我们要感谢阿尔文·韦尔奇和拉里·庞的技术援助。这项工作是由部分由美国国立卫生研究院（NIH）奖P50 CA086306，加州再生医学研究所（CIRM）补助RC1-00149-1和RS1-00203-1 CIRM新进教师奖RN2-00902-1 ，加州理工学院，加州大学洛杉矶分校联合转化医学中心，加州大学洛杉矶分校艾滋病研究中心（CFAR）NIH / NIAID AI028697，加州大学洛杉矶分校艾滋病研究所，CIRM工具和技术奖RT1-01126，和加州大学多校区研究计划和计划从加利福尼亚州中心抗病毒药物的发现（数量MRPI 143226）。

A.生成的BLT小鼠代的BLT小鼠分为三（3）部分:（1）处理的CD34人类造血干细胞的胎儿组织和制备，（2）移植的人体组织，和（3）二次移植。对于所有的协议，我们已经提供了表1与试剂信息。 1。组织处理和CD34造血干细胞的分离新鲜的胎儿组织快递的冰从各机关采购机构或组织都可以使用。胎儿组织通常是没有出息的，证明1000菌落每毫升血液琼脂周围介质的生产。我们经常在组织洗两次，在40毫升的无菌PBS中。虽然这并不能消除所有的细菌，它可以使一个区别，其中最屈从于细菌中的受体小鼠移植成功的系列和结果fection。作为一个附加的步骤，以进一步消毒的组织，培养细胞悬浮液中抗生素的存在。 1.1胎儿胸腺组织的处理<ol><li>浇灭介质进入的漂白剂的烧杯中，并使用手术刀滑入漂白剂，以防止组织洗净胸腺。填充管，用PBS。盖上盖子，颠倒几次，洗胸腺。弃去上清液漂白剂。重复以上步骤。 </li><li>将胸腺组织在8毫升的RPMI + 10％小牛血清，在60毫米的培养皿和剪切成1.5至2毫米的片具有两个解剖刀。剪切与手术刀，最大限度地减少撕裂和破坏的组织位。受损的组织会变得肿胀，发粘，难以绘制成的留置针。获得的比特的数量是第一上限时，对移植的小鼠。 </li><li>暂停位与25毫升移液管（以尽量减少组织损伤）转移到一个T25烧瓶中。加入70微升piptazo（Zosyn）瓶和文化过夜， <s仲&gt;在37℃下，5％CO 2。这大大减少了细菌污染。如果你想这样做组织配型或任何其他的表型分析，取出1毫升的细胞。 </li></ol> 1.2胎肝组织的处理<ol><li>在步骤1.1.1的肝洗净。 </li><li>加入10毫升的Iscove的中，倾出到100毫米的培养皿中的一切。 </li><li>有两个手术刀，骰子的肝脏中转化为小（约3毫米）件。如果您遇到白色结缔组织，刮肝，并丢弃在漂白。 </li><li>均质组织抽吸到12毫升注射器装有一个16号钝针3或4倍，并转移到一个50毫升的试管。 </li><li>准备的酶组织消化如下:向10毫升的Iscove的一个50毫升管中，添加200微升的每个:型胶原酶IV（1毫克/毫升），透明质酸酶（1毫克/毫升），DNA酶I（2 U /毫升）中。 </li><li>绘制到一个12毫升注射器的酶和0.22μ滤波器适合它。过滤enzy我/介质的细胞悬浮液直接进入。 </li><li>盖上盖子，用Parafilm密封的细胞。在37℃下旋转90分钟。 </li><li>设置了一个50毫升的试管100μ细胞过滤网。吸取细胞消化，通过这个过滤器。 </li><li>加入PBS中的细胞中，使体积至50ml。它分成两个25毫升的试管。 </li><li>下垫细胞用10ml的Ficoll。 20分钟没有刹车，转速为2,400 RPM。 </li><li>接口应该是厚的。 5毫升吸管转移到另一离心管中吸出来。你应该有两个管子的接口。加入50毫升PBS中到每个和自旋在1,200 rpm，10分钟。 </li><li>合并到一个管中的颗粒，并用PBS含2％FCS中洗3次以上。 </li><li>最后悬浮在50毫升的RPMI + 10％FCS和使用台盼蓝计数细胞。根据组织的大小，你可以得到10 8 10共有9个肝细胞。 </li><li>将细胞转移至T150烧瓶中，并添加30毫升的RPMI + 10％FCS和0.8米●的piptazo。 </li><li>孵育1至1.5小时，在37℃以除去贴壁细胞。 </li></ol> 1.3排序和转导的CD34造血干细胞<ol><li>连接步骤1.2.15细胞后，轻轻的来回摇动烧瓶中，一分钟，以清除松动的细胞。将有一个特定的成纤维细胞和例如卡住的表面。 </li><li>计数再次使用一个1至20的稀释液，并记录细胞的总数。保存一毫升零点几秒后染色。你应该少约30％的细胞从初始细胞计数（1.2.13）。 </li><li>降速和洗两次，MACS缓冲液40毫升。 </li><li> CD34 +细胞进行排序使用美天旎生物技术公司的CD34直接包。我们完全遵循制造商的协议，由同一家公司提供的LS列。 </li><li>回收的细胞，将在5毫升。计数和计算总的细胞。您的收益应该是CD34 +细胞的细胞总数的3-5％。这会随着日e时代的胎肝。年轻的胎肝往往给予了较高的收益率，CD34 +细胞。 </li><li>的细胞数除以由1×10 6。这给出了对移植的小鼠的第二上限。 </li><li>计数CD34-比例（流动和洗涤）和储备6×10 6个小鼠移植。培养这些细胞在50至80毫升的RPMI + 10％小牛血清中过夜+ 1％piptazo。 </li><li>转导的CD34 +细胞过夜您的慢病毒载体。我们使用Retronectin由Takara的完全制造商的说明。 </li><li>第二天，收获CD34-细胞，转染的CD34 +细胞和计数。 </li><li> CD34 +细胞分割了一半:A部分和B部分。 </li><li>自旋向下的CD34 +转导的细胞，并暂停在4到6毫升的冷冻液（RPMI + 10％FCS + 10％DMSO，0.22μ无菌的过滤）。把1毫升等分冷冻瓶和标签的"huFetalCD34"，在小瓶细胞的数量，日期，和您的姓名缩写。 </li><li>对于每个鼠标混合0.5×10 6部分，乙CD34 +转导的细胞和4.5×10 6 CD34-细胞，在无菌的螺旋盖离心管中，颗粒，并保持在冰上。另外在冰上解冻的Matrigel（BD Biosciences公司）的管。所有这些管子必须置于冰上，直到（步骤2.10）。 </li></ol> 2。组织移植这一步的目标是移植人胎胸腺/ NSG小鼠肾囊肝组织体。这一类器官更好地模仿人体造血干细胞的过程中，人类T细胞的选择和成熟过程将使用人体胸腺不同的T细胞和其他淋巴组织的谱系分化的网站，而不是鼠标。 CD34-细胞在移植过程中使用的是重新生成的胎肝基质和允许更好的植​​入物的移植和生长。 NSG小鼠的年龄是6-8周。 <ol><li>药物的制备苏rgery: <ol class="lalpha"><li>异氟醚:卷起一个2英寸的纱布垫，并把它装到15毫升螺旋盖离心管。倒入约1毫升的异氟醚直接从瓶。 </li><li>氯胺酮/ zylazine:0.52毫升氯胺酮（Ketaset）（100毫克/毫升），和0.52毫升的甲苯噻嗪（Anased）（100毫克/毫升）加入到一个20毫升的小瓶注射生理盐水。出版商的最早到期组件的到期日期，并保持在-20℃冷冻，直到需要。 </li><li>卡洛芬（Rimadyl）。稀释这只是一种外科手术前。用1ml注射器和25G针撤出0.1或0.2毫升的小瓶中稀释1:100。注入一个10或20毫升的生理盐水注射。标签和日期。一天只能存放在冰箱里。装入3毫升25G针头的注射器。 </li></ol></li><li>设置表的手术。把它盖的无菌的蓝色垫，和一个无菌巾，每一个外科医生。每个医生箱的消毒工具:"剪刀，弯钝钳，需要得到LE-鼻子镊子，止血钳，16G减弱套管针，伤口夹具。此外，他们需要的弯曲针薇乔缝合，堆栈异丙醇擦拭数据包，一个35毫米陪替氏培养皿的PBS，和1ml注射器填充有PBS。 </li><li>设置在中间的表玻璃科普林氏罐2厘米的聚维酮碘和棉签。倒入胸腺位到60毫米的培养皿。 </li><li>执行一个单独的表，或用无菌蓝色垫覆盖在发动机罩的麻醉。设置两个小架和一个电子天平，烧杯中举行的老鼠。使用一个机架容纳0.5毫升×28G胰岛素注射器装有氯胺酮/甲苯​​噻嗪和其他持有3毫升注射器卡洛芬。此外，移动的生物危险废物容器附近举行剃皮毛。 </li><li>权衡所有小鼠（6-8周龄），在一个笼子里，与15微升/克氯胺酮/赛拉嗪的剂量得到的平均重量和负载注射器。