从作者实验室的工作已经支持农业部/ CSREES / NRICGP（2007-35203-18213）; NIH / NCRR（R01 RR17359-06），美国国立卫生研究院/ NIEHS的（1 R21的ES014856-01A2）和艾伯塔创新 - 健康解决方案。

A部分胚芽细胞移植在小鼠 1。受体小鼠的制备<ol><li>受助人应是免疫耐受（无论是捐助者或免疫缺陷的基因相匹配的）捐助睾丸细胞。 </li><li>收件人应当要么自然没有精子（ 如 W / 宽 V小鼠）或内源性的生殖细胞耗尽。可以通过辐射或化疗药物，如马利兰生殖细胞耗竭。在这个协议中，我们描述的准备与马利兰受体小鼠的方法。 </li><li>对待受助人的年龄在4-6周，通过腹腔注射白消安。白消安的最佳剂量是应变依赖。在常用收件人株，剂量为40-50毫克/千克足以消耗内源性生殖细胞（如44毫克/千克裸鼠，50毫克/公斤为B6/129 F1的收件人）。 </li><li>溶解在二甲基亚砜马利兰粉，然后加入等量的无菌水净化O的终浓度为4毫克/毫升。保持溶液温在35-40℃前使用，以避免沉淀白消安。丢弃的解决方案，如果降水观察。 </li><li>马利兰治疗后，允许使用收件人之前至少一个月。受助人之间可以使用1个月和3个月后马利兰治疗。 </li></ol> 2。显微注射移液器的制备<ol><li>选择外径1.0毫米，0.75毫米内径，长度为3英寸的高硼硅玻璃吸液管（毛细管）。 </li><li>硅化（siliconize）Sigmacote玻璃吸管，用甲醇冲洗移液器和吹干。 </li><li>用吸管拔拉移液器。不同的吸管拔机有不同的设置，因此，测试一些设置。一般来说，用于眼球摘除移液器类似的设置工作。 </li><li>打破解剖显微镜下的枪头前使用，以实现一个直径约为50微米尖端。 </li></ol> 3。用于移植的供体细胞的制备<ol><li>供体菌的体例选择是依赖于实验研究的问题。如果精子量化为端点使用，使用一个容易辨认的遗传标记，如紫胶-Z的捐助者（如B6.129S7-GT（罗莎）26Sor“/ J从杰克逊实验室）或GFP（如C57BL/6-Tg（ CAG-EGFP）/ 1Osb从杰克逊实验室十）。 </li><li>准备在1毫克/毫升，4毫升的胰蛋白酶-EDTA（0.25％胰蛋白酶加1mM的EDTA），2毫升的DMEM的DNase在7毫克/毫升，7毫升培养液胶原酶。这一数额是2体小鼠睾丸消化。 </li><li>无菌条件下收集睾丸和去除白膜。传播的曲细精管细镊子轻轻以促进消化酶。 </li><li>传输管将胶原酶，在37°C孵育5-10分钟，并经常鼓动。 </li><li>洗管两次旋转（200-300克）为3分钟，重新悬浮在PBS W / O 钙+ </s达&gt;。 </li><li>重新挂起管在胰蛋白酶-EDTA和动摇，直到他们成为粘性和阴天。监测的37°C的，它应该出现1-2分钟内消化管。 </li><li>加入DNase和摇匀。孵育1-2分钟。 </li><li>胎牛血清加入3毫升，以阻止酶的作用。 </li><li>过滤细胞悬液，用40-70微米孔径的尼龙网，以消除细胞/组织块。 </li><li> 600Ğ5分钟收集细胞重新悬浮细胞在培养液（100微升）少量。 </li><li>计数细胞和音量调整到理想的最终浓度（通常为100×10 6）。在冰上保持细胞悬液后才能使用。 </li><li>将充满每个马利兰治疗的小鼠睾丸细胞悬液只有10-15微升。由于潜在的浪费和泄漏，旨在有30-50％睾丸液。 </li></ol> 4。移植过程（图1） <ol><li>麻醉受助人按照批准的协议。 </li><li>背地点斜卧和手术准备的腹部。使了〜1厘米中线腹部切口暴露腹壁。提起腹壁用在白线的小镊子，以避免意外受伤的腹部器官，进行了〜0.5厘米的切口，暴露腹腔，腹壁中线。 </li><li>使用一个虹膜钳举行腹壁，并使用另一个虹膜钳对搜索连接到附睾和睾丸在腹腔的脂肪垫。脂肪垫轻轻一拉，直到睾丸形象化和睾丸动脉和附睾都清晰可见。一个睾丸在工作时间。 </li><li>将薄的无菌悬垂性从蒸压指数卡下方的脂肪垫/睾丸更好的视觉识别（可选）。悬垂性也吸收液。 </li><li>添加一台盼蓝染料进入细胞悬液下降仔细到polyethy加载细胞悬液lene的油管连接到1毫升注射器。连接进油管拉移液器和移液器轻轻迫使细胞悬液，通过施加压力的注射器。 </li><li>确定传出管道（连接睾丸附睾），轻轻地取下导管周围的脂肪组织。管道和他们周围的膜是半透明的，工作认真。 </li><li>小心地插入导管传出管道包吸管，轻轻线程向睾丸数毫米。 </li><li>同时避免移动的注射针，注射器，达到轻轻压下注射器的柱塞，以确保暂停进入睾丸曲细精管流开始填写。 </li><li>注射率和细胞悬液流受拇指压力。避免突然增加的压力;监测肾小管悬挂在运动。 </li><li>停止注射时，几乎所有的表面管已填补前睾丸开始BEC青梅缺血。 </li><li>返回睾丸腹腔。对侧睾丸的重复过程。缝合用6-0丝线缝合腹壁，关闭与金属伤口剪辑的皮肤。监测小鼠变暖垫，直到完全恢复。 </li></ol> 5。收件人睾丸的分析<ol><li>允许分析前2-4个月后移植。 </li><li>安乐死的收件人鼠标根据动物护理和使用准则，并收集至PBS的睾丸和附睾。当乳糖-Z的转基因供体菌已被使用，附睾可以用来作为阳性控制，因为它有内源性β-半乳糖苷酶的活性。 </li><li>为紫胶-Z的染色检测供体细胞，修复睾丸1-2小时，在4°C的4％多聚甲醛（PFA）的PBS中。冲洗3×30分钟，在室温中的lacZ冲洗缓冲区。 </li><li>在37℃孵育过夜LacZ染色解决方案。睾丸作为一个整体或分散后可染色肾小管。 </li><li>修复并存储在10％中性福尔马林。如果需要部分使用中性的快速反染色的红色。 </li></ol> B部分异位异种移植小鼠 （从Dobrinski和Rathi 2008 15，罗德里格斯·索萨等 2011 16）。 1。收集组织的捐助<ol><li>获得通过阉割睾丸组织活检从捐助者男性。 </li><li> PBS或培养基中的活组织切片检查，保持无菌条件下的地方睾丸。 </li><li>保持实验室的冰和运输组织收集。 </li></ol> 2。捐助组织的制备<ol><li>在组织文化引擎盖执行，以保持无菌。 </li><li>每个睾丸洗净，在冰冷的PBS含有抗生素转移到文化与PBS菜前的两到三倍。在活检的情况下，洗睾丸碎片的两到三倍，与冰冷的文化的Medi嗯含有抗生素，由纺纱在150克的2分钟，新鲜冰冷的培养基重新悬浮。 </li><li>对于完整的睾丸，消除沿表面切开鞘膜，挤出睾丸。从睾丸附件结构（精索，附睾，结缔组织）。成菜用PBS文化睾丸一次洗冷PBS和转让。 </li><li>用手术刀刀片和一把剪刀，小心地取出睾丸白膜。如果睾丸很小，挤压睾丸组织的外膜，而通过一个小切口一端与另一端的一个小镊子对控股的鞘膜，鞘膜可以被删除。 </li><li>根据睾丸的大小，无论是整个睾丸组织可以切成小块的大约1 - 2毫米大小用弯钳和手术刀刀片，或睾丸组织的大块可以首先从睾丸中删除，然后切细件。这一切应做在冰冷的培养基和无菌条件下，在一个小培养皿（60 X15毫米）。 </li><li>转让冰冰冷的小文化，美食培养基准备组织碎片，直到嫁接。 </li></ol> 3。阉割的收件人鼠标<ol><li>麻醉鼠标如上，并准备通过修剪头发（没有必要在裸鼠），用70％乙醇和优碘溶液擦拭无菌医疗器械领域。 </li><li>使0.5 -1厘米腹正中皮肤切口，显露腹壁。 </li><li>仔细地暴露睾丸和附睾睾丸动脉在4.2-4.3 A部，所述。 </li><li>尾引带epidydimis，钝性分离。 </li><li>结扎睾丸动脉和输精管连同血管与丝绸，部分切割之间的睾丸和结扎，结扎结构。 </li><li>重复塞科议事第二睾丸。 </li><li>与一个或​​两个手术针缝合腹壁。 </li><li>关闭一个或两个米歇尔剪辑的皮肤切口。 </li></ol> 4。异位异种移植<ol><li>鼠标放置在腹卧，并准备在其上面的回无菌医疗器械领域。 </li><li>根据移植是多少要插入（一般4-8/mouse），使鼠标背面的中线两侧约0.5厘米长的皮肤切口。 </li><li>使用镊子，以保持皮肤切口的边界，使皮下腔外的结缔组织，用剪刀戏弄。 </li><li>使用虹膜钳地方的一块睾丸组织深入到皮下腔，持有边境的皮肤切口与另一虹膜钳。 </li><li>关闭一个米歇尔剪辑的皮肤切口，并保持在加热垫的鼠标，直到它开始从麻醉中恢复。 </li><li>额外的绝缘和合作，将鼠标笼子VER和小鼠显示器直到完全康复。 </li></ol> 5。