这项工作是由赠款美国国立卫生研究院（5K22A1119151-01和1R56DK104903-01），以美国职棒大联盟，糖尿病研究中心（P30-DK079638）在BCM的试点/可行性方案，JDRF 1-FAC-2014-243-AN APF，ADA支持1-15-JF-07，免疫学奖学金到MB，罗伯特和Janice捷基金会AAI招聘。

伦理学声明:尽一切努力在辐射和尾静脉注射到动物的不适或压力保持在最低水平。小鼠被用作这些实验中的细胞源;因此不存在程序或操作除了安乐死。小鼠将CO 2吸入颈椎脱位确认死亡安乐死。这个过程是在美国兽医协会的安乐死小组的建议相一致。 1.准备逆转录病毒构建<ol><li>子克隆的T细胞受体（TCR）的α和β链，由一个2A接头分开，到基于MSCV逆转录病毒11载体（例如，pMIAII或pMIGII）11。 注:2A链接允许从单个开放阅读框的α和βTCR链的化学计量和一致表达。该载体包括用于将IRES荧光蛋白盒（紫黄或绿色荧光蛋白）跟踪转导的细胞和转染效率。对于一个详细的协议，请参阅霍尔斯特等。11 </li><li>使用商购获得的血浆prep试剂盒或类似的产品分离质粒DNA。使用1工作浓度- 2微克微升-1。 </li></ol> 2.代逆转录病毒生产细胞系的<ol><li>利用两步过程，以产生反转录病毒生产细胞系。 注:对于一个详细的协议，请查看霍尔斯特等[11]。 <ol><li>通过生成293T细胞有三个质粒（pVSVG，PEQ-PAM3（-E）和利息11向量）的瞬时转染逆转录病毒，并采取逆转录病毒上清从293T细胞转导的GP + E86嗜性逆转录病毒生产者细胞系。 </li><li>通过FACS分离荧光表达GP + E86生产细胞的顶端40％和传播transdcuced-GP + E86生产细胞作为病毒的来源。 注:代的高滴度生产为骨髓细胞的有效转导的关键。对于一个完整的协议，请查看霍尔斯特等。11 </li></ol></li></ol> 3.逆转录病毒介导的干细胞基因转移（天-5） <ol><li>解冻并在完全DMEM各GP + E86生产者细胞系的培养0.5 -1.0×10 6 10％（体积/体积）胎牛血清，以允许两个汇合150毫米板足够的生长通过该协议的第0天。 注:解冻0.5 -在10cm平板各GP + E86生产者细胞系的1.0×10 6将允许30 - 50％汇合在5天这还允许培养5天，以扩大在GP + E86生产者细胞系两个融合150毫米盘由0天。 <ol><li>在含有5％羊小牛血清，和环丙沙星（10微克/毫升），以避免支原体污染的完整组织培养基培养反转录病毒生产细胞系。 注意:当generatin停止使用环丙沙星克骨髓转导的病毒上清。避免生产细胞系的过度增长，因为这会产生负面影响骨髓的转导效率。 </li></ol></li></ol> 4.注入供体小鼠与5-氟尿嘧啶（5-FU）（-3天） <ol><li>称量供体小鼠（5 - 19周龄），以确定的5-FU所需的量。利用老鼠超过5周低骨髓产量的年龄结果年轻。为了确保单个TCR表达，使用T细胞缺陷的小鼠品系，如SCID，抹布或TCR阿尔法缺陷小鼠的骨髓捐赠者。 </li><li>在组织培养罩工作，淡化的5-FU的库存用无菌PBS以使足够量的10毫克毫升-1的5-FU工作溶液每克体重的递送0.15毫克的5-FU。 </li><li>用1ml注射器用37克针，腹膜内注射每克供体小鼠体重0.15毫克的5-FU。 注:每一个收件人使用1捐助鼠标启动和调整根据细胞产量的供体小鼠的数目。的5-FU处理的骨髓祖细胞的富集和分离的另一种方法是如在Viret 等人所述谱系阳性细胞的阴性选择。 </li></ol> 5.骨髓提取程序（第0天） <ol><li>获得清扫剪刀和镊子骨隔离。制备培养基（HBSS补充了5％FCS（体积/体积））收集在50ml锥形管中的骨头。保持含冰介质50毫升锥形。通过安乐死CO 2小鼠吸入颈椎脱位确认死亡。捏爪子，以确保没有检测到反射。 </li><li>消毒手术部位，并用乙醇或甲醇手术工具。首先切割和骨盆带下小心地取出股骨，胫骨，肱骨，和骨盆带。