注入所有的老鼠在笼子里。 </li><li>后小鼠变得缓慢剃光LEFT侧从臀部到肩膀之间的背部和腹部奥斯特第40号刀片剪的中心。记录他们的体重，编号，冲自己的耳朵。 </li><li> 0.3毫升卡洛芬注入皮下的肩膀上。 </li><li>检查麻醉的鼠标，通过挤压它的爪子。如果鼠标退缩，管理异氟醚管约12秒。然后再次尝试的爪子紧缩。请给短镜头的异氟醚，直到爪子反射消失。奠定了其面临的右侧左鼠标。 </li><li>一个16号的癌症留置针，用于插入组织提出的尖端圆。冲洗套管针几次的菜PBS。握住杆内的尖端水平。添加一块胸腺尖嘴钳，吸它。 </li><li>冷Matrigel的一个辅助增加5微升的Eppendorf容积式移液管与管细胞（1.3.12），并轻轻搅拌混合。外科医生HOLDS的套管针和水平方向上拉杆慢慢向后，作为辅助移液管的基质胶混合成的套管针。混合凝胶在室温下。 </li><li>用优碘擦拭裸露的皮肤。然后用异丙醇擦拭擦拭。重复以上步骤。 </li><li>皮肤下找到健脾。它是在最黑暗的地方。老鼠的肾脏位于约5毫米背脾，可以看出，通过刮胡后的肌肤。 </li><li>拿起这点与直率的镊子和皮肤作一个切口平行于脾约15毫米长。在下面的肌肉层腹膜做一个类似的削减。在男性中，肾是直接可见的，你只需要挤压腹部弹出。在女性中，您可能不得不拿起卵巢止血钳拖出肾。这可能有助于保留肾脏以外的身体。对于男性，你会用你的左手，保持一定的压力，腹部，保持肾脏暴露。 </li><li>拨动小洞的posterioR端的肾囊中。它可能流血了一点。 </li><li>滑动套管针插入此孔沿肾脏直到由肾脏胶囊被完全覆盖的孔的套管针。然后用你的右手小指，注入组织。 </li><li>封闭止血钳轻轻推入肾。在并列的双重知道的腹膜，将一针一针。挤压皮肤像一个钱包和投入在两个伤口剪辑。 </li><li> PBS每只眼睛一滴奠定了鼠标在其上笼床上用品。确保所有的小鼠在离开之前他们回到了他们的肚子上铺设。 </li><li>第二天，给每个鼠标的卡洛芬再出手。 </li></ol> 3。二次移植在此步骤中生成的BLT小鼠的目标是填充的moue的人类造血干细胞的骨髓。从步骤（2）移植的植入物是不够的支持重建的第Ë人体的免疫系统，在这些小鼠。二次移植期间，我们亚致死照射小鼠耗尽小鼠骨髓细胞，从而产生"空间"的人CD34 +细胞的植入。 <ol><li>四到六周后2.7 Gy的照射小鼠。同一天，部分转导的CD34 +细胞的小鼠被注射。根据你的/ LIV植入的移植步骤2中的建立和发展的时间表。通常情况下，在此期间中，植入物已生长显着允许我们继续进行到下一步骤。 </li><li>解冻，A部分的CD34 +转导的细胞，洗涤并悬浮在介质中的浓度为5×10 6个 / ml。 </li><li>负载0.5毫升×28G胰岛素注射器用0.1毫升的细胞（10 5到鼠标每0.1毫升，每5×10 5个细胞）的一个笼子里的老鼠（每笼最多可包含5只），并将其放置在机架上背对。 </li><li>麻醉与异氟醚管通过scruffing，并持有其鼠标约20秒，在管中的鼻子。计数的时间跟踪。 </li><li>然后面对它的权利，并在其脖子的左手拇指和食指紧拉扯皮肤。它的下颚下按你的第三个取景器，以便其右眼突出。持注射器鼠标的头部的长轴平行，后面的眼睛和滑针，轻轻滑动，直到它停止。不要按任何进一步的，但，注入负荷在约2秒。 </li><li>二至三个月后小鼠流血retroorbitally使用EDTA镀膜玻璃吸液管。您可以只需要200微升的血液每月每鼠标最大的。 50-100微升血液应该是足够的FACS分析。出血的替代战略，包括面部静脉出血和尾静脉出血。后者会给你的血液可能不足以为多个测定的非常小的体积。 </li><li>血液样本被染色的表达的人类淋巴细胞标记CD45，以评估重建。乙载量的样品加入到1毫升红血细胞裂解缓冲液（160 mM的NH 4 Cl的 ，100mM的KHCO 3，0.01毫摩尔EDTA）中，并在室温下孵育5分钟。 </li><li>洗净的细胞在裂解缓冲液在1,200 rpm，5分钟，并在必要时重复。 </li><li>离心细胞在3.8和补充有3％胎牛血清（FACS-PBS）在PBS中清洗。 </li><li>去除上清液，并重新悬浮于100μlFACS-PBS粒料。 </li><li>增加抗体，以评估人体淋巴细胞的重建。我们通常包括以下CD45（人体淋巴细胞标记），CD8，CD4，CD14和CD16的巨噬细胞和单核细胞的B细胞，CD19和CD56 NK细胞。 </li><li>在4℃下孵育30分钟。 </li><li>在步骤3.9中进行清洗，并重新悬浮细胞的FACS分析的2％多聚甲醛。 </li><li>如果小鼠重组，我们继续与肿瘤注射。的重组人淋巴细胞水平变化，从低于1％至40％的总血细胞。级别和类型的淋巴细胞&gt;YTE谱系是什么人会期​​望从健康人的血液捐赠者。然而，预期的非T细胞谱系数的波动。如果数字是不能接受的，在12周的时间内，你可以重复从步骤3.1二次移植手术。 </li><li>肿瘤细胞系进行培养，根据制造商的或实验室的建议。收集细胞，洗涤，再悬浮在基质胶以1:1的比例（50微升cells/50微升基底膜）。样品在任何时候都保持在冰上。 </li><li>小鼠麻醉，剃的区域处注射。的区域进行杀菌使​​用异丙醇垫。预加载一个辅助细胞悬浮液冷却23G 1毫升注射器。皮下注射的肿瘤细胞。治疗的部位注射抗生素软膏。的肿瘤应建立和在4周开始增长。 </li></ol> B. 在体内正电子发射断层扫描（PET）。 在我们的研究中，我们考试国家统计局葡萄糖的摄取（[18 F]脱氧葡萄糖（[18 F] FDG）小鼠禁食4-6小时之前。MicroPET的成像/ CT扫描是使用微正Inveon扫描仪"（西门子临床前解决方案）和MicroCATII CT扫描仪的（西门子PreclinicalSolutions）进行图像分析使用OsiriX（Pixmeo，瑞士）软件。目的是测量的肿瘤的代谢活性和最终使用PET显像作为物理测量肿瘤消退的替代，正如在图2中看到，我们所遇到的物理外观和大小的肿瘤，根据目标，但现场的PET成像显示广泛的组织坏死。这种方法可以作为一个更加敏感和准确的指标肿瘤消退。 <ol><li>指定一腔麻醉（2％异氟烷）的小鼠，60分钟摄取FDG PET扫描之前（"等待室"）和一室。将蓝色垫在两院。 </li><li>的动物是anesth，多长时间etized，它们应保持温暖，在30℃通过使用装满水的手套，在微波炉中加热，热垫和放置的笼子。 </li><li>此室，关闭异氟醚的流动。允许异氟烷以填补前放置到腔室中的任何小鼠麻醉箱为约5-10分钟。确保盖子紧紧锁定和关闭，以避免泄漏异氟醚。 </li><li>将鼠标移动到麻醉室和锁定的盖子盖紧。允许5-10分钟或直到鼠标是无意识的。 </li><li>取出小鼠从麻醉室，标签在鼠标的尾部（不同的颜色，每笼），，并注入腹腔鼠标[18 F] FDG（80微居里）。注意的小鼠注射的时间。鼠标将再次被麻醉50分钟，从这个时间点，让PET扫描（总60分钟10分钟[18 F] FDG摄取前PET扫描）。 </li><li>将注入的鼠标进入等待室，让[18 F] FDG摄取1小时前PET扫描。不要锁定盖。保持宽松的盖子，让空气流通。 </li><li>立即将鼠标FDG注射麻醉室。随后的小鼠注射剂应间隔10分钟，，因为PET扫描的过程中，是10分钟。后立即被扫描时，一个鼠标60分钟FDG摄取要扫描的下一个鼠标将准备正好相隔10分钟。 </li><li>继续麻醉和注射小鼠中所述步骤7-9。 </li><li> 50分钟后的第一只老鼠注射的时间点，将首先注入鼠标再次进入麻醉室（同时仍继续麻醉和注射小鼠）。 </li><li>将鼠标床的氧气和异氟醚线，并开启异氟醚流在床上。将蓝色垫在床上。 </li><li>将鼠标准备PET扫描在床上，伸展双臂，腿，并用胶带担任该职务。将润滑油的眼睛。这一步是最关键的，因为定位的动物可能会影响你的成像质量。 </li><li>断开异氟醚和氧气线，快速安装在床的PET扫描机。将异氟醚和氧气供应管道。确保异氟醚的流动是PET扫描机。 PET扫描线连接，并开始扫描。 </li><li>一旦PET扫描完成，然后将动物的CT扫描仪。这个扫描后的动物移动到其各自的笼子。 </li></ol>