收集，分析和精子收获睾丸移植瘤<ol><li>安乐死的主机鼠标，根据动物护理和使用指南，并从尾部至颈部皮肤和开放的背面皮肤作正中皮肤切口。这暴露了它可以位于皮下组织或附着在皮肤移植。 </li><li>小心取出移植，使用一对钳子和一把剪刀。 </li><li>移植康复，个别移植的大小和重量的记录数。 </li><li>检索从腹部鼠标的精囊，并记录自己的体重，睾酮生产的指示，通过嫁接的组织。 </li><li>组织学分析<ol class="lalpha"><li>暂停到卷中的样品中含有的Bouin的解决方案（或其他固定液）的小瓶的裸鼠~~移植的10倍，，标签小瓶适当的。 </li><li>孵化在70％乙醇洗涤，最好在24小时的间隔至少3次冰箱过夜。 </li><li>进行处理和石蜡包埋。 </li></ol></li><li>精子的收获<ol class="lalpha"><li>洗纺纱他们在300克1分钟，重新悬浮在培养基中含有抗生素，他们的异种移植。 </li><li>切成小块，移植和百果精心组织文化包含3菜钳 - 5毫升培养基。 </li><li>通过40微米的细胞过滤器的过滤器的碎组织。 </li></ol></li></ol> C部分代表性成果 1。生殖细胞移植如果移植的供体细胞悬浮液含有精原​​干细胞携带的LacZ基因的捐助者在接受睾丸精原干定植，如遗传标记，可以通过X-gal染色观察独特的蓝色部分的SEMiniferous肾小管后2-3个月后移植3（图2A）。一套行之有效的殖民地应该完全填充的段长的暗蓝色舒展，两端有两个或更多层的蓝色细胞向中间靠拢，相对较弱的染色地区单个，成对或小团体的蓝色细胞网络很明显（图2B）。一个深蓝色的曲细精管区的横截面应揭示以及建立和有组织的精子与蓝色的生殖细胞在不同分化阶段（图2C）。 2。睾丸组织异种移植睾丸组织移植后的生存能力呈负相关与捐赠人的发展阶段;从新生男婴的组织使用时，得到最好的结果，而成年组织的退化和死亡17-21显示了较高的倾向。一般来说，移植成功降低捐助组织经历我福特iosis的精子发生的第一次浪潮。时间不成熟的捐助组织和完整的精子的全面发展是种属特异性，往往有些原位睾丸比较短。根据物种的完整的精子的曲细精管的数量是可变的。虽然在绵羊，山羊和猪的数量大于50％，牛，猫，它是小于15％14（图3）。 <img alt="图1" src="/files/ftp_upload/3545/3545fig1.jpg" /> 图1。生殖细胞移植手术。广场受助人在麻醉后，背卧，并作出了〜0.4英寸中线腹部切口（一）。公开撤回脂肪垫附睾和睾丸的睾丸和更好的视觉识别（二）脂肪垫/睾丸下方放置一个薄的无菌悬垂。定位，使埋在脂肪垫传出管道可辨（的C，D，E），睾丸和附睾。 ID实体传出管道，轻轻地清除管道周围的脂肪组织。一张彩纸或塑料，可以放在下面更好的可视化（G）的管道。打破或磨枪头，根据管道和负载细胞悬液，将移液器（高）的大小。小心地插入导管传出管道束的吸管，轻轻地向睾丸（H中的箭头显示的吸管注射液和线程的方向）线程数毫米。睾丸曲细精管与成功注入（一）所示。 TS：睾丸; EP：附睾。 <img alt="图2" src="/files/ftp_upload/3545/3545fig2.jpg" /> 图2。生殖细胞移植 ）注射用X-gal的染色睾丸有代表性的成果 。曲细精管的蓝色部分代表从转基因捐助的SSC（LacZ基因的转基因）建立殖民地。 b）高倍在移植后3个月的SSC殖民地。日Ë深蓝色区域中心代表完整的精子在两端和淡蓝色区域代表日益扩展殖民地。 C）深蓝色的曲细精管组织切片（移植后3个月），显示了良好的组织的精子。在B和C的比例尺为200微米和30微米，分别。 （图每年牧师细胞开发生物学改编自2008年24:263-86和生物学复制业1999年六月60（6）:1429-36）。 <img alt="图3" src="/files/ftp_upload/3545/3545fig3.jpg" /> 图3。异位异种移植到免疫缺陷小鼠的大型动物的睾丸组织发育不成熟，不成熟的捐助睾丸片段（约1毫米3）移植免疫缺陷小鼠背部皮肤（一）下都能够生存和响应鼠标促性腺激素。作为一个结果，睾丸组织经历了完整的开发，包括施肥主管（二）精子的形成。一旦睾丸移植瘤收集（三）它们可用于分析或获得精子受精（E）和胚胎生产（六）（四）。酒吧等于50微米（B，E和F）的10微米（四）。 （罗德里格斯索萨和Dobrinksi的2009 14个从修改）。

