This work is supported by the V Foundation Scholar (AGF) and the MPN Research Foundation (AGF).

这项研究获得批准，按照机构动物护理和使用委员会在加州大学尔湾分校的建议进行的。异氟烷麻醉下进行所有的程序并作出一切努力，尽量减少痛苦。 1.代亲嗜性逆转录病毒<ol><li>制备高品质的质粒与使用一个商用马克西prep试剂盒或氯化铯纯化的至少1微克/微升的浓度。 注意:这些包括编码感兴趣的基因（在这个情况下，CALR 7,8）的MSCV骨架载体和选择（绿色荧光蛋白，新等 ）的标志物，以及亲嗜性包装质粒，编码的gag-POL-ENV（简称这里质粒）。 </li><li>补充有10％FBS和L-谷氨酰胺青霉素/链霉素（D10）在DMEM解冻和扩大低通道293T细胞。板5×10 6个细胞在10cm的组织培养处理过的皿中加湿在37℃组织培养孵化和5％的CO 2。 </li><li>扩大293T细胞中培养。转染前一天，通过加入1ml PBS中并吸出上清液到真空烧瓶中洗涤细胞。 </li><li>在37℃3分钟和5％的CO 2 -向trypsinize细胞，在2的潮湿组织培养箱加2ml的0.05％胰蛋白酶和孵化的菜肴。 </li><li>要停止胰蛋白酶，加3毫升D10抛出和转移细胞到15毫升锥形管。旋转细胞以400×g离心10分钟。 </li><li>吸上清到2毫升D10保温瓶和悬浮细胞沉淀。 </li><li>算使用血球通过台盼蓝排除细胞。 </li><li>种子2×10 6细胞每皿成每病毒产生的3个10厘米的组织培养处理的菜肴。 注 :为了达到最佳效果，不要让这些细胞超过30 -转染，也可以得到一个贫穷的病毒产量的50％的时间汇合。 </li><li>转染前随即，抽吸介质放入保温瓶和替换用5毫升新鲜的D10。 </li><li>对于每一个10cm培养皿待转染制备个别无菌1.5毫升离心管中。 </li><li>吸移管760微升减少血清培养基到每个微量离心管中，然后小心地添加40微升转染试剂的直接进入介质。孵育在组织培养罩管在室温下5分钟。 </li><li>添加MSCV向量的30微克与感兴趣基因（15微克如果使用空载体），以及5微克质粒各相应管，并通过上下抽吸轻轻混匀。孵育在组织培养罩室温进行15分钟。 注意:较小的载体往往有较高的病毒产量。 </li><li>小心添加每个反应（约800微升）的总容积逐滴成其各自的10cm培养皿，轻轻地左右倾斜菜一边混合。返回细胞加湿组织立方米lture孵化器。 </li><li>继在组织培养孵化器的8小时或过夜培养，吸媒体进入一个真空瓶中，轻轻替换用10毫升新鲜D10的。 </li><li>在48小时后转上清收集到35毫升注射器，并通过0.45微米的滤膜过滤，除去细胞碎片。 </li><li>可选:将10mL的293T细胞和其他病毒的新鲜D10背面可在72小时转染后收获。 </li><li>收集没有额外的病毒时在生物危险废物293T细胞的处理。 </li><li>分装病毒上清成一次性使用的卷（例1毫升病毒滴度或6.5毫升BM感染）。闪光冷冻病毒与在-80℃的液氮和存储病毒。 注:避免重复冷冻/解冻循环，因为它们大大降低病毒滴度。 </li></ol> 2.通过流式细胞仪确定相对病毒滴度<ol><li>解冻3T3细胞并维持在D10。培养中的细胞在10cm组织培养，在37℃处理盘在湿润的组织培养箱和5％的CO 2。 </li><li>一式三份进行病毒滴度。为了制备细胞，通过加入1ml PBS中并吸出上清液到真空烧瓶中洗涤细胞。 </li><li>到trypsinize细胞，在37℃下在湿润的组织培养箱中加2ml的0.05％胰蛋白酶和孵化菜和5％ 的 CO 2。 </li><li>要停止胰蛋白酶，加3毫升D10抛出和转移细胞到15毫升锥形管。旋转细胞以400×g离心10分钟。 </li><li>吸上清到2毫升D10保温瓶和悬浮细胞沉淀。 </li><li>算使用血球通过台盼蓝排除细胞。 </li><li>种子3T3细胞以密度1×10 5个细胞的成15 60mm组织培养处理过的菜和孵育过夜。 </li><li>使1:3，1:10，1:30和1:在总共1毫升用D10解冻病毒上清100稀释液。还包括免费的病毒控制。 </li><li>吸我直径从3T3细胞到真空烧瓶中并用4ml D10的替换。 </li><li>叠加3T3细胞用病毒上清液的稀释液，并添加聚凝胺至8微克/毫升至每个培养皿的最终浓度。孵育在37℃在组织培养培养箱48小时和5％的CO 2。 </li><li>确定通过流式细胞仪相对病毒滴度: <ol><li>为了洗涤细胞，吸媒体进入一个真空瓶中，加入5毫升的PBS。 </li><li>吸出PBS成真空瓶。加入1ml 0.05％胰蛋白酶并在37℃下孵育2 - 5分钟以从培养皿分离细胞。 </li><li>添加3毫升D10和吸管上下分离从板的任何剩余的细胞。转移细胞在FACS管中并离心，在400×g离心10分钟。 </li><li>滗上清，洗涤细胞用3ml PBS中。离心机在400×g离心10分钟，并倾析上清液。重悬在200μlPBS中。 </li><li>运行在细胞流式细胞仪9流动。收集20,000 - T内部30000事件他活细胞门（由FSC 与 SSC确定），并计算GFP +和GFP的百分比-细胞。 注:病毒滴度被认为是可以接受的，当1:30稀释的病毒上清液产率至少为50％ 的 GFP + 3T3细胞。如果再次优转导的结果可以得到小于50％的GFP +细胞这种稀释的结果。以确定病毒滴度计算改编自利蒙等。 ， 血 （1997年）9。 </li><li>要确定相对的病毒滴度，用公式:病毒滴度=颗粒形成单位（PFU）/毫升= [（的3T3细胞×GFP +细胞的频率数）上清液（毫升）/音量]×稀释因子。 </li></ol></li></ol> 3.转导供体骨髓细胞<ol><li>为了制备用于骨髓供体小鼠（BM）收获，第一麻醉通过异氟烷蒸发器的小鼠，用5％结合的补充的流率氧气以1升/分。捏脚来评估响应。 注:供体小鼠（小于8周龄）应骨髓收获前5天准备（移植前8天的时间）。一个供体小鼠将提供足够的细胞至少2收件人。 </li><li>注入150毫克/公斤使用27 G×½"针5-氟尿嘧啶（5-FU），通过在麻醉小鼠眶后注入。 警告！ 5-FU的抗肿瘤化疗剂。总是处理5-FU经过认证的化学通风柜或管道生物安全柜内。与该机构的实验室和动物安全委员会进行磋商，以确定与5-FU治疗的小鼠适当的标签。 <ol><li>在其一侧麻醉鼠标定位，并通过同时使用拇指和食指使得眼睛从头部稍微突出约束。 </li><li>用针外侧向内侧眼角，并与朝上锥开始，插入针以45°角在内侧方向，当针被中途插入停止。缓缓注入细胞，并删除小心取出针。 注:如果正确执行了这种技术，眼稍应该收回和无阻力应在进针感觉到。 </li><li>检查鼠标任何受伤的迹象，如肿胀或出血。 </li></ol></li><li>嘉实骨髓后第5天5-FU（移植前3天）的治疗。 <ol><li>麻醉通过异氟烷蒸发器的小鼠在1升/分钟的5％组合补充氧气的流速。捏脚来评估响应。 </li><li>颈椎脱臼牺牲鼠标。通过用70％乙醇慷慨喷洒直到毛皮变成湿消毒鼠标。 </li><li>使用解剖剪刀，使皮肤切口和解剖筋膜从腿部肌肉了。接着，通过切断在臀部股骨和在踝胫骨从小鼠解剖腿部程。 </li><li>弯曲腿成"L"形，并通过在与解剖剪刀膝关节切削分隔每个腿骨。 </li><li>在每个腿骨的远端移除软骨，使用解剖剪刀直到软骨脱开轻轻刮去肌肉朝向腿骨的一端。 </li><li>使用具有27国祥½英寸针头的注射器，冲洗各腿骨用PBS在50ml锥形管中补充有2％FBS的超过100微米的尼龙膜。冲洗骨直到骨变白最大化收获的细胞的数目。 </li><li>使用注射器的柱塞通过尼龙膜醪细胞进50毫升锥形管，以获得单细胞悬浮液。与其他鼠标腿重复。 </li></ol></li><li>离心细胞，在4℃下在400 xg离心10分钟，弃上清。悬浮的BM在5毫升氯化铵钾（ACK）缓冲液以裂解红血细胞。孵育细胞在冰上10分钟。 </li><li>加油管与PBS阻止细胞溶解。 