We would like to thank Ms. Jessica Selander in providing artistic assistant in making our figures. This work was funded by grants from the NIAID/NIH, grant no. RO1AI078806, the UCLA Center for AIDS Research (CFAR), grant no. P30AI28697, the California Institute for Regenerative Medicine, grant no. TR4-06845, the American Federation for AIDS Research (amfAR), grant no. #108929-54-RGRL, and the UC Multi-campus Research Program and Initiatives, California Center for Antiviral Drug discovery (CCADD)

伦理声明:人类胎儿组织是从高级Biosciences的资源或从Novogenix获得并未经识别信息，不需要为它的使用的IRB批准得到。在这个手稿中描述的动物研究是加州大学洛杉矶分校和分校（UCLA）的动物研究委员会（ARC）的按照所有联邦，州和地方的指导方针书面同意下进行。具体来说，这些研究是在严格按照该指南的关怀和全国研究委员会实验动物的使用和认可，并为实验动物的评估和认可委员会（AALAC）协会的指导原则中的指导原则进行国际加州大学洛杉矶分校在ARC协议编号2010-038-02B。所有手术均在氯胺酮/甲苯​​噻嗪和异氟醚麻醉下进行并作出一切努力，以尽量减少动物的痛苦和不适。 1.人性化小鼠的构建CD4嵌合抗原受体工程化<ol><li> 处理胎儿胸腺和胎肝隔离CD34 +造血干细胞 <ol><li> 胸腺处理 <ol><li>轻轻洗磷酸盐缓冲盐水（PBS），pH值7.4，胸腺在15ml锥形管中。重复洗涤步骤3 - 4倍。 </li><li>添加7毫升RPMI培养基+ 10％FBS +青霉素/链霉素。倒出一切进入无菌百毫米培养皿。 </li><li>使用解剖刀到胸腺切成小块的约1mm 2。放置在单个井的每一个胸腺片在96孔板中。利用转印胸腺件到96孔板时弯曲的钝钳。 <ol><li>的媒体少量添加（100 - 200微升）到所有的孔中，使得所述组织不干燥。显微镜下可视化（thymi有裂片应该像细胞的麻袋）。 注:放弃以任何方式看起来可疑的任何部分;常常有结缔组织和这不应该被植入。 </li></ol></li><li>取下证实胸腺件，并将它们全部进入T25细胞培养瓶中。新增7毫升补充有10％FBS的RPMI培养基和450微克/毫升哌拉西林/三唑巴坦和两性霉素B轻轻岩石烧瓶以混合。培养在37ºC/ 5％CO 2的烧瓶中过夜。 注:需要这个步骤，以防止组织的细菌污染。 </li><li> （可选步骤）冷冻以供将来使用胸腺。平衡用10％二甲亚砜（DMSO）中的90％人AB血清的组块10分钟。以1ºC/分钟的速率到-50ºC，然后快速冷却到-150ºC冷冻。当准备解冻，解冻迅速在37ºC水浴在RPMI完全培养基轻轻地洗3次无DMSO。 </li></ol></li><li> 肝脏处理 <ol><li>轻轻洗肝在PBS在50毫升锥形管中。重复洗涤步骤3 - 4倍。 </li><li>加入10毫升（Iscove氏修改的Dulbecco氏媒体）的IMDM培养基的50ml锥形管中。倒出一切都变成100毫米的无菌培养皿。 </li><li>均质化使用两个手术刀肝组织。割肝小块约3mm 2。剪下并丢弃任何白色的结缔组织。 </li><li>使用10个毫升注射器配有一个16号钝针占用肝片和媒体。然后转移到50毫升锥形管中。 </li><li>轻轻重悬媒体和组织悬架和驱逐5-7次完全均匀的组织。 </li><li>制备出10毫升的IMDM培养基补充有酶:500U / ml胶原酶2400单位/毫升hyluronidase，和300 U / ml的DNA酶以及450微克/毫升哌拉西林/三唑巴坦和两性霉素B通过一个0.22微米的过滤器过滤介质，然后媒体添加到肝脏停牌。 </li><li>帽含有肝悬浮液的50ml锥形管中并用自密封膜，如石蜡膜Ť封紧Ø防止泄漏。旋转在培养箱试管旋转器在37ºC90分钟。 </li><li>过滤通过100微米的细胞滤网消化的细胞悬液到一个新的50ml管中。 注:加入的PBS悬浮液中，使总体积达50毫升分成各含25 ml的细胞悬浮液50ml试管的两个管这一点。 </li><li>慢慢地和轻轻下垫细胞每管10毫升密度离心介质（ 如，聚蔗糖）。转速为1200 XG 20分钟不带刹车。注意:在这个协议中提到的所有离心是在室温（25℃）进行。 </li><li>小心地从每管和传输接口（ 即 ，血沉棕黄层），以两个独立的50ml试管。带来的接口的每个管的体积至50ml用PBS。旋转300 XG 7 - 10分钟。小心吸上清。 </li><li>将二者结合起来的颗粒。用含2％FBS的50毫升PBS洗三次。旋转300 XG 7 - 10米每次在同时认真吸取上清液。 </li><li>重悬在50ml RPMI培养基+ 10％FBS沉淀。在进行细胞分选之前计数使用血球此时细胞。 </li><li>排序CD34 +细胞立即用CD34排序试剂盒（ 例如 ，CD34微珠），根据制造商的协议。 注:可替代地，细胞可以在RPMI培养基+ 10％FBS 100万过夜培养/ ml的。 </li><li>节省CD34 + CD34-和分数。 注:在这一步骤中，CD34 +和CD34-的细胞可以使用Bambanker冷冻介质或其它冷冻媒体被冻结。冻结1毫升4-的-每管6×10 6个 CD34 +细胞，并冷冻加入1ml 40 -每管60×10 6个 CD34 -细胞。 </li></ol></li></ol></li><li> CD34 +细胞转导 <ol><li>计算6孔组织培养板的所需转导井的数量。 1以及可用于转导至8×10 6个细胞。对于各BLT鼠标，0.5×10 6个 CD34 +将与CD34-细胞和肾囊下胸腺和0.5×10 6个 CD34 +细胞一起被植入将静脉内注射。 CD34 +细胞使用的数量是由小鼠（每鼠标1百万个细胞）的数目来确定。 </li><li>涂层的非组织培养的孔的所需要的数处理6孔板1.25 ml重组人纤连蛋白溶液（ 例如 ，Retronectin进行）（20微克/ ml，在PBS中）到每个孔中。覆盖板，并允许它放置在室温下2小时在一个干净的生物安全柜中。 </li><li>从井抽吸纤连蛋白溶液，并添加1.25毫升FACS缓冲液（PBS中的4％FBS）到每个孔用于阻断。允许该板在室温下（25℃）放置30分钟。 </li><li>吸FACS缓冲液和洗涤液井一次，用PBS。 </li><li>保持的PBS中包被的孔，直到该板是准备使用。在4ºC存储板过夜如果不立即使用。 </li><li>板CD34 +细胞在感染培养基（2％人血清白蛋白在Yssel的无血清T细胞培养基）中纤连蛋白溶液包被的孔（〜2×10 6个细胞/ ml）孵育37ºC1小时。 </li><li>使用移液管，以于感染复数（MOI）之间慢病毒载体添加至孔2 - 10轻轻地混合，并在37ºC孵育过夜。 注意:使用的慢病毒载体的效价应预先确定。 </li><li>通过用细胞刮刀轻轻刮孔的底部收集细胞，第二天早晨。收集细胞并用此时血球计数。 </li><li>为了制备细胞植入物，以每只小鼠，等分试样4.5×10 6个 CD34 -细胞结合0.5×10 6个转导的CD34 +细胞进入无菌1.5毫升螺丝帽试管中。旋转细胞下来300 XG沉淀其中，吸上清。在300 XG再次旋转它们，使用P10吸管吸任何剩余的上清液并且吸取非常CArefully。保持冰的干燥颗粒在整个研究。注:为了确保细胞是可行的，使用沉淀的细胞和2-3小时内进行手术。 </li><li>为了准备细胞注射，旋转0.5×10 6每只小鼠转CD34 +细胞沉淀其中，吸上清。重悬在每鼠标100微升RPMI媒体细胞保持在冰上。 </li><li>签转导效率，等分试样〜1×10 5非转导，并从在200μl细胞因子培养基（RPMI培养基含10％FBS的每个条件和培养转导的CD34 +细胞，补充有100纳克/ ml人IL-3，IL-6的，SCF）中进行5 96孔板 - 7天37ºC。 注意:在这个步骤中使用的细胞不用于手术，但可以确保转导是成功的，该载体是无毒性干细胞存活和更新。转导效率可以通过寻找该载体的基因表达进行检查（ 例如 ，绿色荧光蛋白与CD4）并通过流式细胞术分析。 </li></ol></li><li> 组织移植构建遗传工程小鼠 <ol><li>就在同一天手术前进行NOD.Cg- PRKDC SCID IL2RG牛逼m1Wjl / SZJ（NSG）免疫受损的小鼠与铯-137辐射器和2.7戈瑞（270Rad）的剂量全身照射。 注:核供应国集团小鼠是严重免疫。因此，他们的住房和维护必须符合最高标准的健康和训练有素的工作人员处理。 </li><li>从倒入烧瓶胸腺片和中成60毫米的菜。倾PBS到另一个60mm培养皿，将用于清洁套管针并保持肾湿。 </li><li>通过将它们放置在开1.5毫升无菌螺丝帽管上的冰浴容积枪头。保持与干燥的细胞沉淀和胶状蛋白质混合物如基质胶的冰。 注:保持胶状蛋白质混合物和任何管或提示是很重要的直到留置针装入将触摸它的冷在任何时候。 </li><li>麻醉小鼠:单独称量小鼠和记录的权重;耳冲小鼠数点。氯胺酮的15微升（2.6毫克/毫升在盐水中）/赛拉嗪每克体重（100毫克/盐水中毫升））腹膜内注射它们。把老鼠放回笼子里，等待它被完全麻醉。 注:通过挤压爪子检查鼠标的麻醉水平。如果鼠标本能退缩，辖氯胺酮/甲苯​​噻嗪的原始量的25-50％，进一步麻醉小鼠。等到它不退缩条件反射来进行手术。 </li><li>使用奥斯特削波器（剃须刀），从臀部剃每只小鼠的左侧的背部和腹部的中心之间承担。注入皮下0.3毫升稀释卡洛芬（6毫克/千克）到动物的肩膀或腹股沟三角（腿窝）。用棉签，把人工泪液一小滴到每只眼睛和侧放鼠标回到笼子里。 注:限制手术准备一个笼子 - 在同一时间（约4 5只）。 </li><li>冲洗16号癌植入针（套管针），用PBS的插管。使用一对钝弯钳，放置从60毫米的菜一块胸腺入套管与套管刚刚开口内开幕，再拉回来的套管针的组织吸进套管。 </li><li>使用正位移移液器和冷藏尖把5微升冷凝胶状蛋白混合物进入管干细胞团（CD34 + CD34-和混合物植入的细胞），并轻轻搅动产生细胞悬液。不要移液器上下。吸取胶状蛋白质混合物/细胞悬液到套管的开口和慢慢拉回来的套管针加载针。 注:建议有一个助手来加载吸管而一个操纵留置针。 </li> &lt;利&gt;拭鼠标的剃区域用聚维酮碘，随后的擦平与醇的区域擦拭三次。确定皮肤下的最黑暗的地方。这表明脾脏的位置。肾是约5mm背侧脾脏。抬起与弯钳皮肤并与平行于脾皮肤手术剪15毫米长的切口。然后作出类似的削减腹膜下方层。在男性中，肾应该很容易可见的，并且可以通过按压上腹部简单地挤出。可以支持用止血钳或一对弯曲钝钳的肾。在女性，卵巢往往会阻止从易提取肾脏。用止血钳，拿起卵巢并仔细暴露出来的肾脏。 </li><li>使用针尖镊子在肾囊的后端掐一个微小的孔。 注意:不要使用这些针尖镊子处理生物危害的材料。 </li><li>滑动植入针插入该孔并沿着肾脏直到套管的开口完全被肾囊所覆盖。 </li><li>轻轻挤压肾囊下的组织，并拔出针​​退了出去。胸腺片可粘所以用一个弯钳以确保胸腺片不与针出来。 </li><li>抬起用钳子腹膜，轻轻用止血钳推肾脏回原位。使用4-0薇乔可吸收缝线腹膜领带一张双人床打结针。使用两个Autoclips伤口剪辑关闭皮肤。 </li><li>混合被预留用于注射该被转导的CD34 +细胞和摄取100微升（0.5×6个细胞）进胰岛素注射器。注入这些细胞进入通过眶后静脉注射或静脉注射的其他途径的鼠标。用棉签，把人工泪液一小滴在每个眼睛，躺在鼠标在其一侧回来关在笼子里。 </li><li>一旦所有的小鼠都bEEN植入，确认该动物苏醒，离开他们面前走动正常。 </li><li>后操作方式:在手术后的第二天，皮下注入0.3毫升稀释卡洛芬（6毫克/千克）和1.2毫升无菌的生理盐水到每只小鼠。图2和3天后手术，皮下注入1.5毫升无菌盐水到每只小鼠。