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+genes+important+for+spermatogonial+stem+cell+self-renewal+and+survival.">Identifying genes important for spermatogonial stem cell self-renewal and survival.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (25), 9524-9524 (2006).</li><li>Dobrinski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+transplantation+and+testis+tissue+xenografting+in+domestic+animals.">Germ cell transplantation and testis tissue xenografting in domestic animals.</a> Animal Reproduction Science. 89 (1-4), 137-137 (2005).</li><li>Dobrinski, I., Avarbock, M. R., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+germ+cells+from+rabbits+and+dogs+into+mouse+testes.">Transplantation of germ cells from rabbits and dogs into mouse testes.</a> Biology of Reproduction. 61 (5), 1331-1331 (1999).</li><li>Honaramooz, A., Snedaker, A., Boiani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+from+neonatal+mammalian+testes+grafted+in+mice.">Sperm from neonatal mammalian testes grafted in mice.</a> Nature. 418 (6899), 778-778 (2002).</li><li>Rodriguez-Sosa, J. R., Dobrinski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+testis+tissue+xenografting.">Recent developments in testis tissue xenografting.</a> Reproduction. 138 (2), 187-187 (2009).</li><li>Dobrinski, I., Rathi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+grafting+of+mammalian+testis+tissue+into+mouse+hosts.">Ectopic grafting of mammalian testis tissue into mouse hosts.</a> Methods in Molecular Biology. 139, 450-450 (2008).</li><li>Rodriguez-Sosa, J. R., Schlatt, S., Dobrinski, I., Seli, E., Agarwal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testicular+tissue+transplantation+for+fertility+preservation.">Testicular tissue transplantation for fertility preservation.</a> Fertility Preservation: Emerging Technologies and Clinical Applications. , 331-331 (2011).</li><li>Rathi, R., Honaramooz, A., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+fate+and+seminiferous+tubule+development+in+bovine+testis+xenografts.">Germ cell fate and seminiferous tubule development in bovine testis xenografts.</a> Reproduction. 130 (6), 923-923 (2005).</li><li>Rathi, R., Honaramooz, A., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+development+in+equine+testis+tissue+xenografted+into+mice.">Germ cell development in equine testis tissue xenografted into mice.</a> Reproduction. 131 (6), 1091-1091 (2006).</li><li>Kim, Y., Selvaraj, V., Pukazhenthi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+donor+age+on+success+of+spermatogenesis+in+feline+testis+xenografts.">Effect of donor age on success of spermatogenesis in feline testis xenografts.</a> Reproduction, Fertility, and Development. 19 (7), 869-869 (2007).</li><li>Arregui, L., Rathi, R., Zeng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenografting+of+adult+mammalian+testis+tissue.">Xenografting of adult mammalian testis tissue.</a> Animal Reproduction Science. 106 (1-2), 65-65 (2008).</li><li>Schlatt, S., Honaramooz, A., Ehmcke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+survival+of+adult+human+testicular+tissue+as+ectopic+xenograft.">Limited survival of adult human testicular tissue as ectopic xenograft.</a> Human Reproduction. 21 (2), 384-384 (2006).</li><li>Shinohara, T., Avarbock, M. R., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta1-+and+alpha6-integrin+are+surface+markers+on+mouse+spermatogonial+stem+cells.">beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (10), 5504-5504 (1999).</li><li>Kanatsu-Shinohara, M., Toyokuni, S., Shinohara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD9+is+a+surface+marker+on+mouse+and+rat+male+germline+stem+cells.">CD9 is a surface marker on mouse and rat male germline stem cells.</a> Biology of Reproduction. 70 (1), 70-70 (2004).</li><li>Ryu, B. Y., Orwig, K. E., Oatley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+aging+and+niche+microenvironment+on+spermatogonial+stem+cell+self-renewal.">Effects of aging and niche microenvironment on spermatogonial stem cell self-renewal.</a> Stem Cells. 24 (6), 1505-1505 (2006).</li><li>Costoya, J. A., Hobbs, R. M., Barna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+Plzf+in+maintenance+of+spermatogonial+stem+cells.">Essential role of Plzf in maintenance of spermatogonial stem cells.</a> Nature Genetics. 36 (6), 653-653 (2004).</li><li>Morrow, C. M., Hostetler, C. E., Griswold, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ETV5+is+required+for+continuous+spermatogenesis+in+adult+mice+and+may+mediate+blood+testes+barrier+function+and+testicular+immune+privilege.">ETV5 is required for continuous spermatogenesis in adult mice and may mediate blood testes barrier function and testicular immune privilege.</a> Annals of the New York Academy of Sciences. 1120, 144-144 (2007).</li><li>Zeng, W., Snedaker, A. K., Megee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+and+transplantation+of+porcine+testis+tissue.">Preservation and transplantation of porcine testis tissue.</a> Reproduction, Fertility and Development. 21 (3), 489-489 (2009).</li><li>Jahnukainen, K., Ehmcke, J., Hergenrother, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cold+storage+and+cryopreservation+of+immature+non-human+primate+testicular+tissue+on+spermatogonial+stem+cell+potential+in+xenografts.">Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts.</a> Human Reproduction. 22 (4), 1060-1060 (2007).</li><li>Rathi, R., Zeng, W., Megee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+testicular+tissue+from+infant+monkeys+after+xenografting+into+mice.">Maturation of testicular tissue from infant monkeys after xenografting into mice.</a> Endocrinology. 149 (10), 5288-5288 (2008).</li><li>Honaramooz, A., Li, M. W., Penedo, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+maturation+of+primate+testis+by+xenografting+into+mice.">Accelerated maturation of primate testis by xenografting into mice.</a> Biology of Reproduction. 70 (5), 1500-1500 (2004).</li></ol>

