV基因区域触发DNA双链断裂的承诺。 CRISPR-Cas9是RNA-引导核酸酶参与打击入侵的噬菌体等外来遗传物质18-20适应性免疫。有在一个范围内的微生物物种21三CRISPR-CAS系统。 II型CRISPR-CAS系统广泛用于编辑真核细胞和大的病毒的基因组。它由RNA导向Cas9核酸内切酶（由化脓性链球菌 ）中，单个导的RNA（因组）和反式激活crRNA（tracrRNA）22-24。所述Cas9 /因组复杂识别在哺乳动物细胞22 5&#39;-NGG-3&#39;Protospacer相邻基序（PAM）的序列，23之前的互补20-核苷酸基因组序列，已被成功地用于有效产生遗传修饰的细胞，病毒和动物模型22-32。 所述CRISPR-Cas9系统已被证明是对基因组中的VV的细胞质与同源重组结合靶向的有效工具感染的CV-1细胞s至产生突变牛痘病毒33,34。为了延长该方法的潜在应用，我们提出有关此系统的方法的详细信息。所述协议可用于创建与特定的基因缺失的突变体VV和/或与治疗性转基因臂的突变病毒。

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:343px;">Plasmid #41824</td>
			<td style="width:212px;">41824</td>
			<td style="width:137px;">Addgene</td>
			<td style="width:225px;">gRNA cloning vector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pST1374&#160;</td>
			<td>13426</td>
			<td>Addgene</td>
			<td>Cas9 cloning vector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pGEMT easy</td>
			<td>A1360</td>
			<td>promega</td>
			<td>repair donor vector cloning</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">One Shot TOP10 Chemically Competent&#160;E .Coli</td>
			<td>C4040-10</td>
			<td>Invitrogen</td>
			<td>transformation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAprep Spin Miniprep Kit&#160;</td>
			<td>27106</td>
			<td>Qiagen</td>
			<td>plasmid extraction</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dulbecco&#8217;s Eagle&#8217;s Medium (DMEM)</td>
			<td>11965-092</td>
			<td>Life Technologies</td>
			<td>cell culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fetal bovine serum (FBS)&#160;</td>
			<td>SH30088.03HI</td>
			<td>Hyclone</td>
			<td>cell culture serum</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">penicillin, streptomycin</td>
			<td>15070-063</td>
			<td>Thermo Scientific</td>
			<td>antibiotics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Effectene</td>
			<td>301425</td>
			<td>Qiagen</td>
			<td>transfection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermo Scientific Nalgene Cryogenic vial; 2.0 mL</td>
			<td>W-06754-96</td>
			<td>Thermo Scientific</td>
			<td>collect virus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fluorescence microscope&#160;</td>
			<td>Olympus BX51 Fluorescence&#160;</td>
			<td>Olympus</td>
			<td>find RFP-positive plque</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNeasy Blood &#38; Tissue Kit</td>
			<td>69506</td>
			<td>Qiagen</td>
			<td>DNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Extensor Long PCR ReddyMix Master Mix</td>
			<td>AB-0794/B</td>
			<td>Thermo Scientific</td>
			<td>PCR reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10cm culture dish</td>
			<td>Z688819-6EA</td>
			<td>sigma</td>
			<td>cell culture&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plate</td>
			<td>Z707759</td>
			<td>sigma</td>
			<td>cell culture&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cell scraper</td>
			<td>C5981</td>
			<td>sigma</td>
			<td>scrap cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1XTrypsin-EDTA solution</td>
			<td>T4299</td>
			<td>sigma</td>
			<td>trypsinize cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Virkon</td>
			<td>95015661</td>
			<td>Anachem Ltd</td>
			<td>desinfectant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon tube</td>
			<td>CLS430791-500EA</td>
			<td>Sigma</td>
			<td>hold cell suspension</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N1L forward 5&#8217;-TATCTAGCAATGGACCGT-3&#8217;</td>
			<td></td>
			<td>Sigma</td>
			<td>PCR primer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N1L reverse&#160; 5&#8217;-CCGAAGGTAGTAGCATGGA-3&#39;</td>
			<td></td>
			<td>Sigma</td>
			<td>PCR primer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">A52R forward 5&#8217;-ATAGGATTGTGTGCATGC-3&#8217;</td>
			<td></td>
			<td>Sigma</td>
			<td>PCR primer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">A52R reverse 5&#8217;-TTGCGGTATATGTATGAGGTG-3&#8217;</td>
			<td></td>
			<td>Sigma</td>
			<td>PCR primer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RFP forward 5&#8217;- GCTACCGGACTCAGATCCA-3&#8217;&#160;&#160;</td>
			<td></td>
			<td>Sigma</td>
			<td>PCR primer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RFP reverse 5&#8217;-CGCCTTAAGATACATTGATGAG-3&#8217;</td>
			<td></td>
			<td>Sigma</td>
			<td>PCR primer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Argarose</td>
			<td>A7431</td>
			<td>Sigma</td>
			<td>resolve PCR product</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">G:BOX F3</td>
			<td>G:BOX F3</td>
			<td>Syngene</td>
			<td>UV gel doc</td>
		</tr>
	</tbody>
</table>

a simple and efficient approach to construct mutant vaccinia virus vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