监测小鼠和手术后10-14天的切口。除去Autoclips和后10-14天称量小鼠。注:小鼠不畅辐射和生理盐水注射防止脱水变成动物后。 </li><li> 8后 - 10周放血小鼠和外周血进行FACS分析，染色标记如CD45，CD3，CD4，CD8和载体应表达任何基因检查植入。 </li></ol></li></ol> 2.分化的表征和基因修饰细胞的发育<ol><li> 表征从外周血基因修饰细胞<ol><li> 8 - 从眼眶出血100微升小鼠血 - 移植10周后，获得50。在含10微​​升EDTA的离心管的地方。离心细胞，在350×g离心3分钟。收集血浆。在-80用于ELISA分析或血浆病毒载量检测冻结如果鼠标是艾滋病感染者。 </li><li>加2ml 氯化铵 （83％）溶液以裂解红血细胞。孵育在室温（25℃）5分钟。孵育后，加入10 mL RPMI 10％FBS，以填补管。旋在300×g离心5分钟。小心吸上清。细胞准备用于免疫染色和流式细胞仪分析等测定。 注:关于各种造血细胞谱系，活化，以及用于幼稚或记忆细胞表型标志物标记可以每2周前和HIV感染通过流式细胞后进行测量。盖茨是通过染色细胞的同型对照创建。它建议以染色healtHY人PBMC和使用，作为阳性对照。或者，鼠标可以安乐死和至多1毫升外周血可以从心脏穿刺将用于流分析17的多个面板提供足够的细胞而获得。 </li></ol></li><li> 脾脏，胸腺，骨髓基因鉴定修饰细胞 <ol><li> 收获脾脏，胸腺和骨髓从人性化的BLT小鼠 <ol><li>通过使用异氟醚过量安乐死小鼠。确认继发颈椎脱位安乐死。喷用70％乙醇的胎体的表面，以保持皮毛粘到组织中。牵制在解剖蜡盘四肢。 </li><li>用手术剪，剖开皮肤，然后通过腹膜层切开，以暴露在体腔内。 </li><li>使用镊子取出脾脏。取出使用镊子和剪刀肾脏胸腺植入物。把植入胸腺和脾脏中标记的试管CONTA进不去5毫升的PBS。 </li><li>从中间腹部切割并从小鼠覆盖下肢的末端部分去除皮肤。 </li><li>切断由下肢肌肉用剪刀，小心地从脱位髋关节髋臼。取出股骨和胫骨，并将其放置于含有5毫升的PBS的管。 </li></ol></li><li> 脾细胞的分离 <ol><li>放置在50ml锥形管中的100微米的细胞滤网。湿用3ml RPMI培养基中补充有10％FBS和青霉素/链霉素（RPMI完全培养基）的过滤器。 </li><li>放置脾到细胞过滤。使用10毫升注射器中的橡胶柱塞，捣碎脾通过过滤器，以分离入50ml管中。 </li><li>冲洗用5毫升的RPMI完全培养基4至5倍的细胞过滤网。 </li><li>旋转细胞以300×g离心10分钟。 </li><li>吸上清，重悬沉淀在5ml红细胞裂解缓冲液。孵育在室温彩画TURE 10分钟。在300 xg离心加20毫升RPMI完全培养基和旋转7分钟。 </li><li>抽吸上清液，并在10毫升的RPMI完全培养基重悬沉淀，并通过100微米的细胞滤网再次进行过滤。使用血细胞计数器细胞。 注:细胞可用于体外测定法，流动分析，并且可以可行地冷冻供以后使用。细胞计数之前，台盼蓝可用于确定存活细胞的数量。 </li></ol></li><li> 从植入人体胸腺细胞的分离 <ol><li>放置胸腺到5毫升的PBS在6孔板的孔中。使用清洁钝钳和剪刀剪胸腺小块。 </li><li>将消毒的不锈钢网方入井。使用10毫升注射器中的橡胶柱塞，捣碎在不锈钢丝网胸腺件掰开胸腺。 </li><li>重悬细胞井和应变通过100微米的细胞滤网。旋转细胞以300×g离心10分钟。暂停在10毫升RPMI完全培养基中的细胞。如果团块可见，再次通过悬浮细胞通过细胞过滤。 </li><li>使用血细胞计数器细胞。 注:细胞可用于流分析和可行地冷冻供以后使用。 </li></ol></li><li> 从骨髓细胞的分离 <ol><li>清洁和消毒砂浆并用70％的乙醇杵。放置股骨和胫骨成砂浆。加入5毫升冷的PBS。使用杵粉碎的骨头，直到PBS变为淡粉色，多云。 </li><li>吸取从砂浆和过滤器中的流体通过放置在50ml锥形管中的50微米的细胞滤网。用5毫升PBS洗涤砂浆，重复五次，直到PBS变得清晰。 </li><li>旋转细胞向下在300×g离心7分钟。吸上清，重悬在10毫升的RPMI完整的媒体。 </li><li>使用血细胞计数器细胞。 注:细胞可用于体外测定法，流动分析，并且可以是可行地冷冻˚F或以后使用。 </li></ol></li></ol></li></ol> 3.基因修饰细胞功能研究<ol><li> 艾滋病毒感染和病毒复制随着时间的推移监控 <ol><li>人体免疫系统重建的确认后，通过使用胰岛素注射器眼眶静脉注射注入HIV病毒所需的量。 HIV感染后，经眼眶出血，每2周收集100微升外周血。 </li><li>收获血浆以下部分2.1.1步骤 - 2.1.2。对于HIV病毒负荷测定中，使用根据制造商的指导RNA提取试剂盒提取病毒RNA，并测量通过实时RT-PCR检测病毒载量与合适的引物和探针组用于4,8,18,19 HIV病毒。 </li><li>为了测量细胞相关的HIV RNA并识别正在积极受艾滋病毒感染的细胞，先执行以下步骤2.1.2 RBC裂解</li><li>在1悬浮细胞悬液;毫升的PBS，均匀地划分成两个管。旋在300×g离心5分钟。吸上清。 </li><li>为了测量细胞相关的RNA，提取使用RNA RNA提取试剂盒根据制造商的指令，并使用合适的引物和探针组进行实时RT-PCR法。 注:该引物/探针不能识别用于修饰干细胞的慢病毒是非常重要的</li><li>以测量的HIV流动感染细胞，悬浮细胞在另一管在50μlFACS缓冲液，并与如抗CD45，抗CD4和抗CD3所需抗体进行表面染色。表面染色后，修复和使用抗Gag的抗体（克隆KC57）通透细胞和细胞内染色的插科打诨的表达。执行流式细胞仪。 </li></ol></li><li> 基因体外细胞因子检测修饰细胞从脾 <ol><li>从小鼠脾细胞收获如第2.2.2所述。 </li><li>准备靶细胞。为了测试功能CD4嵌合抗原受体修饰的T细胞性，用HIV感染T1细胞作为靶细胞。感染的T1细胞与HI​​V前3天的细胞因子测定法中，通过用抗HIV的gag抗体（克隆KC57）细胞内染色细胞证实HIV感染。使用未感染细胞T1作为对照靶细胞。 </li><li> 1共孵育的目标或控制的脾细胞:1的比例过夜。对于最好的结果，进行滴定（1:1，1:3,1:9）的效应的（脾细胞）对靶细胞（感染的T1）。例如，重悬90万脾于0.25ml RPMI完整的媒体，加入悬浮于0.25ml RPMI完整的媒体0.9，0.3或0.1万受感染的T1或未受感染的T1线路。 </li><li>第二天早上，添加蛋白转运抑制剂6小时，以抑制蛋白质的运输和进行染色细胞外标记和如在8先前所述细胞因子的胞内表达。 </li></ol></li></ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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> Nature medicine. 12 (11), 1316-1322 (2006).</li><li>Shultz, L. D., Brehm, M. A., Garcia-Martinez, J. V., Greiner, D. L. <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+for+immune+system+investigation:+progress,+promise+and+challenges.">Humanized mice for immune system investigation: progress, promise and challenges.</a> Nature Reviews: Immunology. 12 (11), 786-798 (2012).</li><li>Vatakis, D. N., Bristol, G. 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The authors have nothing to disclose.