没有利益冲突的声明。

1。生殖细胞移植生殖细胞移植提供了明确确认的精原干细胞存在于细胞的人口（SSCS）的唯一功能检测。只有共享服务中心可以回家和拓殖在基底膜SSC的利基，并发起捐助源性精子。生殖细胞移植使人们有可能以前所未有的方式SSC的研究和操纵。该技术已用于生产转基因动物通过SSCS 4的基因操纵澄清定植3,8南南合作的模式，效率和动力学研究信号通路调节SSC的?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>胶原酶（IV型） </td><td>西格玛</td><td> C5138 </td></tr><tr><td>胰蛋白酶EDTA </td><td> Invitrogen公司</td><td> 25200-056 </td></tr><tr><td> DNaseI </td><td>西格玛</td><td> DN25的</td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 31053-028 </td></tr><tr><td>台盼蓝染色</td><td> Invitrogen公司</td><td> 15250-061 </td></tr><tr><td>尼龙网状细胞过滤</td><td> BD公司</td><td> 352340（40微米） 352350（为70μm） </td></tr><tr><td>马利兰</td><td>西格玛</td><td>具推拔孔</td></tr><tr><td>薄壁玻璃毛细管</td><td>世界精密仪器</td><td>台湾100-3 </td></tr><tr><td>的屋宇署intramedic plyethylene管材（PE100的） </td><td>屋宇署</td><td> CA-63018-725 </td></tr><tr><td>爱惜康6-0丝线缝合</td><td>爱惜康</td><td> 706G </td></tr><tr><td>伤口剪辑</td><td>屋宇署</td><td> 427631 </td></tr><tr><td> Sigmacote </td><td>西格玛</td><td> SL2的</td></tr><tr><td> X-gal的</td><td>西格玛</td><td> B4252 </td></tr><tr><td>亚铁氰化钾</td><td>西格玛</td><td> P9387 </td></tr><tr><td>铁氰化钾</td><td>西格玛</td><td> P3667 </td></tr><tr><td>氯化镁</td><td>西格玛</td><td> 208337 </td></tr><tr><td>去氧胆酸钠</td><td>西格玛</td><td> D6750 </td></tr><tr><td> ，N，N-二甲基甲酰胺</td><td>西格玛</td><td> D4551 </td></tr><tr><td> IGEPAL的CA-630 </td><td>西格玛</td><td> 18896 </td></tr></tbody></table>