CENTRI夫格细胞在4℃下在400 xg离心10分钟，弃上清。重复裂解步骤，如果BM颗粒具有红色。 </li><li>悬浮在5ml PBS中并计数使用血细胞计数器通过台盼蓝排除细胞。 </li><li>重悬的细胞以每平方米2×10 6个细胞与含有DMEM补充有20％FBS，L-谷氨酰胺，青霉素/链霉素2X-STIM预鸡尾酒，IL-3（14毫微克/毫升）中，IL-6（24纳克/毫升）和SCF（112毫微克/毫升）中。板2毫升，每孔在6孔板中。 </li><li>孵育细胞过夜，在37℃和5％的CO 2。 </li><li> 2毫升病毒上清液添加到每个孔含有BM细胞，并添加凝聚胺（8微克/毫升）。在30℃下90分钟旋板在1500 xg离心然后返回板于组织培养培养箱中过夜。 </li><li>吸管细胞悬浮到锥形管中，并在4℃离心机在400 xg离心10分钟，不丢弃该板。每孔的2ml悬浮细胞新鲜2X预STIM鸡尾酒，并返回到原来的井。 </LI&gt; <li>通过重复步骤3.7-3.9执行第二 spinoculation。 </li></ol> 4.移植供体细胞到收件人小鼠<ol><li>准备同基因受体小鼠与致死剂量全身照射&lt;骨髓细胞注射24小时之前移植。 注 :致死剂量照射BALB / c小鼠通常是800-900 cGy的和1,000-1,200 cGy的为C57B / 6 10。剂量典型地分成隔开至少3-4小时的照射两个会话。 </li><li>轻轻吹打成上清液锥形管从步骤3.11，从6孔板收获转供体细胞继续。用1毫升的PBS，收集残余细胞，并添加至50ml锥形管中洗每个孔中。 </li><li>将0.5ml 0.05％胰蛋白酶的至孔中，并在组织培养培养箱中于37℃孵育5分钟。 </li><li>加入1 ml D10到水井和轻柔吹打分离任何剩余的细胞。添加细胞到各管期从步骤4.2和沉淀通过离心在4℃下在400×g下10分钟。 </li><li>用PBS洗并离心细胞在4℃下在400×g下10分钟。使用血细胞计数器通过台盼蓝排除悬浮在5ml PBS中并计数细胞。 注意 :如果该矢量具有GFP标签然后量化GFP阳性细胞的百分比用流式细胞。 </li><li>悬浮5×10 5 - 2×10 6个细胞在50 - 100微升的PBS注射到小鼠中。 </li><li>注射用无菌27国祥½"针经眼眶细胞注射到麻醉小鼠。 注意:其他的注射技术包括尾静脉，脾，或股。 <ol><li>使用异氟醚和捏脚，以评估响应麻醉鼠标。 </li><li>在其一侧鼠标的位置，并通过同时使用拇指和食指使得眼睛从头部稍微突出约束。 </li><li>随着斜角朝上，将第needle外侧成45°角的内眦。 注:如果正确执行了这种技术，眼稍应该收回和无阻力应在进针感觉到。 </li><li>检查鼠标任何受伤的迹象，如肿胀或出血。 注:移植后的小鼠会产生数周的低血球计数，因此很容易受到感染。可用于预防与抗生素或使用的酸化水。 </li></ol></li><li>一个月后移植，测量外周血％GFP为用于通过流式细胞术植入供体细胞的指标。 注:单独的GFP不能确定细胞的植入的成功。在病毒转导可能已经失败还可能发生植入。在这种情况下，没有GFP +细胞会被检测到，但在移植细胞的植入是成功的。 <ol><li>取10微升外周血B的Ÿ用21g的×1½"注射器穿刺隐腿部静脉，并在PBS为100毫米EDTA以防止凝血。 </li><li> 1毫升ACK添加到血液和裂解在冰上10分钟。沉淀细胞，在400×g离心10分钟离心。 </li><li>吸去上清液在真空烧瓶中并加入1毫升的PBS补充有2％FBS的停止裂解。沉淀细胞，在400×g离心10分钟离心。 </li><li>在在100μlPBS中的真空烧瓶，重悬沉淀吸去上清液补充有2％FBS的。 </li><li>在流式细胞仪运行样品，收集活细胞门（由FSC 与 SSC确定）范围内30万的事件和计算GFP +和GFP的百分比-细胞9。 </li></ol></li><li>另外1个月后移植，执行使用自动兽医血液分析仪18来监控疾病的进展全血细胞计数（CBC）。 注:继续监测％的疾病GFP外周血和CBC每月。 </li><li>要终止实验中，用异氟醚和捏脚，以评估响应麻醉鼠标。通过颈脱位和收获脾和骨髓组织病理学牺牲小鼠。 注:在实验的长度是主观和终止日期应该根据预期的疾病表型进行评估。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Hematopoietic+Stem+Cell+Identification+And+Analysis.">Mouse Hematopoietic Stem Cell Identification And Analysis.</a> Cytometry A. 75 (1), 14-24 (2009).</li><li>Ergen, A. V., Jeong, M., Lin, K. K., Challen, G. A., Goodell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+mouse+side+population+cells.">Isolation and characterization of mouse side population cells.</a> Methods Mol. Bio. (Clifton, N.J.). 946, 151-162 (2013).</li><li>Weksberg, D. C., Chambers, S. M., Boles, N. C., Goodell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD150-+side+population+cells+represent+a+functionally+distinct+population+of+long-term+hematopoietic+stem+cells.">CD150- side population cells represent a functionally distinct population of long-term hematopoietic stem cells.</a> Blood. 111 (4), 2444-2451 (2008).</li><li>Oguro, H., Ding, L., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+markers+resolve+functionally+distinct+subpopulations+of+hematopoietic+stem+cells+and+multipotent+progenitors.">SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.</a> Cell Stem Cell. 13 (1), 102-116 (2013).</li><li>Li, J., Kent, D. G., Chen, E., Green, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+myeloproliferative+neoplasms:+JAK+of+all+grades.">Mouse models of myeloproliferative neoplasms: JAK of all grades.</a> Dis. Model. Mech. 4 (3), 311-317 (2011).</li><li>Mullally, A., Lane, S. W., Brumme, K., Ebert, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloproliferative+neoplasm+animal+models.">Myeloproliferative neoplasm animal models.</a> Hematol. Oncol. Clin. North Am. 26 (5), 1065-1081 (2012).</li></ol>