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	<li>McCune, J. M., Namikawa, R., Kaneshima, H., Shultz, L. D., Lieberman, M., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SCID-hu+mouse:+murine+model+for+the+analysis+of+human+hematolymphoid+differentiation+and+function.">The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function.</a> Science. 241 (4873), 1632-1639 (1988).</li><li>Bristol, G. C., Gao, L. Y., Zack, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+maintenance+of+SCID-hu+mice+for+HIV+research.">Preparation and maintenance of SCID-hu mice for HIV research.</a> Methods. 12 (4), 343-347 (1997).</li><li>Mosier, D. E., Gulizia, R. J., Baird, S. M., Wilson, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+a+functional+human+immune+system+to+mice+with+severe+combined+immunodeficiency.">Transfer of a functional human immune system to mice with severe combined immunodeficiency.</a> Nature. 335 (6187), 256-259 (1988).</li><li>Melkus, M. W., Estes, J. D., Padgett-Thomas, A., Gatlin, J., Denton, P. W., Othieno, F. A., Wege, A. K., Haase, A. T., Garcia, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+mice+mount+specific+adaptive+and+innate+immune+responses+to+EBV+and+TSST-1.">Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1.</a> Nat. Med. 12 (11), 1316-1322 (2006).</li><li>Denton, P. W., Estes, J. D., Sun, Z., Othieno, F. A., Wei, B. L., Wege, A. K., Powell, D. A., Payne, D., Haase, A. T., Garcia, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiretroviral+pre-exposure+prophylaxis+prevents+vaginal+transmission+of+HIV-1+in+humanized+BLT+mice.">Antiretroviral pre-exposure prophylaxis prevents vaginal transmission of HIV-1 in humanized BLT mice.</a> PLoS Med. 5 (1), e16 (2008).</li><li>Sun, Z., Denton, P. W., Estes, J. D., Othieno, F. A., Wei, B. L., Wege, A. K., Melkus, M. W., Padgett-Thomas, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrarectal+transmission,+systemic+infection,+and+CD4++T+cell+depletion+in+humanized+mice+infected+with+HIV-1.">Intrarectal transmission, systemic infection, and CD4+ T cell depletion in humanized mice infected with HIV-1.</a> J. Exp. Med. 204 (4), 705-714 (2007).</li><li>Shimizu, S., Hong, P., Arumugam, B., Pokomo, L., Boyer, J., Koizumi, N., Kittipongdaja, P., Chen, A., Bristol, G., Galic, Z., Zack, J. A., Yang, O., Chen, I. S., Lee, B., An, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+efficient+short+hairpin+RNA+potently+down-regulates+CCR5+expression+in+systemic+lymphoid+organs+in+the+hu-BLT+mouse+model.">A highly efficient short hairpin RNA potently down-regulates CCR5 expression in systemic lymphoid organs in the hu-BLT mouse model.</a> Blood. 115 (8), 1534-1544 (2010).</li><li>V, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+HIV+Latency+in+Humanized+BLT+Mice.">Generation of HIV Latency in Humanized BLT Mice.</a> J. Virol. 86 (1), 630-634 (2012).</li><li>Vatakis, D. N., Koya, R. C., Nixon, C. C., Wei, L., Kim, S. G., Avancena, P., Bristol, G., Baltimore, D., Kohn, D. B., Ribas, A., Radu, C. G., Galic, Z., Zack, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+activity+from+antigen-specific+CD8+T+cells+generated+in+vivo+from+genetically+engineered+human+hematopoietic+stem+cells.">Antitumor activity from antigen-specific CD8 T cells generated in vivo from genetically engineered human hematopoietic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 108 (51), 1408-1416 (2011).</li></ol>