切割沿股骨胫骨回落。删除使用剪刀和镊子获得干净的尸骨周围的组织。保持骨头在HBSS + 5％（体积/体积）FBS的水合。 </li><li>通过在关节处切割，确保两头切掉每块骨骼分离的骨头。插入连接到在含HBSS + 5％（体积/体积）10毫升注射器FBS的25号针头。施加压力，并通过在50毫升的锥形70微米的过滤休息刷新所有的骨髓。 </li><li>周期性地，通过使用柱塞从1毫升或3毫升注射器的70微米的过滤器推骨髓。冲洗用HBSS + 5％（体积/体积）FBS的过滤。这将确保单细胞悬浮液，是免费的骨碎片和组织。保持细胞在无菌罩执行该过程无菌的。 </li><li>离心机的骨髓细胞在300×g离心在4℃下10分钟。 </li></ol> 6.骨髓培养（第0天） <ol><li>倒出或抽吸的介质，并在每50毫升的锥形管1毫升氯化铵根据红细胞裂解液（RBC或裂解液当量）悬浮细胞沉淀。后1分钟温育在室温温度，通过加入10ml完全DMEM的20％（体积/体积）FBS的骤冷红细胞裂解液。 </li><li>离心细胞，在300×g离心在4℃下10分钟。 </li><li>滗析或在5ml完全DMEM 20％（体积/体积）FBS的抽吸上清，重悬的骨髓。算用Neubauer计数室中的细胞。 注意:一般屈服应为约5 - 10％小鼠×10 6细胞;然而，收率小鼠不同菌株而异。 </li><li>在30ml完全DMEM的20％（体积/体积）FBS补充有IL-3的板的骨髓细胞以每150毫米板4×10 7个细胞的密度（20克毫升-1），IL-6（50克·毫升-1）和SCF（50克毫升-1）。 注:使用刚解冻分装细胞因子。不使用已经为2周以上冷冻和解冻一次以上或保持在4℃的细胞因子。 </li><li>在37°C孵育骨髓过夜（约24小时），用5％ 的 CO 2。 </li></ol><ol><li>板3×10每150毫米板6的逆转录病毒生产细胞的18ml完全DMEM的20％（体积/体积）FBS。 </li><li>孵育逆转录病毒生产细胞在37℃下过夜。预计1500万的骨髓细胞中一种150毫米板（约2 - 3供体小鼠）。 </li></ol> 8.逆转录病毒上清液（1天） <ol><li>第二天（约24小时后），通过与用0.45 10毫升注射器和过滤器直接绘制了逆转录病毒上清液关150毫米板的收获从生产者板（在步骤7中建立）的逆转录病毒上清液-μm注射器过滤。 </li><li>小心以18毫升新鲜完全DMEM 20％（体积/体积）胎牛血清代替除去的逆转录病毒上清液。 </li><li>补充有IL-3的经过滤的逆转录病毒上清液（20克毫升-1），IL-6（50克毫升-1），MSCF（50克毫升-1），聚凝胺（聚凝胺）（6＆＃956;克·毫升-1）。 注:聚凝胺要新鲜制成每2个月，以避免逆转录病毒转导的疗效下降。 </li></ol> 9.骨髓收获（1天） <ol><li>完成步骤8.3之后，收获来自步骤6.4的骨髓细胞和包含非粘附细胞介质转移到无菌的50毫升管中。 </li><li>用10毫升的PBS洗涤大力板和步骤9.1在50毫升锥形收集。如果有必要，重复清洗步骤。 </li><li>离心将细胞在300×g离心在4℃下10分钟，倒出上清液，在小体积重悬细胞（1 - 3毫升）完全DMEM 20％（体积/体积）FBS和计数。预期50 -从第0天悬浮骨髓以每1毫升含细胞因子和聚凝胺的反转录病毒上清液的1×10 6个细胞的75％的恢复。 </li></ol> 10.骨髓转导（1天） <ol><li>板逆转录病毒上清液/马骨以每井3毫升6孔组织培养板中的体积rrow混合物。裹保鲜膜板，以尽量减少在步骤10.2离心过程中的气体交换。 </li><li>旋缠绕6孔板在1000 xg离心60分钟37℃。 </li><li>自旋完成后，取出保鲜膜，并放置在板在37℃ 的 CO 2培养箱中24小时。 </li></ol> 11.逆转录病毒上清液和转导（第2天） <ol><li> 24小时后，收集病毒生产者细胞系新鲜病毒上清。使用10毫升注射器和0.45微米的注射器过滤器过滤该反转录病毒上清液。