The authors have nothing to disclose.

这个协议提供了如何在小鼠进行骨髓移植，以概括与进展中原发性血小板增多症样疾病对骨髓纤维化与CALRdel52突变疾病的驾驶员的详细描述。表达增加血小板，巨核细胞的扩张和骨髓纤维化CALRdel52结果细胞的移植成功。骨髓移植是一个多步骤的过程，它确认，其中可避免技术误差，以防止表达感兴趣甚至小鼠死亡的基因的细胞的植入差的步骤是重要的。 以一个常常被忽视一?...

转导移植是小鼠恶性血液病建模的有用方法。该技术已用于研究骨髓恶性肿瘤追溯到了BCR-ABL1的异位表达可以忠实地概括小鼠1慢性粒细胞白血病首次证明特别有价值。这一技术随后促进JAK2 V617F和MPL W515K / L的广泛研究突变骨髓增殖性疾病（MPN）。 MPN是一组恶性血液病特征在于成熟骨髓细胞和骨髓纤维化的生产过剩。这些疾病通常由造血干细胞的克隆扩增已在任一JAK2，MPL，或CALR获得的体细胞突变产生的。转导移植JAK2 V617F和MPL W515K / L车型展览真性红细胞增多症和骨髓纤维化2的临床特点- 5 &lt;/ SUP&gt;。最近，也已用转导移植方法6产生钙网蛋白突变MPN的小鼠模型。这些小鼠发展的原发性血小板增多症样疾病具有增加血小板，巨核细胞的数量增加，和骨髓纤维化。一起，这些模型不仅提供了机会来洞察MPN的分子发病机制，而且要开发和临床前设置研究治疗剂的能力。 这份手稿提供转导移植方法，重点对CALRdel52突变的详细描述。该技术包括表达该突变体构造成照射同基因受体小鼠逆转录病毒转导骨髓细胞移植。

<table><tbody><tr><td>&lt;strong&gt;Cell Lines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>MT-10-013-CV</td><td></td></tr><tr><td>293T cells</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>3T3 cells</td><td>ATCC</td><td>CRL-1658</td><td></td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EcoPak, also known as pCL-Eco</td><td>Addgene</td><td>12371</td><td>Retroviral packaging cell lines, such as EcoPack 2-293, may be used in place of the EcoPak plasmid and standard 293T cells. Additional &amp;gamma;-retrovirus envelope and packaging plasmids are available from Addgene and others.</td></tr><tr><td>MSCV-IRES-GFP (MIG)</td><td>Addgene</td><td>20672</td><td>Additional &amp;gamma;-retroviral transfer plasmids are available from Addgene and others.</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>27 G x 1/2&quot; needles</td><td>BD</td><td>305620</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Corning</td><td>MT-35-010-CV</td><td></td></tr><tr><td>Penicillin/streptomycin/L-glutamine</td><td>Corning</td><td>MT-30-009-CI</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>Corning</td><td>MT-25-052-CI</td><td>Can be homemade</td></tr><tr><td>PBS</td><td>Corning</td><td>MT-21-031-CV</td><td></td></tr><tr><td>10 cm dishes</td><td>Fisher</td><td>172931</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Fisher</td><td>12565268</td><td></td></tr><tr><td>60 mm dishes</td><td>Fisher</td><td>150288</td><td></td></tr><tr><td>Polybrene</td><td>Fisher</td><td>NC9840454</td><td></td></tr><tr><td>5-FU</td><td>Fisher</td><td>A13456-06</td><td></td></tr><tr><td>100 &amp;#181;m cell strainers</td><td>Fisher</td><td>22363549</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Fisher</td><td>12565270</td><td></td></tr><tr><td>6-well plate</td><td>Fisher</td><td>130184</td><td></td></tr><tr><td>FACS tubes</td><td>Fisher</td><td>14-959-5</td><td></td></tr><tr><td>0.45 &amp;mu;m syringe filters</td><td>Fisher</td><td>0974061B</td><td></td></tr><tr><td>Opti-MEM</td><td>Gibco</td><td>31985-070</td><td></td></tr><tr><td>ACK buffer</td><td>Lonza</td><td>10-548E</td><td>Can be homemade</td></tr><tr><td>Recombinant murine IL-3</td><td>Peprotech</td><td>213-13</td><td></td></tr><tr><td>Recombinant murine IL-6</td><td>Peprotech</td><td>216-16</td><td></td></tr><tr><td>Recombinant murine SCF</td><td>Peprotech</td><td>250-03</td><td></td></tr><tr><td>X-tremeGENE 9</td><td>Roche</td><td>6365809001</td><td>Transfection reagent</td></tr><tr><td>1.5 ml centrifuge tubes</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BD Accuri C6</td><td></td><td></td><td></td></tr><tr><td>X-ray irradiator</td><td></td><td></td><td></td></tr></tbody></table>

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.

转导移植技术导致与细胞中表达感兴趣的基因受体小鼠的造血重建。 图1显示了突变的MPN钙网蛋白的转导移植小鼠模型的概述。简言之，将表达CALRwt或CALRdel52逆转录病毒被用于从一个C57B / 6供体小鼠感染BM细胞。转导的细胞移植到照射C57B / 6受体小鼠和供体细胞植入的第一个月后移植过程中发生。一个疾病表型的证据是显而易见后3个月，移植，其中小鼠表现出?...

