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是健壮病毒生产者确定病毒滴度。此外，从所收获的骨髓中去除红血球的提高转导效率。确保骨髓祖细胞的健康是协议的成功是至关重要的。为此，总是用新鲜的培养基（少于4周4℃补充有FCS之后）和新鲜的细胞因子（以下超过2周时在4℃保存）。聚凝胺也是降解敏感，应使新鲜，一旦它是在溶液中，每2个月。其他细节，以确保最佳的骨髓健康和传导包括预暖旋转前的离心机。在37℃旋转转导，敷板用保鲜膜，以减少气体交换尽可能。另外，不要让骨髓坐在37℃培养箱外或冰上长时间，因为这会增加细胞死亡和减少骨髓转导和重建。骨髓细胞已在第4天（第13步）收获之后，只有立即注射小鼠之前装入注射器。最后，确保小鼠置于上抗生素日照射的前一天或。 此逆转录病毒介导的转导协议的利用允许导致骨髓祖细胞的转导生成T细胞受体retrogenic小鼠。 Retrogenic小鼠更快地产生比TCR转基因小鼠和成本比生成单个TCR转基因相当少。然而，retrogenic小鼠不能繁殖，并与每个实验10产生必须重新。外周T细胞的数目是在retrogenic小鼠低于TCR转基因小鼠中观察到，不过TCR的调节是TCR retrogenic和TCR转基因10之间可比。 TCR的retrogenic系统允许多个的TCR的相同群体内，甚至在同一实验中的表达。利用这里描述的TCR retrogenic协议允许一个研究者来测试和筛选众多新近确定的MHC I或II类限制性TCR的发展和功能。用"人源化小鼠"模型18,19的发展，所述TCR retrogenic可以进一步利用克隆的人TCR的扩大。此外，2A链接结构Ç一个也可用于研究其他多蛋白，包括CD3链，白细胞介素和嵌合抗原受体20-24。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM, high glucose + glutamine</td>
			<td>Corning Cellgro</td>
			<td>10-013-CV</td>
			<td>Dulbecco&#39;s Modification of Eagle&#39;s Medium with 4.5 g/L glucose, L-glutamine &#38; 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 um 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 dH2O</td>
		</tr>
		<tr>
			<td>Ciprofloxacin&#160;</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 1M 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 um 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>&#160;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>27G x 1/2 BD Precision Glide Needle</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>30G 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2B所有TCR阳性细胞一致地表达了紫黄晶荧光蛋白。 <img alt="图1" src="/files/ftp_upload/54196/54196fig1.jpg" /> Figu再1. 骨髓转导和外设TCR Retrogenic性T细胞的例子。A）转导骨髓细胞的效率进行分析的基础上对骨髓移植日荧光标记物（紫黄晶）。收获的骨髓的约10微升加入到PBS 100微升和紫黄晶表达进行分析。一个成功的骨髓转导将产生25％和70％。B）的外周血后6周的骨髓重建的TCR retrogenic T细胞的实施例之间的荧光阳性细胞的百分比。获得的外周血和红血细胞溶解用RBC裂解缓冲液中。剩余的细胞进行染色TCRβ。代表分析紫黄晶表达和反TCRβ染色后6周的骨髓重建。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54196/54196fig1large.jpg" target="_blank">请点击此处查看T的放大版本。</a>他的身影。

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

Nanyang Technological University

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

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 ...

科研

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 ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

鼠后肢长骨解剖和骨髓隔离

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

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, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

使用胶原酶E从毛里结肠分离拉米纳普里亚单核细胞

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

骨髓移植平台研究树突状细胞在移植与宿主疾病中的作用

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...