补充有IL-3的经过滤的逆转录病毒上清液（20克毫升-1），IL-6（50克毫升-1）和SCF（50克毫升-1），聚凝胺（6克毫升-1）。 </li><li>接着，小心地用移液管除去或从6孔板的每个孔中含有的骨髓细胞吸出前2毫升媒体。 注:工作小心不要吸出骨髓，这通常是集中在井的中间。可替代地，除去上清液可纺向下收回任何丢失的骨髓细胞。 </li><li>向每个孔中的骨髓6孔板加入3毫升新鲜病毒含有上清液的细胞因子和聚凝胺的。包裹在塑料板，并重复旋转以1000 xg离心60分钟37℃。解开的板，并在37℃和5％CO 2的过夜（约24小时）孵育细胞。 </li></ol> 12.加入新鲜DMEM加味（3日） <ol><li> 24小时后，从含有骨髓细胞，在6孔板的各孔中除去前2毫升媒体如步骤11.2。用新鲜的DMEM的20％（体积/体积）FBS补充有IL-3的最终浓度替换除去的介质（20克毫升-1），IL-6（50克毫升-1）和SCF（50克毫升-1） 。 </li><li>孵育骨髓在37℃ 的 CO 2培养箱中另外24小时平板。 </li></ol> 13.收获转导骨髓（4日） <ol><li> 24小时后，收集来自6孔板的骨髓细胞。用PBS大力洗涤板以去除所有非贴壁细胞。 <ol><li>离心细胞，在300×g离心10分钟，在4℃，倒出上清液。 </li><li>重悬在PBS + 0.5％（体积/体积）的小体积（1ml）中的骨髓热灭活的FBS。保持细胞在冰上。 </li></ol></li><li>以每骨髓条件的10微升样品和计数使用Neubauer计数板细胞。假设1 -每个收件人2捐助，产量将足以传输每个收件人鼠标至少4×10 6个细胞。悬浮骨髓中的PBS + 0.5％（体积/体积）的热灭活FBS足够体积注入200 - 每只小鼠300微升。 <ol><li>注入至少2×10 6个细胞与转导效率的25％（可以注入多达8×10 6个细胞）每只小鼠静脉注射。 注:每骨髓细胞的两个6孔平板我们经常注入5受体小鼠。如果产率是相当低，应使用更供体小鼠直到能得到足够的骨髓数目。为了达到最佳的重组，至少5×10 5个转导（荧光阳性）应注入每个收件人。 </li></ol></li><li>用微吸移管，采取的每骨髓条件10微升样品和流式细胞术基于所述荧光标记物（ 图1A）11的表达通过流式细胞分析转导效率。 注意:通常情况下，荧光阳性的细胞的百分比是25％和70％之间，这取决于构建体和逆转录病毒滴度。转导的骨髓细胞的必要数量以重构一个子致死量照射到鼠标可以根据转导效率而变化。该TRAN较低sduction效率，需要越多的骨髓细胞，相反，骨髓传导效率越高，所需用于重建细胞的数量较少。低于25％的转导效率是不理想的，并且可能导致重建和存活不足。为了确保最佳的骨髓恢复和转导效率，使用新鲜制备的组织培养基和细胞因子。当表达新的TCR利用retrogenic系统中，T细胞受体在体内的选择是未知的。根据每个人TCR亲和力，TCR选择在胸腺和外周T细胞表达会有所不同17。 </li></ol> 14.老鼠照射，骨髓注入和护理<ol><li>照射小鼠骨髓注射前一天（当天为第13步）。使用以下剂量:C57BL / 6小鼠（以及其它野生型株），900拉德; RAG1 - / -小鼠，500拉德; SCID小鼠，300拉德）。 <ol><li>阿莫西林地方受体小鼠（50毫克/公斤/天）或辐照前另一种抗生素水处理，并保持对抗生素骨髓注射后的头两个星期。 注:最优辐射剂量可能不得不凭经验确定。 </li></ol></li><li>对于注射，加载来自步骤13.1细胞成使用钝针立即注射前，则针改变到27号注射，驱逐任何空气，以避免空气栓塞1-ml注射器。加热灯下热老鼠，注入0.2毫升每只小鼠的骨髓的静脉注射。 注意:不要让细胞坐在注射器任何时间长度或细胞将定居。 </li><li>监测小鼠每周，以确保它们保持健康。 </li><li>后5 - 6周，流血小鼠检查重构的GFP + /紫黄晶+和TCR共表达（ 图1B）11的基础上。 </li></ol>