没有利益冲突的声明。

加上在体内的 PET成像BLT人性化的小鼠模型研究人类慢性疾病的有力工具。该系统以BLT小鼠模型及垫款超出限定范围为艾滋病研究。此外，它是一个伟大的系统中，我们可以研究各种基因治疗方案以及诊断技术，才可以达到临床上。后者成本低，再加上利用老鼠与灵长类动物，这是一个非常有用的模型。 PET成像技术使我们能够评估我们的方法的疗效。如果我们完全依赖...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> PBS </td><td> Invitrogen公司</td><td> 10010049 </td><td></td></tr><tr><td> RPMI 1640 </td><td></td><td> A1049101 </td><td></td></tr><tr><td> Iscove氏</td><td></td><td> 12440061 </td><td></td></tr><tr><td>胎牛血清</td><td></td><td> 16000085 </td><td></td></tr><tr><td>胶原酶IV型</td><td> Sigma Aldrich公司</td><td> C5138 </td><td></td></tr><tr><td>透明质酸酶</td><td></td><td> H3506 </td><td></td></tr><tr><td> DNA酶I </td><td>罗氏诊断</td><td> 10104159001 &lt;/ TD&gt; <td></td></tr><tr><td> Piptazo（Zosyn） </td><td>辉瑞公司</td><td></td><td> <a href="http://labeling.pfizer.com/showlabeling.aspx?id=416" target="_blank">哌拉西林/他唑巴坦组合</a> </td></tr><tr><td> FICOLL（的Ficoll-Paque加） </td><td>通用电气医疗集团生命科学</td><td> 17-1440-02 </td><td></td></tr><tr><td> MACS缓冲</td><td>美天旎生物技术</td><td>查看评论</td><td> MACS缓冲液:PBS补充有0.5％胎牛血清和2mM柠檬酸葡萄糖式-A </td></tr><tr><td> CD34微珠试剂盒</td><td></td><td> 130-046-702 </td><td></td></tr><tr><td> LS列</td><td></td><td> 130-042-401 </td><td></td></tr><tr><td> Retronectin进行</td><td>宝</td><td> T100A </td><td></td></tr><tr><td&gt;基底膜</td><td> BD Biosciences公司</td><td> 354263 </td><td></td></tr><tr><td>异氟烷</td><td>巴克斯特</td><td></td><td> <a href="http://www.baxter.com/healthcare_professionals/products/forane.html" target="_blank">http://www.baxter.com/healthcare_professionals/products/forane.html</a> </td></tr><tr><td>卡洛芬（Rimadyl） </td><td>辉瑞动物保健</td><td></td><td> <a href="https://www.rimadyl.com/default.aspx" target="_blank">https://www.rimadyl.com/default.aspx</a> </td></tr></tbody></table>