对于建设一个新的VV载体，关键出发点是设计和建造，可以针对基因组的特定区域供体穿梭载体。在这项研究中，VV N1L基因被用作例子的目标。施主穿梭载体的重组，并在VV目标区域的盒中示出在图1中，为了提高同源重组的效率，表达Cas9和N1L特异性gRNA质粒共转染入CV -1细胞48小时之前，执行所述同源重组33。一天（24小时）转染后，RFP在CV-1细胞（ 图2）表示。 CV-1细胞的感染与来自同源重组整个裂解物（步骤5）后，RFP阳性和阴性噬菌斑均在CV-1细胞（ 图3）观察到。以下所描述的纯化方案，一个纯粹的噬斑共<html>经过3-5轮的净化ULD获得。如在图4中所示，感染与来自与突变牛痘病毒纯菌斑的细胞裂解物后所有斑块提出了RFP信号。为了验证是否纯突变的VV具有正确的基因修饰，用PCR，确认没有靶N1L基因的， 如图5的改性病毒的PCR显示为RFP阳性信号和用于N1L缺席信号（ 图5车道AG），而N1L为控制病毒（CTR）具有完整N1L区域的PCR检测结果为阳性。 A52R的控制DNA片段呈阳性所有的重组病毒和CTR病毒。在RFP阳性斑块人力资源的效率是在这个实验中（这是高达94％，在以前的工作34）×100％（6/6）。本文所描述的方法已超过100倍相比传统协议33,34提高了复合效率。 内容"FO:保together.within页="1"&gt; <img alt="图1" src="/files/ftp_upload/54171/54171fig1.jpg" /> 图1:供体穿梭载体的同源重组的盒，并在VV基因组中的目标区域的纯化标记基因RFP由H5启动子驱动。针对在RFP基因N1L区域结果修复供体载体被纳入同源重组后N1L区域。 "X"表示在修复供体载体，将取代在牛痘病毒基因组相同的序列同源序列。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54171/54171fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54171/54171fig2.jpg" /> 图2:同源重组后的CV-1细胞一天成像一天AFTE- [R同源重组，感染VV和维修供体载体转染的细胞是RFP阳性答 :相衬图像（放大100X）B:荧光显微镜图像（放大100倍）。比例尺= 50微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54171/54171fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54171/54171fig3.jpg" /> 图3:在突变病毒的第一轮的纯化的RFP阳性噬菌斑在突变病毒的第一轮纯化，将RFP阳性斑块（用红线中圈出）与目标区缺失是通过由形成斑块包围野生型病毒（与黄线盘旋。）A:相衬图像（放大100X）B:荧光MICRoscopy图像（放大100倍）。比例尺= 50微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54171/54171fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54171/54171fig4.jpg" /> 图4:纯突变VV后3-5轮的净化，纯净的斑块获得所有斑块RFP阳性答 :相衬图像（放大100X）B:荧光显微镜图像（放大100X） 。比例尺= 50微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54171/54171fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54171/54171fig5.jpg" /> 图5:Verificatio纯的突变体VV的DNA从VV萃取的n个感染CV-1细胞。 N1L通过PCR使用引物N1L验证了目标区域的缺失，RFP的插入N1L区通过PCR用引物的RFP证实。泳道AF对应六个纯化的噬菌斑，泳道G是N1L缺失先前的工作33验证一个控制斑块，泳道CTR（对照）是野生型牛痘病毒（与N1L区完整）。 A52R基因扩增为所有样品对照基因。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54171/54171fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors at JoVE.com

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

牛痘病毒（VV）已被广泛用于生物医学研究和人体健康的改善。本文介绍使用CRISPR-Cas9系统编辑VV基因组的简单，高效的方法。

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

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JoVE Journal

Immunology and Infection

11.1K 次观看.  Queen Mary University of London. 牛痘病毒（VV）已被广泛用于生物医学研究和人体健康的改善。本文介绍使用CRISPR-Cas9系统编辑VV基因组的简单，高效的方法。 

视频: A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi筛选参与宿主因素牛痘病毒感染使用果蝇细胞

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

科研

免疫与感染

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

简单而强大在体内和在体外方法研究病毒大会

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

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 ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

一种简单有效的方法，通过流式细胞仪检测核转录因子激活人体中性粒细胞

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

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. ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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 ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...