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L., 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=Analysis+of+the+aphthovirus+2A/2B+polyprotein+'cleavage'+mechanism+indicates+not+a+proteolytic+reaction,+but+a+novel+translational+effect:+a+putative+ribosomal+'skip'.">Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'.</a> J Gen Virol. 82, 1013-1025 (2001).</li><li>Atkins, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+case+for+"StopGo":+reprogramming+translation+to+augment+codon+meaning+of+GGN+by+promoting+unconventional+termination+(Stop)+after+addition+of+glycine+and+then+allowing+continued+translation+(Go).">A case for "StopGo": reprogramming translation to augment codon meaning of GGN by promoting unconventional termination (Stop) after addition of glycine and then allowing continued translation (Go).</a> RNA. 13, 803-810 (2007).</li><li>Doronina, V. A., 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=Site-specific+release+of+nascent+chains+from+ribosomes+at+a+sense+codon.">Site-specific release of nascent chains from ribosomes at a sense codon.</a> Mol Cell Biol. 28, 4227-4239 (2008).</li><li>Bettini, M., 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=TCR+affinity+and+tolerance+mechanisms+converge+to+shape+T+cell+diabetogenic+potential.">TCR affinity and tolerance mechanisms converge to shape T cell diabetogenic potential.</a> Journal of immunology. 193, 571-579 (2014).</li><li>Brehm, M. A., Wiles, M. V., Greiner, D. L., Shultz, L. D. <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+improved+humanized+mouse+models+for+human+infectious+diseases.">Generation of improved humanized mouse models for human infectious diseases.</a> J Immunol Methods. 410, 3-17 (2014).</li><li>Brehm, M. A., Shultz, L. D., 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+mouse+models+to+study+human+diseases.">Humanized mouse models to study human diseases.</a> Curr Opin Endocrinol Diabetes Obes. 17, 120-125 (2010).</li><li>Chaplin, P. J., 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=Production+of+interleukin-12+as+a+self-processing+2A+polypeptide.">Production of interleukin-12 as a self-processing 2A polypeptide.</a> J Interferon Cytokine Res. 19, 235-241 (1999).</li><li>Collison, L. W., 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=The+inhibitory+cytokine+IL-35+contributes+to+regulatory+T-cell+function.">The inhibitory cytokine IL-35 contributes to regulatory T-cell function.</a> Nature. 450, 566-569 (2007).</li><li>Holst, J., 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=Scalable+signaling+mediated+by+T+cell+antigen+receptor-CD3+ITAMs+ensures+effective+negative+selection+and+prevents+autoimmunity.">Scalable signaling mediated by T cell antigen receptor-CD3 ITAMs ensures effective negative selection and prevents autoimmunity.</a> Nature immunology. 9, 658-666 (2008).</li><li>Kalos, M., 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=T+cells+with+chimeric+antigen+receptors+have+potent+antitumor+effects+and+can+establish+memory+in+patients+with+advanced+leukemia.">T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.</a> Sci Transl Med. 3, 95ra73 (2011).</li><li>VanSeggelen, H., 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=T+Cells+Engineered+With+Chimeric+Antigen+Receptors+Targeting+NKG2D+Ligands+Display+Lethal+Toxicity+in+Mice.">T Cells Engineered With Chimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice.</a> Mol Ther. 23, 1600-1610 (2015).</li></ol>

