1。手术之前执行，做到心中有数，有大约动物的研究和教学使用的法律和伦理要求<ol><li>所有手术过程中，必须经当地道德和法律的权威（IACUC或伦理委员会或民政厅等）。 </li><li>确保在开始前的地方是这个审批。 </li><li>如果有任何使用动物进行手术或批准的批准问题，不要继续。 </li></ol> 2。了解无菌技术<ol><li>作为一个概念的不育<ol><li>无菌技术的目标是在可能的情况下对动物和环境中的正常细菌负担，才开始手术，以减少或消除。这意味着，仪器消毒，手术工作表面消毒，动物和外科医生的手消毒。 </li><li>外科，正确执行，并不需要预防性应用抗生素或任何其他治疗，缓解疼痛以外。 </li><li>即使有良好的无菌技术操作，来自手术受的细菌仍然可能被引入创建手术伤口。适当的无菌技术，限制了风险的传染性病原体的引入，主要是细菌，到手术部位。 </li></ol></li><li>无菌区<ol><li>无菌领域包括手术区，该地区的动物准备，和前面的外科医生。 </li></ol></li><li>符不育<ol><li>不育中断发生时，外科医生接触的无菌领域以外的东西。这可能是她的脸，一个灯具，毫无准备的动物区，或一个非无菌的文书。 </li><li>这是预防性应用抗生素可用于的原因。 </li><li>请记住，一个特殊的活动正在发生。要冷静，周到，精确。 </li></ol></li></ol> 3。手术做准备<ol><li>鼠类手术并不需要一个专门的手术套房。理想的情况下，应该进行手术，在实验室内一个独立的区域，如一个罩或层流柜。无论如何，选择的地区应整洁，光线充足，出流量。鼠类手术往往是在实验室工作台进行。在安排手术区前，用消毒干净的台式。 </li><li>这是有利于外科医生有一个助理。这个助理可以帮助麻醉，准备动物和监测恢复。这使得外科医生作好准备，而动物准备，从而缩短动物在麻醉状态下的时间，也许更要手术。 </li><li>安排手术区，以便有三个不同的区域：准备，手术和恢复。 </li><li>对于准备区，组装所需的用品： <ol><li>润滑眼药膏</li><li>快船或剃刀</li><li>纱布</li><li>手术擦洗解决方案<ol><li>常用的解决方案是手术擦洗碘伏或洗必泰/ </li><li> 70％的异丙醇还用作手术擦洗的一部分。 </li></ol></li></ol></li><li>手术区，组装，并安排所需的物资。 <ol><li>为保持灭鼠温暖的热源<ol><li>理想的热源是一个循环水加热垫。电热垫不接受手术。用温水或化学手保温填充手术手套也可以使用。如果使用后两者之一，物体的温度必须在手术过程中仔细监测。 </li></ol></li><li>手术平台<ol><li>这可能是一个无菌的防水垫，放置在热源。 </li></ol></li><li>一次性用品<ol><li>这些可能包括：无菌手术窗帘，纱布，棉花施药，政府的啮齿类动物，组织灌溉解决方案的体液。 </li></ol></li><li>无菌仪器<ol><li>文书的补充，将因手术而异，但一般包括手术刀，组织拉钩，镊子，止血，和剪刀。 </li><li>仪器灭菌或消毒的解决办法，如果同样的仪器将被用于一个以上的手术受<ol><li>使用的一个热点珠灭菌消毒与动物之间的仪器是很常见的。 </li></ol></li></ol></li><li>关闭手术切口的材料。 <ol><li>所用的材料，将取决于手术的站点和应用程序，但可能包括：缝合线和针，钉书针和主食撒施，或组织胶</li></ol></li><li>任何其他特殊需要的设备（立体定位仪，植入物，手术显微镜） </li></ol></li></ol> 4。准备手术工具<ol><li>文书应保持清洁，在无菌手术开始。这通常是通过创建完整的手术，蒸压包。 </li><li>工具可用于一个以上的灭鼠手术无需高压灭菌和重新包装，但之间的啮齿类动物，必须清洗和消毒。 <ol><li>如果热珠消毒柜使用，一定要允许的文书，灭菌后冷却下来的时间。仪器可在无菌水浸泡冷却。 </li><li>也可用于化学消毒剂，但需要一个较长的接触时间（〜10分钟，可能是30-60分钟，需要漂洗）。酒精是不灭菌。 </li></ol></li><li>排列顺序，他们将使用无菌防水悬垂文书。 </li></ol> 5。准备手术的动物<ol><li>术前管理的三种麻醉，镇痛，抗生素。 <ol><li>麻醉手术所必需的。麻醉剂可能是通过吸入或注射给药。 <ol><li>目前最常用的吸入麻醉异氟烷。 </li><li>注射麻醉剂最常见的是氯胺酮和甲苯噻嗪的组合，可能与其他镇静添加。 </li><li>适当的剂量，请咨询您的设施的兽医人员。 </li></ol></li><li>先发制人的止痛药，建议，应在手术开始前管理。 <ol><li>常用的非甾体抗炎药包括氟尼辛和卡洛芬。 </li><li>可用的阿片类药物可能包括丁丙诺啡，吗啡或芬太尼。 </li><li>适当的剂量和给药途径，请咨询您的设施的兽医人员。 </li></ol></li><li>适当地进行手术，抗生素一般需要，除了一些高风险的胃肠手术。 <ol><li>如果使用抗生素的，用来提供围手术（第一切口前）。 </li><li>适当的抗生素，剂量，给药途径，请咨询您的设施的兽医人员。 </li></ol></li></ol></li><li>切口部位的手术准备<ol><li>剃除切口部位。一般剃须面积约3倍的建议切口的大小。剃光和准备区的大小可能是有限的动物或动物的面积大小。 </li><li>剃光面积消毒的手术擦洗溶液和异丙醇中使用的多个应用程序。应用程序通常手术擦洗第一，再用酒精。擦洗解决方案的一个最终的应用程序结束。 </li><li>手术擦洗是一个圆形图案，开始在该中心，并向外，或在较小的动物，一个单向的方式不断上升。 <ol><li>如果擦洗单向，地点在剃光面积crainial保证金无菌纱布，覆盖动物的毛发。轻轻地把握和稳定的动物通过无菌纱布。应用解决方案和酒精擦洗单向远离无菌纱布。 </li></ol></li></ol></li><li>手术区移动的动物<ol><li>向上，且无联系准备现场，准备手术部位移动的动物。 </li><li>手术区的动物的位置，使手术部位是向上的，面临的外科医生。 </li></ol></li><li>悬垂的动物<ol><li>如果动物是身披助理，助理必须记住，悬垂的外表面被认为是无菌区的一部分，不能由助理处理。悬垂往往是左边的外科医生，和悬垂性，包括手术包。 </li><li>一个标准的防水一次性纸悬垂性，一般建议。 </li></ol></li></ol> 6。准备手术的外科医生<ol><li>外科医生的制备应强调一个特殊的活动将采取地方，在其中的外科医生，必须认识到他们的每一个移动的无菌领域。 </li><li>的头发和脸一般都是一顶帽子和面罩，分别覆盖。在手术会议开始，这些应该是新的，但不需要将动物之间的改变。 </li><li>外科医生应该穿一次性手术衣，洁净的一次性白大褂，或者至少一个干净的白大褂。这并不需要改变动物之间，但应手术会话之间改变。 </li><li>手术前应清洗双手。应采用外科手术手套或考试手套。如果考试使用手套，他们应该用手术前正在使用的消毒剂。手套应改变与动物之间。 </li></ol> 7。在手术过程中<ol><li>维修的无菌领域。 <ol><li>一旦你的手是无菌的经营领域是无菌的，你将不得不将仔细，记得不要打破不育。这包裹体DES有一个无菌的领域上，您可以设置仪器，记住不要划伤你的鼻子，只有通过无菌的悬垂性的手术病人交接。 </li></ol></li><li>组织处理<ol><li>把手轻轻的组织。保持组织湿润，特别是如果手术延长。最小化创造组织的口袋和不必要的夹层。 </li><li>适当的组织处理，减少了术后感染，愈合速度的机会和风险，并减少手术后的疼痛。 </li></ol></li><li>更换液体丢失<ol><li>在啮齿类动物中的流体管理通常的路线皮下或腹腔内。 ，例如使用小动物- 1 - 2毫升腹腔注射对小鼠的小卷。 </li><li>温暖的液体可能有助于防止体温下降。小鼠和大鼠的体温比人类高，所以流体必须温暖。 </li></ol></li><li>保暖<ol><li>以下的动物在手术过程中的一个加热垫和笼在恢复过程中的位置是一个标准的方式来维持动物的体温。 <ol><li>小心加热垫的使用只有那些标记手术。其他可能诱发的热损伤。 </li></ol></li><li>温暖的液体，腹腔注射，也可以帮助温暖的动物。 </li></ol></li><li>操作显微镜可方便一​​些程序（微血管手术，年纪较大的外科医生等，也可使用的放大镜，放大镜一样，等取得） </li></ol> 8。手术后的恢复注意事项<ol><li>动物应恢复在一个平面的纸张床上用品（消毒纸巾等），而不是标准的畜牧业床上用品。 </li><li>保持动物的温暖。温暖，将有助于早日康复。这通常是通过放置回收笼半加热垫，使动物可以选择自己喜欢的的温度，因为他们恢复。 </li><li>不要返回动物区的动物或笼，直到所有的动物出现正常。任何已做了手术的动物必须有恢复能力的权利本身在笼子里，并能够移动之前被返回到持有约通常。 </li><li>监控动物手术后意外的疾病或感染的迹象。动物通常会失去了手术后的重量的少量，但适当的镇痛和提供食物，他们应该迅速恢复重量。 </li></ol> 9。手术后的监测是手术成功的最后一部分<ol><li>手术后5至7天，监测动物的一般状况。 <ol><li>手术后，动物应亮，警报和积极的。他们应该与cagemates通常交互，进食和饮水，并能够实现正常的特定物种的姿势。 </li><li>应审查这种变化的原因是抑郁，厌食，或呆滞的动物。考虑未经处理的疼痛或感染的可能性。 </li></ol></li><li>食品/液体摄入量也是重要的复苏。 <ol><li>手术后会有一些落客消费。这可以减轻，从而使食物和水更容易访问。 <ol><li>较长的吸管管可能是有用的的。 </li><li>每日提供在笼底的湿食品也可能吸引动物吃。 </li></ol></li><li>在大鼠异食癖导致一些止痛药，所以要知道，动物可以吃不恰当的项目（如床上用品）受伤。 </li></ol></li><li>监控动物切口感染的迹象。危险期是第7天，应每天检查的动物。 <ol><li>感染的症状包括红肿，放电（脓性或浆液性），疼痛，或切口（裂开）开幕。 </li></ol></li></ol> 10。完成手术后，最后一步就是衡量你的手术的成功<ol><li>模型诱导/删除/部分功能恢复？ </li><li>是有尽可能少的疼痛？ </li><li>有没有感染？ </li><li>成功！ </li></ol>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> American College of Laboratory Animal Medicine Position on Rodent Surgery. , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Animal Welfare, 9 CFR, Parts 1, 2, and 3. , .</li><li>Bradfield, J. F., Schachtman, T. R., McLaughlin, R. M., Steffen, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+and+physiologic+effects+of+inapparent+wound+infection+in+rats.">Behavioral and physiologic effects of inapparent wound infection in rats.</a> Lab Anim Sci. 42, 572-578 (1992).</li><li>Brown, M. J., Pearson, P. T., Tomson, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+animal+surgery+in+research+and+teaching.+AVMA+Panel+on+Animal+Surgery+in+Research+and+Teaching,+and+the+ASLAP+(American+Society+of+Laboratory+Animal+Practitioners).">Guidelines for animal surgery in research and teaching. AVMA Panel on Animal Surgery in Research and Teaching, and the ASLAP (American Society of Laboratory Animal Practitioners).</a> Am J Vet Res. 54, 1544-1559 (1993).</li><li>Brown, P. A., Hoogstraten-Miller, S., Reuter, J. D., Suckow, 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=Principles+of+Aseptic+Rodent+Survival+Surgery:+Parts+I+&#38;+2.">Principles of Aseptic Rodent Survival Surgery: Parts I &#38; 2.</a> Laboratory Animal Medicine and Management. , (2004).</li><li>Institute of Laboratory Animal Resources, National Research Council. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals. , 556-570 (1996).</li><li>Popp, M. B., Brennan, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+vascular+access+in+the+rat:+importance+of+asepsis.">Long-term vascular access in the rat: importance of asepsis.</a> Am J Physiol. 241, H606-H612 (1981).</li><li>Rutala, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APIC+guideline+for+selection+and+use+of+disinfectants.">APIC guideline for selection and use of disinfectants.</a> Am J Infect Control. 24, 313-342 (1996).</li></ol>

