笔者想涡移植的宝贵意见，以及过去和现在的桑切斯实验室成员感谢千代子小林和Kiyokazu阿加塔对这些技术的发展过程中的宝贵讨论。这项工作是由美国国立卫生研究院训练津贴（5T32 HD0791）主计长办公室和美国国立卫生研究院R37GM057260亚撒支持。 ASA是霍华德·休斯医学研究所的研究员。

注：此协议表明，潜在的有害物质的使用（铅和chloretone的）。收购，读，并按照对所有潜在的有害物质的MSDS。 1。动物文化，选择和制备<ol><li>动物养殖和处理使用涡水（1X 9 Monjuic蒙特惠奇盐）和塑料转让移液器。 </li><li>粉虱Schmidtea地中海性可用于在实验室中提出，在正常培养条件10。无性标本到生产所需的大小， 与地中海提出在正常情况下，在室温下，应投喂在使用前一至两个月的一倍至三倍的正常频率（每周2-3次）。另外，在正常频率无性动物喂食，可保持在10度摄氏无限期，以增加他们的平均规模。 </li><li>选择1至2厘米之间的动物长度和宽度超过2毫米，然后饿死的动物3-7天前使用。 </li><li>如果目的主机上，捐助者，或局部照射动物进行任何药物或放射治疗，在这一点上执行的处理（S）。 </li><li>如果进行药物治疗，需要喂养的动物，饿死动物额外的3至7天前使用。 </li></ol> 2。解决方案和材料的制备<ol><li>涡水溶解在0.1-0.2％W / V chloretone和寒心的解决方案上的冰准备，轻度局部麻醉剂，chloretone解决方案。 </li><li>如果进行局部照射，继续加强2.7。组织移植，继续加强2.3。 </li><li>使用本生灯，0.75 mm内部直径，用来切割移植组织，和0.7毫米外径，用于创建一个主机将收到1-2厘米的移植，毛细管90°角的孔弯曲从EA的结束CH管。为了节省材料，每个毛细管两端弯曲，打破他们的一半产生两个工具。小心不要管非常两端火焰。 </li><li>削减到指定大小的下列文件： <ul><li>黑色滤纸（切成长方形约2.5厘米×1.5厘米） </li><li>滤纸＃3滤纸（切成长方形约2厘米×0.5厘米） </li><li> kimwipe（折叠切成WADS约。3厘米×0.5厘米×4层） </li><li>卷烟纸（清除口香糖的条状，切成长方形约3厘米×2厘米） </li></ul></li><li>准备修改Holtfreter的解决方案（3.5克/ L的NaCl，0.2 g / L的碳酸氢钠 ，0.05 g / L的氯化钾，0.2 g / L的硫酸镁 ，0.1 g / L的氯化钙 ，pH值7.0-7.5）和酪蛋白饱和Holtfreter的解决方案和寒冷同时4°C。 </li><li>将折叠Kimwipe佩尔蒂埃冷却器板或其它冷却设备，坐落在解剖显微镜下的封口膜广场和地方。饱和的与冰鲜Holtf Kimwipereter的解决方案和地方两个黑色的Kimwipe滤纸矩形。 </li><li>线滤纸＃2滤纸的培养皿。浸湿的滤纸与Holtfreter的解决方案和冷藏冰菜肴。一个更大的盘子和滤纸班轮局部照射也可使用。 </li></ol> 3。麻醉和固定<ol><li>填写入菜与冰鲜chloretone解决方案和吸管虫的培养皿。仅用于移植的麻醉一台主机和一个捐助一次。许多动物（N&gt; 10），局部照射可以是麻醉一次。 </li><li>允许蠕虫，直到他们成为一动不动在chloretone溶液浸泡（5-10分钟）。 </li><li>冲洗吸取到他们与冰鲜Holtfreter的解决方案，填补了菜虫。 </li><li>吸取到黑色过滤纸浸透与冰鲜Holtfreter的解决方案和东方钳腹朝下固定动物。