没有利益冲突的声明。

在协议中，我们详细介绍几个关键步骤，以确保最佳的骨髓健康，传递效率和重建。第一个关键步骤是产生和GP + E86病毒生产细胞的适当的维修。使用早期传代生产细胞系，并在80％汇合或使用较低前保持。当制作新鲜的GP + E86病毒生产细胞，保证了293T细胞是早期的通道，并在培养24成长 - 48小时。在转步电镀太多的GP + E86细胞会降低病毒滴度。如在霍尔斯等 11中所述，以确保GP + E86?...

人类和小鼠的T细胞受体（TCR）剧目估计为1×10 8 2×10 6个独特的TCR分别1,2。这个大多样性使T细胞能够识别来自自身肽，以及从由抗原呈递细胞（APC）的主要组织相容性复合体（MHC）颁发的病原体衍生的抗原表位的繁多。在这个独特的肽MHC复合物的TCR的相互作用的细微差别决定T细胞是否发生凋亡，无能，活化，分化，细胞因子的产生和细胞毒性。但是，由于大的T细胞受体库的一个特异性TCR将如何对特定抗原作出反应的分析需要使用单一的TCR系统。 各种TCR转基因小鼠已为了研究单一的TCR的功能在体内模型3-9生成的。然而，有警告到TCR转基因小鼠，包括成本，时间，以产生一个单一的转基因小鼠和随机转基因插入的所谓的创始人作用到种系的DNA 10的长度。因此，已经产生相对少的TCR转基因小鼠为任何给定抗原和高和低TCR的亲和力为相同的表位的功能影响很少讨论。为了满足迅速的方法来屏的需要，单独地或组合研究多种的TCR，（来自逆转录病毒的"逆向"和从转基因"基因"）retrogenic小鼠已被利用作为替代TCR转基因小鼠的11-13。 内几种病毒中发现的2A肽共有基序包含一个2A-ASP-缬氨酸/异亮氨酸 - 戊二醛-X-ASN-Pro的基-Gly-2B-Pro的，其中的2A的甘氨酸和2B的脯氨酸之间发生裂解从顺式短效水解酶活性，导致核糖体跳绳翻译10,14-16中。有关详细的图描绘的C各种2A肽（F2A，E2A，T2A和P2A）的leavage请参阅参考文献10 - 12以这种方式，2顺反子（TCR的α和TCR测试版）可以链接导致单个载体化学计量的翻译。利用这种方法，我们都能够表达和体内直接比较多个抗原特异性的TCR。