所有作者的工作查尔斯河，手术改变为研究动物的主要供应商。

啮齿类动物实验的手术是由兽医或医生很少开展。相反，它往往很少或根本没有正规的训练，在手术的原则和无菌技术的生物医学研究人员。在研究设置，手术通常是从同事了解到，自己平时没有受过正式训练任。在研究实验室进行的手术也可能被视为主要是由于准备产生一组动物为研究目标的动物模型的方法。虽然任何特定的手术是通常一个手眼coordination和技术技能的问题，都可​​以被收购通过实践，原则和的无菌技术的概念（和因此手术无感染）相关的技能可以不被收购通过实践完全和常常没有充分的研究人员转达。 手术原则的正规培训的情况下，可能会导致缺乏技术上是正确的手术前和手术后的爱护动物的平等意义的理解。手术本身和动物的围手术期护理的代价，将导致动物福利的增加，从手术模型获得的数据质量。例如，手术后感染的亚临床可以混淆的结果，可以在行为和生理疼痛看到的变化。作为猎物，大多数啮齿类动物有着惊人的坚忍的，虽然从手术后的疼痛或感染的痛苦，可能会出现一些临床症状。在啮齿类动物中的疼痛或窘迫的微妙迹象进一步的承认在啮齿类动物的这种迹象的生物医学研究人员的培训，普遍缺乏遮蔽。此外，鼠类是最活跃的的在黑暗的周期，当工作人员一般不以查看手术后疼痛的临床和行为的迹象。此影片不以适当的手术训练，这将理想的双手和他们的机构提供，但目的是让研究人员共同关心的问题的概述，并接受无菌程序和技术开发所需的最低限度的能力的视觉示范在啮齿类动物的生存手术的性能。

A correction to author list has been made for article Principles of Rodent Surgery for the New Surgeon.

Erratum: Principles of Rodent Surgery for the New Surgeon.

principles of rodent surgery for the new surgeon

科学和动物福利的原因，应采取手术基本概念和技术培训，不断寻求执行啮齿动物手术前。学生，博士后学者，鼠类作为一项研究协议的一部分在执行手术有兴趣的人可能不会有正式外科培训作为其所需课程的一部分。手术本身是一个技术性的技能，并与实践，这将提高。无菌技术操作原则，但是，往往仍然原因不明或untaught。对于大多数新的外科医生，这个重要的信息是在头痛医头，脚痛医脚的方式，或在工作中了解到，都不是理想的。它也可能使学习如何执行一个特定的手术难度大，新的外科医生学习外科手术技术和无菌操作的原则，在同一时间。本文总结和灭鼠手术成功所必需的基本手术技能和技巧的建议。本文的目的是通过用户的机构培训，以补充动手。

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Principles of Rodent Surgery for the New Surgeon

一个新的外科医生尝试手术之前，应该有基本的手术技术和概念的培训。本文将目前对啮齿类动物为重点的基本手术的考虑。

鼠类外科的原则，为新的外科医生

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

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111.9K 次观看.  Research Models and Services. 一个新的外科医生尝试手术之前，应该有基本的手术技术和概念的培训。本文将目前对啮齿类动物为重点的基本手术的考虑。

视频: Principles of Rodent Surgery for the New Surgeon

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Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

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Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

特定地点的重组酶（SSR）技术的原则

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

形成免疫突触和Kinapse研究的支持平面双层膜

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

鼠类全胚胎培养方法，使用的肩型瓶培养系统

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

恒化器中微生物系统生物学中的应用

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

肿瘤细胞球体的高通量图像分析：一个用户友好的软件应用来测量球体的大小自动准确

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...