与汽车和走向临床研究基于HSC工程免疫力蓄势待发，它有一个合适的动物模型来仔细研究这些工程细胞的分化和功能是非常重要的。在这个协议中，我们描述的构建和测试与设计的抗HIV基因修饰的干细胞小鼠人性化的方法。有前移植干细胞的有效转导是重要的。然而，由于T细胞在识别靶细胞增殖的能力，干细胞修饰的低含量足以产生针对HIV复制8健壮响应。 然而，要?...

尽管联合抗逆转录病毒治疗的成功，HIV感染仍然是一个终身疾病。抗HIV细胞免疫反应在控制HIV复制非常重要的作用。在干细胞处理的最新进展已经允许基因治疗的飞速发展途径HIV治疗1-3。其结果是，它有一个适当的动物模型，它允许在抗HIV基于细胞的疗法的效力的体内研究是非常重要的。 在动物模型中与HIV的工作是由一个事实，即病毒只感染人类细胞复杂。为了规避这一限制，科学家们开始借助疾病模型像猕猴4,5猴免疫缺陷病毒（SIV）。不幸的是，在此模型中，由于跨物种的固有差异和SIV和HIV之间的差异主要限制。此外，只有高度专业化的设施是C支持工作，非人类灵长类动物，每个猕猴apable需要大量的投资。因此，为模型中的迫切需要，利用人的免疫系统，这是易受HIV感染/发病机制，并且是较少财政望而却步。 非肥胖糖尿病（NOD） -严重联合免疫缺陷（SCID）-commonγ链基因敲除（三γ - / - ）（或NSG）血液/肝脏/胸腺（BLT）的人源化小鼠模型日益被证明是一个重要的工具以研究艾滋病病毒感染。通过注入的造血干细胞（HSC）以及胎胸腺，小鼠能够开发和概括一个人免疫系统1-3。基于干细胞的基因治疗的一种类型涉及&#39;重定向&#39;的外周T细胞重新编程造血干细胞（HSC）能够分化为抗原特异性T细胞为目标的HIV。我们以前曾表明，工程造血干细胞与分子克隆抗HIV特异性T细胞再受体（TCR）对抗原SL9（氨基酸77-85; SLYNTVATL）HIV-1的Gag可以干细胞重定向到形成的抑制人性化的NSG-BLT小鼠模型6 HIV复制成熟T细胞。使用分子克隆的TCR的需要注意的是，它被限制为特定的人白细胞抗原（HLA）亚型，这将限制这种疗法的应用。嵌合抗原受体（CAR），另一方面，可普遍适用于所有的HLA亚型。进行最初的研究利用具有融合于细胞内ζ信令的CD3域人CD4的细胞外和跨膜结构域构成的汽车（称为CD4ζCAR）。 CD4ζCAR上的CD8 T细胞上表达能够识别HIV包膜并引发细胞毒性T细胞应答是类似于由T细胞受体7介导的。我们最近证明人肝星状细胞可以与CD4ζCAR，然后可以分化成多种造血里进行修改neages，包括能够抑制在人源化小鼠模型8 HIV复制的功能的T细胞。在嵌合抗原受体疗法的快速进步癌症9，和有效的宽的中和抗体10-12抗HIV，允许单链抗体车的结构的持续表征，它是可感知的，许多新的候选构建体，除了CD4ζCAR ，将产生与艾滋病毒和其他疾病的干细胞基于基因疗法试验。另外，含有这些抗原特异性的CARs人源化NSG-BLT小鼠模型也可提供一个有用的工具仔细检查体内人T细胞应答。重要的是，我们的协议不同于先前描述的结构的人源化的BLT小鼠13-15在于，造血干细胞在胶状蛋白质混合物代替胎肝中继线16的使用方法。本协议描述:1）humani建设与CD4ζCAR工程捷思BLT小鼠;和2）的遗传修饰的细胞的分化的表征;和3）的经遗传修饰的细胞的功能性表征。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CD34 microbead kit</td>
			<td>miltenyi</td>
			<td>130-046-702</td>
			<td>For sorting human CD34+ progenitor cells</td>
		</tr>
		<tr>
			<td>Bambanker</td>
			<td>Wako</td>
			<td>302-14681</td>
			<td>for freezing cells</td>
		</tr>
		<tr>
			<td>QIAamp Viral RNA kit&#160;</td>
			<td>Qiagen</td>
			<td>52904</td>
			<td>For measuring viral load in the serum</td>
		</tr>
		<tr>
			<td>MACSQuant Flow Cytometer</td>
			<td>Miltenyi</td>
			<td></td>
			<td>For flow analysis</td>
		</tr>
		<tr>
			<td>BD LSRFortessa&#8482;</td>
			<td>BD biosciences</td>
			<td></td>
			<td>For flow analysis</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H6254-500MG&#160;</td>