<table><tbody><tr><td>DMEM, high glucose + glutamine</td><td>Corning Cellgro</td><td>10-013-CV</td><td>Dulbecco&#039;s Modification of Eagle&#039;s Medium with 4.5 g/L glucose, L-glutamine &amp;amp; sodium pyruvate</td></tr><tr><td>FBS</td><td>Atlanta Biological</td><td>S11550</td><td></td></tr><tr><td>Trypsin-Versene</td><td>Lonza</td><td>17-161F</td><td></td></tr><tr><td>0.45 &amp;#181;m syringe filter</td><td>Thermo Scientific</td><td>194-2545</td><td></td></tr><tr><td>polybrene</td><td>Sigma</td><td>H9268-10G</td><td>Sterile Filtered in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Ciprofloxacin&amp;nbsp;</td><td>VWR</td><td>AAJ61970-06</td><td></td></tr><tr><td>5-fluorouracil (5-FU)</td><td>VWR</td><td>AAA13456-06</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Corning Cellgro</td><td>25-000-CI</td><td></td></tr><tr><td>MEM nonessential Amino Acids</td><td>Corning Cellgro</td><td>25-025-CI</td><td></td></tr><tr><td>HEPES 1 M solution</td><td>Corning Cellgro</td><td>25-060-CI</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Gibco by Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Pen/Strept</td><td>Corning Cellgro</td><td>30-002-CI</td><td></td></tr><tr><td>L-glutamine</td><td>Corning Cellgro</td><td>25-005-CI</td><td></td></tr><tr><td>150 mm tissue culture dishes</td><td>Greiner Bio-one</td><td>639160</td><td></td></tr><tr><td>Tisue culture-treated 6-well flat plate</td><td>Greiner Bio-one</td><td>657160</td><td></td></tr><tr><td>70 &amp;#181;m nylon cell strainers</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>Mouse IL-3</td><td>Invitrogen</td><td>PMC0033</td><td></td></tr><tr><td>Human IL-6</td><td>Invitrogen</td><td>&amp;nbsp;PHC0063</td><td></td></tr><tr><td>Mouse Stem Cell Factor</td><td>Invitrogen</td><td>PMC2113L</td><td></td></tr><tr><td>10x PBS</td><td>Corning Cellgro</td><td>46-D13-CM</td><td></td></tr><tr><td>HANKS Buffer</td><td>Corning Cellgro</td><td>21020147</td><td></td></tr><tr><td>BD 10 ml Syringe</td><td>BD</td><td>300912</td><td></td></tr><tr><td>BD 1 ml Syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>27 G x 1/2 BD Precision Glide Needle</td><td>BD</td><td>305109</td><td></td></tr><tr><td>30 G x 1/2 BD Precision Glide Needle</td><td>BD</td><td>305106</td><td></td></tr></tbody></table>

retroviral transduction of bone marrow progenitor cells to generate t-cell receptor retrogenic mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

骨髓转导效率在协议的步骤13.3检查骨髓注入尾静脉静脉之前以代表骨髓转导的图（ 图1A），收获的骨髓的约10微升加至100微升PBS和紫黄晶表达分析。通常荧光阳性的细胞的百分比是25％和70％之间，这取决于构建体和逆转录病毒滴度。 6周骨髓注射后，将小鼠放血，以确定骨髓重建（ 图1B）。大约外周血25微升红细胞裂解并用抗TCRβ染色。在图...

Watch this Scientific Journal Video about Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice at JoVE.com

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

retroviral-transduction-bone-marrow-progenitor-cells-to-generate-t

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

9.5K 次观看.  Baylor College of Medicine. We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

视频: Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

骨髓增殖性疾病的转导移植小鼠模型

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

科研

癌症研究

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

与预先定义的T细胞受体特异性Transnuclear小鼠对弓形虫获得通过体细胞核移植

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

免疫与感染

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

重组逆转录病毒的生产和B细胞的感染

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

瞬时转染293T细胞复制缺陷型逆转录病毒生产

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

生物学

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

发电<em&gt;德诺</em&gt;抗原特异性人T细胞受体通过中心的Hemichain的逆转录病毒转导

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

The current protocol combines single cell paired human TCR alpha and beta chain sequencing with streamlined generation of retroviral vectors compatible with in vitro and in vivo TCR expression.

简化的单细胞 TCR 分离和逆转录病毒载体的生成, 用于体外和体内表达人类 TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

CXCR4在Murine B-1a细胞上的逆转录病毒过度表达和靶向B-1a细胞向骨髓和IgM生产转移的采用转移

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

小鼠胚胎的逆转录病毒感染干细胞衍生胚体细胞造血分化的分析

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...