using the blt humanized mouse as a stem cell based gene therapy tumor model

如老鼠的小动物模型已被广泛用于研究人类疾病和开发新的治疗干预措施。尽管从这些研究中获得的大量信息，别具特色的种属特异性的病毒引起的疾病，如人类免疫缺陷病毒（HIV）感染小鼠免疫功能以及人性化的小鼠模型的发展。早期型号使用C. B 17 SCID / SCID小鼠体内移植的人胎胸腺，胎肝称为你的/ LIV（SCID-胡）1，2或过继的人外周血白细胞（SCID-huPBL ）3。这两种型号，主要用于HIV感染的研究。 这两种模式的主要限制之一是缺乏稳定的重建人体免疫细胞的外围，使他们更有关生理模型来研究HIV疾病。为此，BLT嗡嗡声anized小鼠模型的开发。 BLT代表骨髓/肝/胸腺。在这个模型中，6〜8周老NOD.Cg Prkdcscid Il2rgtm1Wjl / SZJ（NSG）免疫功能低下小鼠收到你的/ LIV植入的SCID-hu的小鼠模型，但随后第二次人类造血干细胞移植4。这个系统的优点是在外围人体免疫系统的完整的重组。这个模型已被用于研究艾滋病病毒感染者和潜伏期5-8。 我们已经产生了一个修改后的版本，这个模型中，我们使用转基因造血干细胞（HHSC）构建你 ​​的/ LIV植入的自体注射转HHSC的7,9。这种方法会导致产生的转基因谱系。更重要的是，我们适于此系统，以检查表达黑素瘤特异性T细胞受体的功能性细胞毒性T细胞（CTL）产生的电位。使用这个模型，我们能够评估我们的转基因CTL利用的现场正电子发射断层扫描（PET）成像，以确定肿瘤消退（9）的功能。 此协议的目的是描述的过程中，这些转基因小鼠，用活PET成像在体内的疗效和评估。作为一个说明，因为我们使用的人体组织和​​慢病毒载体，我们的设施符合C​​DC美国国立卫生研究院生物安全2级（BSL2）的指引，有特殊的预防措施（BSL2 +）。此外，核供应国集团的小鼠有严重免疫功能低下，因此，他们的住房和维护必须符合最高的健康的标准（ <a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a> ）。

移植过程的流程图是在图1A中所示。 如图1B所示的图片，你的/ LIV植入的。胸腺组织发展通常并具有生理人类CD4 +和CD8 + T细胞的分布。经过重组，动物携带人体免疫系统的与正态分布的CD4，CD8 T细胞和其他免疫细胞谱系。 肿瘤大小和活组织之间的差异，是在图2中所示。虽然CT扫描（灰色区域）显示肿瘤的生长， 在体内 PET成像显示，...

Watch this Scientific Journal Video about Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model at JoVE.com

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

这里描述的是使用人源化小鼠的肿瘤特异性T细胞的产生和表征。人胸腺组织和转基因的人类造血干细胞移植到免疫功能低下小鼠。这将导致在一个精心设计的人体免疫系统在体内的抗肿瘤免疫反应的检查允许的重组。

BLT人性化的鼠标使用一个基于干细胞的基因治疗肿瘤模型的建立

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

using-blt-humanized-mouse-as-stem-cell-based-gene-therapy-tumor

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JoVE Journal

Immunology and Infection

18.6K 次观看.  David Geffen School of Medicine at UCLA. 这里描述的是使用人源化小鼠的肿瘤特异性T细胞的产生和表征。人胸腺组织和转基因的人类造血干细胞移植到免疫功能低下小鼠。这将导致在一个精心设计的人体免疫系统在体内的抗肿瘤免疫反应的检查允许的重组。

视频: Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

科研

免疫与感染

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

PRP作为一种新的方法，以防止感染的制备及<em在体外</em&gt;抗菌性能的PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

人源化的小鼠模型来研究细菌感染瞄准微血管

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

类器官的模型传染病:人类的文化与小鼠胃组织体与幽门螺杆菌的显微注射

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

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.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

BLT人性化的鼠标使用一个基于干细胞的基因治疗肿瘤模型的建立

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