如果动物能够禄表参，吸收多余Holtfreter的解决方案，略有下降，您的佩尔蒂埃或冷板的温度，或增加chloretone治疗的长度。 </li></ol> 4。局部照射注意：请按照下列步骤准备动物局部照射。如果进行移植，而不是进行到第5。 <ol><li>从步骤2.7冰在冰桶内的顶级源X-射线照射，将适合放置冷冻培养皿。 </li><li>培养皿中的麻醉动物移动的黑色的滤纸上，他们是固定的安排。使用钳直接移动麻醉蠕虫，可能会伤害他们。 </li><li>运输安排顶级源X-射线照射的动物和位于从阴极管动物的距离最小，冰桶等，从而最大限度地有效剂量率。 </li><li>位置铅屏蔽（S）（ 图1）之间的动物和阴极ţUBE需要。盾牌应该是4.5到6毫米厚，以便为97至99 1 325kV X射线束的11％衰减。 </li><li>提供X射线剂量。如果需要完整的非屏蔽地区的干细胞消融，提供30 Gy或使用的X-射线照射。供参考，是30戈瑞相当于3.6分钟，在320千伏和10毫安，在以场源的距离为30厘米的精密X射线公司XRAD320。 </li><li>立即服药后完成后，处理的黑色滤纸，转让动物蠕虫进入冷涡水。允许涡水升温至室温和动物逐出自己的黑色滤纸。局部照射程序现已完成。 </li></ol> 5。组织移植<ol><li>使用移液管，镊子，安排单独的矩形黑色滤纸上Kimwipe麻醉主机和捐助者的蠕虫已冷却的PeltiER或冷却器板的解剖显微镜下。 </li><li>使用0.75毫米内径的毛细管从供体切出移植插件，使用镊子，放置在了主机的方式部分。如果移植材料在毛细管被卡住了，打跑钳。 </li><li>使用0.7毫米外径毛细管从主机中取出一个插件，使用钳位置移植到留下的洞。 </li><li>转让其黑到在步骤2.7准备的培养皿滤纸矩形移植主机。 </li><li>卷烟纸湿了一块酪蛋白饱和Holtfreter的解决方案框图如下图2A，并把它移植主机上。 </li><li>浸泡在酪蛋白饱和Holtfreter的解决方案和四片滤纸包住移植主机框图如下图2B。 </li><li>四切Kimwipe WADS浸泡在酪蛋白饱和Holtfreter的解决方案，并奠定他们步骤5.6滤纸（ 图2B）。替换盖和冰放在培养皿。 </li><li>转移到涡水，恢复，愈合和再生捐助的蠕虫病毒。 </li><li>当所有移植完成后，将移植到10℃培养箱蠕虫过夜。 </li><li>翌日清晨，照顾不来打扰的移植，发现蠕虫和转让（黑色滤纸）培养皿中充满了涡虫水。 </li><li>允许蠕虫逐出滤纸本身或用钳子轻轻取出。 </li><li>一次改变涡水2-3天。 </li></ol> 6。代表结果紧接着会出现局部照射涡虫正常，不受影响。根据交付的剂量和使用屏蔽的几何形状，照射组织可能倒退甚至解体7。屏蔽组织应保持不变。 FOLL由于组织回归和组织完整性的损失，芽将形成和失踪结构的再生（ 图3A）。如果截肢的部分照射的区域，被照射的组织将被救出（即防止倒退或瓦解）（ 图3B）。在健侧和截肢的情况下再生相比，被切除的非辐照涡虫（ 图3C），将被推迟。如果X射线剂量30 Gy的交付和部分照射的动物是无恙，成功的干细胞可以被确认使用的铅屏蔽相应的2至3天，局部照射后原位杂交技术，干细胞的模式消融标记SMED PIWI-1 12（又名smedwi-1）（ 图4）。 移植后的早晨，一个成功的嫁接移植的组织应该是显而易见的瓦特ithin宿主组织，加入到主机（ 图5A）的背部和腹部表面。偶尔，移植将坚持只腹侧或背侧表面。如果移植是彻底失败的，没有移植的迹象将是从主机（ 图5B）背或腹面可见。不久后的非辐照组织的成功移植到已经消融的干细胞原位杂交致死量照射13,14，SMED PIWI-1，就会发现主要在移植干细胞存在（ 图5C主机）。