			<td>For tissue digestion</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Worthington</td>
			<td>LS002006&#160;</td>
			<td>for tissue digestion</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Life technology</td>
			<td>17104-019&#160;</td>
			<td>for tissue digestion</td>
		</tr>
		<tr>
			<td>CFX Real time PCR detection system</td>
			<td>Biorad</td>
			<td></td>
			<td>For measuring viral load and gene expression</td>
		</tr>
		<tr>
			<td>Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ</td>
			<td>The Jackson Laboratory</td>
			<td>5557</td>
			<td>For constructing the humanized mice</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (Pen Strep)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10378016</td>
			<td>For culturing cells</td>
		</tr>
		<tr>
			<td>piperacillin/tazobactam</td>
			<td>Pfizer</td>
			<td>Zosyn</td>
			<td>Anti-fungal</td>
		</tr>
		<tr>
			<td>Amphotericin B (Fungizone antimycotic)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15290-018</td>
			<td>Anti-fungal</td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clips, 9 mm - 1000 units&#160;&#160;&#160;&#160;</td>
			<td>Becton Dickinson</td>
			<td>427631&#160;</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Sterile Poly-Reinforced Aurora Surgical Gowns, 30 per case&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Medline</td>
			<td>DYNJP2707&#160;</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>sutures, 4-0, vicryl&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Owens and Minor</td>
			<td>23000J304H&#160;&#160;</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Alcohol prep pads&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Owens and Minor</td>
			<td>3583006818</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Gloves, surgical, 6 1/2</td>
			<td>Owens and Minor</td>
			<td>4075711102</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Yssel&#8217;s Serum-Free T-Cell Medium</td>
			<td>Gemini Bio-products</td>
			<td>400-102</td>
			<td>For CD34+ cell transduction</td>
		</tr>
		<tr>
			<td>Human Serum Albumin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A9511</td>
			<td>For CD34+ cell transduction</td>
		</tr>
	</tbody>
</table>