Watch this Scientific Journal Video about Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm at JoVE.com

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

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 ...

transduction-transplantation-mouse-model-myeloproliferative

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

11.8K 次观看.  University of California, Irvine. 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. 

视频: Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

科研

癌症研究

Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells (LSCs). An experimental AML rodent model to test therapies based on successfully transplanting these cells via retro-orbital injections in recipient mice is fraught with challenges. The aim of this study was to develop an easy, reliable, and consistent method to generate a robust murine model of AML using an intra-peritoneal route. In the present protocol, bone marrow cells were transduced with a retrovirus expressing human MLL-AF9 fusion oncoprotein. The efficiency of lineage negative (Lin-) and Lin-Sca-1+c-Kit+ (LSK) populations as donor LSCs in the development of primary AML was tested, and intra-peritoneal injection was adopted as a new method to generate AML. Comparison between intra-peritoneal and retro-orbital injections was done in serial transplantations to compare and contrast the two methods. Both Lin- and LSK&#160;cells transduced with human MLL-AF9 virus engrafted well in the bone marrow and spleen of recipients, leading to a full-blown AML. The intra-peritoneal injection of donor cells established AML in recipients upon serial transplantation, and the infiltration of AML cells was detected in the blood, bone marrow, spleen, and liver of recipients by flow cytometry, qPCR, and histological analyses. Thus, intra-peritoneal injection is an efficient method of AML induction using serial transplantation of donor leukemic cells.

Here, intra-peritoneal injection of leukemia cells is utilized to establish and propagate acute myeloid leukemia (AML) in mice. This new method is effective in the serial transplantation of AML cells and can serve as an alternative for those who may experience difficulties and inconsistencies with intravenous injection in mice.

腹膜内移植在小鼠中产生急性髓系白血病

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells ...

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.

This protocol describes the isolation of double-negative thymocytes from the mouse thymus followed by retroviral transduction and co-culture on the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4) for further functional analysis.

小鼠胸腺细胞在δ样4表达基质细胞上的共培养和转导以研究T细胞白血病的癌基因

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line ...

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.

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 ...

免疫与感染

<meta charset="utf8"></head><body>开发高效、精确的脊髓神经胶质瘤小鼠模型

Surgical Transplantation of Tumor Cells into the Spinal Cord of Mice

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and comprehensive data on spinal cord gliomas remain limited due to the lack of suitable preclinical animal models. Developing a simple and reproducible animal model has become essential for advancing basic and translational research. A murine model is ideal, as the murine spinal cord shares structural similarities with the human spinal cord. This protocol describes the generation of a reproducible murine model of spinal cord glioma by directly injecting tumor cells into the intervertebral space using the spinous process of the seventh cervical vertebra as a guide. Compared to other methods, this approach is more effective and convenient, involving a smaller incision, reduced invasiveness and blood loss, faster recovery, and more stable tumor formation. This model is expected to advance the understanding of disease mechanisms, optimize surgical strategies, and support the development of therapeutic drugs for spinal cord gliomas.

<meta charset="utf8"></head><body>肿瘤细胞手术移植到小鼠脊髓中

The present protocol describes the development of a reproducible murine model of spinal cord glioma by injecting tumor cells into the intervertebral space, offering a more effective and less invasive approach for research and therapeutic development.

肿瘤细胞手术移植到小鼠脊髓中

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and ...

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 ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

小鼠骨髓移植程序研究克隆造血症

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

骨髓增殖性疾病的转导移植小鼠模型

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此视频中的章节

nog 小鼠多发性骨髓瘤异种移植的多模态生物发光和正电子发射成形断层扫描成像

腹膜内移植在小鼠中产生急性髓系白血病

小鼠胸腺细胞在δ样4表达基质细胞上的共培养和转导以研究T细胞白血病的癌基因

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

肿瘤细胞手术移植到小鼠脊髓中

肿瘤细胞手术移植到小鼠脊髓中

肿瘤细胞手术移植到小鼠脊髓中

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

小鼠骨髓移植程序研究克隆造血症

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34⁺ Cells Injection, and Cytokine Delivery