germ cell transplantation and testis tissue xenografting in mice

生殖细胞移植是由拉尔夫Brinster博士和他的同事在美国宾夕法尼亚大学在1994年1,2。这些开创性的研究表明，捐助衍生的精子和精子生产由收件人动物2到不孕受体小鼠的结果曲细精管生精细胞注射从肥沃的供体小鼠。的使用捐赠受援国动物携带细菌β-半乳糖苷酶基因，鉴定捐助衍生的精子和捐赠单体的传输后代男性。令人惊讶的是，进入曲细精管的管腔移植后，移植的生殖细胞能够从管腔车厢移动其中精原细胞位于基底膜。它被普遍接受，唯一的SSC能够殖民的利基，并在收件人睾丸重新建立精子。因此，生殖细胞移植提供了一个功能的方法来研究在睾丸干细胞小生境的特点推测的精原干细胞。到今天为止，生殖细胞移植用于澄清基本的干细胞生物学，生殖细胞的遗传操作，通过生产转基因动物移植4,5之前，研究Sertoli细胞的生殖细胞相互作用6,7，SSC的归巢和定植3， 8，以及SSC的自我更新和分化9,10。 11大物种生殖细胞移植也是可行的。主要应用在这些保存生育能力，传播精英在动物种群遗传学，转基因动物精子和SSC生物学研究这一技术是后勤更加困难和昂贵的，比在啮齿类动物中的一代。从大型物种的生殖细胞移植到小鼠结果在colonizat曲细精管离子的供体细胞和精原干细胞扩张，但没有充分分化，大概是由于收件人的体细胞与生殖细胞系统发育遥远的物种12间不相容。另一种方法是从大型物种与周围的体车厢的生殖细胞的移植。我们首次报道于2002年，即从下背部皮肤移植免疫缺陷小鼠的不成熟男性睾丸组织的小碎片能够生存和接受的全面发展与生产的施肥主管精子13。此后睾丸组织异种移植已被证明是成功在许多物种中，并作为一种有价值的替代研究小鼠14大型动物的睾丸发育和精子出现。

Watch this Scientific Journal Video about Germ Cell Transplantation and Testis Tissue Xenografting in Mice at JoVE.com

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

生殖细胞移植和睾丸组织异种移植的协议描述。在捐助源性受援国睾丸精子的生殖细胞移植的结果和鉴定精原干细胞（SSCS）为代表的功能重建法。睾丸组织异种移植在受体小鼠睾丸发育和精子各捐助国物种再现。

生殖细胞的移植和睾丸组织在小鼠异种移植

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

germ-cell-transplantation-and-testis-tissue-xenografting-in-mice

Research

JoVE Journal

Biology

29.6K 次观看.  University of Calgary. 生殖细胞移植和睾丸组织异种移植的协议描述。在捐助源性受援国睾丸精子的生殖细胞移植的结果和鉴定精原干细胞（SSCS）为代表的功能重建法。睾丸组织异种移植在受体小鼠睾丸发育和精子各捐助国物种再现。

视频: Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

分离和造血干细胞移植（造血干细胞）

科研

生物学

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

在成年斑马鱼的全肾骨髓移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

小鼠异位心脏移植

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

小鼠小肠移植

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

人CD40激活的B细胞的生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

异位和原位气管移植小鼠模型研究闭塞性气道疾病的发展

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

涡固定，局部照射，组织移植

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...