此外，非辐照组织的成功移植到致死剂量照射的主机将导致宿主组织的救援和长期生存的主机15。 <img alt="图1" src="/files/ftp_upload/4015/4015fig1.jpg" /> 图1。大会安排的基本组件在顶级定位的X射线源的X射线照射局部照射。（阴极管）麻醉涡位置下方的X射线源直接照射野内。为了最大限度地利用X射线剂量率，涡虫和X射线源之间的距离应尽量减少。铅屏蔽应定位之间的阴极管及麻醉蠕虫，尽可能接近实际的蠕虫。铅屏蔽应设计，制造和定位，所以它盾牌所需的组织，但完全暴露了其余的蠕虫。许多商业生产将产生定制的铅屏蔽，从简单的设计图，我们已经成功地使用了Alpha系统公司（Bluffdale，UT斯达康）。铅应该足够厚的屏蔽，以便需要的金额。例如，如果需要接近完成一个320千伏的X射线束屏蔽，铅应是4.5至6毫米厚的。涡虫和盾牌定位1 Holtfreter浸泡滤纸的培养皿中冰桶在于内衬。给出一个足够大的X射线辐射场和一些相同的铅屏蔽，许多标本可部分照射一次（图）。 <img alt="图2" src="/files/ftp_upload/4015/4015fig2.jpg" /> 图2。建设恢复室 （一）组织移植恢复室爆炸视图，显示所有被浸泡在酪蛋白饱和Holtfreter的解决方案后，顶部彼此分层组件。 （二）一个接近完成恢复室，说明环环相扣的滤纸安置＃3滤纸矩形紧贴包住麻醉涡，防止愈合过程中的运动。恢复室精心施工，防止动物运动和干燥，促进快速愈合和组织移植的更大的功效。 <img alt="图3" src="/files/ftp_upload/4015/4015fig3.jpg" /> 图3。代表成果简单后局部照射。杜波依斯描述7，（a）当涡虫的后半屏蔽铅，然后暴露在X射线照射前组织观察倒退回边界之间的辐射和屏蔽组织在这点未染色blastemas形成和动物开始再生。 （二）另一方面，当动物被斩首后，执行相同的局部照射（一），组织回归没有观察到，其余辐照前组织被救出。断头部分照射的动物再生头（二），然而，再生了显著延迟比未辐照（三）断头控制。 <img alt="图4" src="/files/ftp_upload/4015/4015fig4.jpg" /> 图4。代表outco我偏干细胞消融后局部照射。SMED PIWI-1的干细胞标记的原位杂交（WISH）全安装显示，野生型涡虫干细胞分布整个身体组织的异常与前感光细胞（箭头）和适当的咽（星号）14。 （一）希望SMED PIWI-1照射，但完全屏蔽控制涡虫固定3天照射后显示了干细胞的分布，是区分野生型涡虫。 （二）另一方面，希望为动物，只有部分屏蔽SMED PIWI-1，离开的前部和后部暴露，但也固定三天后照射表明，干细胞是从非屏蔽地区消融。比例尺是500微米。 <img alt="图5" src="/files/ftp_upload/4015/4015fig5.jpg" /> 图5。 EXA组织移植成功和不成功的mples。（一）实时图像成功移植的涡三天后移植的背，腹意见。移植（指示）清晰可见，背部和腹部表面，并与未染色的特点组织包围在移植物抗宿主接口。 （二）相应地，不成功移植显示不可见移植组织在移植网站（指示），而显示，从失败的移植愈合的，未染色的侧切伤口。只背或腹面坚持一个移植，可以像（一）从一个侧面和一个不成功的移植（二）从其他成功的移植。 （C）当野生型（WT）组织居民干细胞移植的干细胞已被消融到照射主机嫁接以后显露希望SMED PIWI-1天移植后，移植的成功清楚地显示干细胞移植物的位置（箭头）或周围的具体存在。比例尺是500微米。