stem-cell based engineered immunity against hiv infection in the humanized mouse model

与基于细胞干基因疗法的快速发展对抗HIV，有迫切要求的用于动物模型来研究所述遗传修饰的细胞的造血细胞分化和免疫功能。人源化的骨髓/肝脏/胸腺（BLT）小鼠模型允许在周围的人的免疫系统，其包括T细胞，B细胞，NK细胞和单核细胞的完整的重建。人胸腺植入物还允许在自体胸腺组织的T细胞的胸腺选择。除了感染艾滋病毒的研究中，模型矗立作为一个强大的工具来研究造血干细胞（HSCs）的细胞的分化，发育和功能。在这里，我们勾勒出人性化的非肥胖型糖尿病（NOD）的建设-严重联合免疫缺陷病（SCID）中的常见γ链基因敲除（Cγ - / - ）-Bone骨髓/肝脏/胸腺（NSG-BLT）小鼠造血干细胞转导与CD4嵌合抗原受体（CD4CAR）慢病毒载体。我们表明，CD4CAR造血干细胞可以成功地分化成多种谱系，并有抗HIV活性。该研究的目的是说明使用NSG-BLT小鼠模型的作为抗HIV工程化免疫的体内模型。值得注意的是，因为慢病毒和人体组织时，实验和手术应该在II级生物安全柜与特殊的预防措施（BSL2 +）设施执行在生物安全等级2（BSL2）。

图1显示了构建具有修饰的干细胞源化的BLT小鼠的轮廓。植入手术10周后，处死小鼠，以评估基因修饰细胞的分化和发育。 如图2中所示，多个淋巴组织（血液，脾，胸腺和骨髓）中从使用CD4ζCAR改性的小鼠收获。在这个协议中使用的CD4ζCAR包含可由抗CD4抗体和GFP 8的表达进行检测的CD4嵌合抗原受体和GFP。细胞分离并用抗人CD45抗体，以及抗CD4...

Watch this Scientific Journal Video about Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model at JoVE.com

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

干细胞基工程免疫抗HIV感染的人性化小鼠模型

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

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JoVE Journal

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10.1K 次观看.  University of California, Los Angeles. This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection. 

视频: Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

主动脉原位移植大鼠模型，研究慢性血管病变的发展

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

科研

医学

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

表皮黑色素和防止晒斑的人性化小鼠模型的诱导药物

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

在脊髓损伤的实验挫伤型号神经干细胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

外科手术切除为鼠标肋骨:模型进行大规模的长骨修复

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

静脉介入型号:研究旁路移植通畅一个合适的模型

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

球囊损伤诱发小鼠腹主动脉 Myointimal 增生的研究

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...