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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=Expression+of+secreted+Wnt+pathway+components+reveals+unexpected+complexity+of+the+planarian+amputation+response.">Expression of secreted Wnt pathway components reveals unexpected complexity of the planarian amputation response.</a> Dev. Biol. 347, 24-39 (2010).</li><li>Salvetti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+stem+cell+plasticity:+neoblast+repopulation+in+non-lethally+irradiated+planarians.">Adult stem cell plasticity: neoblast repopulation in non-lethally irradiated planarians.</a> Dev. Biol. 328, 305-314 (2009).</li></ol>

没有利益冲突的声明。

固定化的重要性固定是迄今为止对这些过程的任何成功完成最关键的一步。如果涡虫是不当的固定部分照射之前，他们可能将下方的铅屏蔽，产生不一致的，混杂的结果。此外，如果动物没有得到充分固定移植后，主机的蠕虫病毒可能会搬走的移植组织，导致移植失败，在妥善医治主机。适当的固定是通过调整浓度或chloretone治疗的长度和滤纸的温度和湿度对涡虫休息。然而，?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>通用移液管</td><td>萨姆肯</td><td> 691 </td></tr><tr><td>毛细管（编号0.75毫米） </td><td>金控</td><td> 30-30-0 </td></tr><tr><td>毛细管（外径0.7毫米） </td><td>金控</td><td> 30-50-08 </td></tr><tr><td>封口膜中号</td><td>厂商VWR </td><td> 52858-076 </td></tr><tr><td> kimwipes 34155 </td><td>厂商VWR </td><td> 500029-891 </td></tr><tr><td>黑色滤纸</td><td>施莱歇尔＆Schuell </td><td> 10310809 </td></tr><tr><td>滤纸＃2过滤纸1002-055 </td><td> Fisher Scientific则</td><td> 09-810B </td></tr><tr><td>滤纸＃3过滤纸1003-185 </td><td> Fisher Scientific则</td><td> 09-820E </td></tr><tr><td>卷烟纸</td><td>锯齿形，原</td><td>不适用</td></tr><tr><td> Petri网菜</td><td>厂商VWR </td><td> 82050-544 </td></tr><tr><td>钳杜蒙，Inox公司＃5 </td><td>福斯特</td><td> 11251-20 </td></tr><tr><td> chloretone </td><td> Sigma Aldrich公司</td><td> 112054 </td></tr><tr><td>酪蛋白</td><td> Sigma Aldrich公司</td><td> C3400 </td></tr><tr><td>铅屏障</td><td> Alpha系统，UT斯达康公司，Bluffdale </td><td>定制设计</td></tr><tr><td> XRAD-320生物照射</td><td>精密的X射线，北布兰福德，CT </td><td>不适用</td></tr></tbody></table>

planarian immobilization, partial irradiation, and tissue transplantation

涡虫，淡水涡虫，已被证明是一个功能强大的系统解剖的后生再生和干细胞生物学1,2。涡虫再生的任何丢失或损坏的组织，是由成人干细胞可能称为neoblasts 3。虽然已明确显示，这些干细胞是多能干细胞和奇能够再造一个完整的动物4，在干细胞的细胞行为的人口和动态异质性在很大程度上仍然悬而未决。由于大量干细胞广泛分布在整个涡虫的身体计划，需要操纵分子和功能的研究在体内的干细胞亚群的先进方法。 组织移植和局部照射两种方法，可以为进一步研究分离涡虫干细胞亚群。每种技术都有明显的优势。 ţ问题移植允许引进的干细胞，成一个天真的主机，是天生的基因不同，或此前已治疗药物。另外，局部照射使干细胞内的一台主机，并列组织的干细胞缺乏的隔离，没有伤口的引进，臀位或任何组织的完整性。使用这两种方法，一个可以探讨的细胞自主和非自主因素，控制干细胞的功能，如增殖，分化和迁移。 5,6两个组织移植和局部照射7已被用于定义许多正在研究的问题，今天依然对涡虫再生的历史。然而，这些技术仍然未得到充分利用，由于以前的方法费时费力，不一致的性质。这里提出的协议，代表一个大的进步，在减少时间ND努力要再现嫁接或部分照射的动物产生大的人数接近100％的功效。我们涵盖文化的大型动物，固定化，局部照射，组织移植的准备，和动物复苏的优化。此外，这里所描述的工作表明局部照射方法为使用最广泛研究的涡，Schmidtea地中海的首次应用。此外，高效的组织移植在涡虫，打开门的功能测试天真或处理的干细胞亚群在复育实验，这早已是金标准方法化验成人干细胞的潜力在哺乳动物8。这些技术的广泛采用，无疑会导致更好地了解的组织稳态和再生过程中的成体干细胞的细胞行为。

Watch this Scientific Journal Video about Planarian Immobilization, Partial Irradiation, and Tissue Transplantation at JoVE.com

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

一个有效的方法是明确和一贯的涡虫之间的大小为嫁接组织描述。还包括如何结合局部照射活体动物，铅屏蔽的固定化技术用于移植，可适应的描述。

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 ...

planarian-immobilization-partial-irradiation-tissue

Research

JoVE Journal

Biology

19.4K 次观看.  University of Utah School of Medicine. 一个有效的方法是明确和一贯的涡虫之间的大小为嫁接组织描述。还包括如何结合局部照射活体动物，铅屏蔽的固定化技术用于移植，可适应的描述。

视频: Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

小鼠皮肤移植

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

科研

生物学

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 ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

药理和功能的遗传分析处理涡虫的再生日本三角涡虫</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

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 ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

快速分离纯化线粒体移植用组织分离和鉴别过滤

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

小肠部分梗阻的小鼠模型

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

平面性皱毛作为有毒刺激感的定量行为读出

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.