我们感谢米勒实验室成员讨论和批判性阅读的手稿。这项工作是支持的弥敦道冲击卓越中心由新研究者奖老化DLM和国家机构的健康奖R00为AG030550到DLM的生物学基础。

1。建设环境试验箱<ol><li>选择最小的室为您的项目范围内的合理量。商会必须由气体（O）的防渗材料。高硼硅结晶菜，Anaeropack盒，或大型铸铁压克力盒（Ellard仪表），都可以使用。我们已经发现，9 50毫米的钢板可以在100×50 Kimex结晶菜适合。玻璃板可以用作耐热结晶菜肴的盖子。 </li><li>在选定的腔钻一个洞，适合塑料软管倒钩接头（科尔帕默）男性露儿。配件可以保证管件或用环氧树脂。上安装了类似的容器，使气体流向和室对面的拟合。如果可能的话，抵消孔，以增加湍流混合。 </li><li>获得压缩气体罐的定义O 2的浓度（平衡与N 2）认证标准为O 2合作的ntent，或缺氧条件下，纯N 2（&lt;10 ppm的氧气 ）。使用自动开关超过监管的长期研究，以避免破坏商会的氧含量。 </li><li>有机体缺氧反应，已被证明是随温度变化的15。放置在一个孵化室，不同温度下得以维持。孵化器内的温度可能是不平衡的，因此，它是审慎的温度数据记录器的使用，不断测量室内的温度。 </li></ol> 2。连接气环境商会<ol><li>对于所有连接，使用的八分之一英寸的外​​直径管材连接的任一单元连接器或压缩配件。油管应该是不透水和反应活性氧 ，如氟化乙烯丙烯（FEP）的或尼龙（科尔帕默）。为完成安装示意图， 见图1。 </li><li>连接的COM压气罐，流量控制设备，如质量流量控制器（塞拉利昂工具）或浮子（奥尔堡）。确保坦克从上游的压力范围内流装置和软管倒钩配件。两个阶段的监管一般都采用与第二阶段设置所需的压力，选择适当的流量第三节] </li><li>天然气水合物通过蒸馏水使用熔块气缸的气体洗瓶由冒泡，然后直接到一个环境试验箱配件，离开尾气开放第二件（ 见图1）。对于短期研究，气体水化防止板dessication的，但湿度监测可能需要长期研究。 </li><li>道康宁真空润滑脂可用于密封腔。室盖的地方权重，以确保不透气的密封。要确认密封和充足的流动，保持水的小水池，在手掌的y我们戴着手套的手出室装修和检查气泡。 </li></ol> 3。选择流量<ol><li>假设完美的混合，有90％的气态大气气体交换室的体积取代（菲克法）的时间。例如，在100毫升与100毫升/分钟的流量室，室在原来的房子空气将被替换为您想要1分钟后气体的90％，接近90％，每分钟将渐近完整的交换其后。 </li><li>较高的流速和较小的容器将更快地达到你所需的氧气浓度。 100×50 Kimex容器（400毫升），120毫升/分钟的流量将达到99.9％，在10分钟的交流（交流）。这个流量是适合大多数氧条件。我们的知识，有没有一个系统的调查O 2浓度的变化速率如何影响反应在 C 线虫 。 </li></ol><ol><li>暴露于缺氧条件下的蠕虫病毒通常逃脱琼脂平板表面。为了防止这种情况，周围板块边缘的棕榈酸（10毫克/毫升乙醇）放置一个环。棕榈酸会出来的解决方案，作为乙醇蒸发，形成了一个物理屏障。棕榈酸障碍，不影响产蛋，繁殖或在 C寿命率线虫 16,17。穴居在缺氧条件下不会发生更频繁，所以额外的预防措施，一般都不是必需的。 </li><li><ol><li>同步生成在碱性漂白液对非种子选手线虫的生长介质（NGM的）板18小滴漂白妊娠成人的人群。对比标准大批量的次氯酸钠漂白协议，挑NGM的钢板表面的漂白剂溶液滴在1-100动物，然后让漂白粉溶液吸收板<suP&gt; 18。至少12小时后，传输同步的L1幼虫与OP50活细菌接种板。另外，可以允许妊娠的成年人，鸡蛋2-3个小时躺在板产生的蠕虫组，将同步发展，或选择从混合的人口L4幼虫。 </li><li>避免暴露漂白胚胎缺氧，因为这可以减少活力13。收集幼胚（2-4细胞），妊娠的成年人被砍伤，在水的体积小，用刀片和移动通过口吸管板胚胎随后暴露缺氧的。 </li></ol></li><li>在环境舱的密封板。在常氧（室内空气）控制动物应保持在相同的温度视为蠕虫。有没有观察到样品留在室内空气，保持室内空气流淌在他们的相同室之间的差异。启动气体流量和维护所需的时间曝光。为了确保在坡道均匀，一定要取代之前曝光的气体洗瓶水。 </li><li>检测胚胎的生存，让蠕虫发展48小时后，返回到室内空气中，在这一点他们应该是第四阶段的幼虫/第一天的成年人。分数为求生存，以审查任何不能占的蠕虫。 </li><li>可视化动物缺氧，移动蠕虫的M9上下降了22毫米2盖玻片，并在M9 18日到2％琼脂糖垫反转。如果必要，左旋咪唑（25毫米）或叠氮化钠（10毫米），可以用作麻醉剂。叠氮化钠和左旋咪唑由于毒性可能混淆了一些意见和应该被明智地使用19。 </li></ol> 5。低氧暴露蠕虫（例如：HIF-1的西方分析的样品制备的快速收获） 许多缺氧诱导效应迅速扭转后，返回到室内空气中，包括恢复产蛋量12，有丝分裂表位的磷酸化13在胚胎发育和退化的HIF-1蛋白9,20。缺氧的动物​​的快速隔离是必需的，在这些条件下获得的重现效果。有了这个设置，动物可以收获和液氮冷冻，在不到两分钟。虽然手套箱缺氧商会在缺氧条件下，允许的样品操纵的成本和实用性比缺氧条件限制其效用。 <ol><li>布里斯托尔氮气蠕虫增长4个10厘米高增长（HG）板，直到大部分的蠕虫病毒是妊娠成人18。 15 mL锥形管中以1:5碱性漂白粉溶液洗蠕虫和轮流孵化，直到蠕虫开始溶解，不超过5分钟9。洗蠕虫与M9的三倍，在1500 XG彼此之间没有制动洗下来纺纱。 </li><li>到8 150毫米NGM的板和板漂白胚胎允许发展到L4幼虫（〜48小时布里斯托尔氮气在22°C）。板块移动环境试验箱和暴露在低氧（百万分之一千，5000 PPM）和缺氧（N2），4个小时的条件。根据实验设计，曝光时间会有所不同。虽然暴露缺氧对产蛋率有立竿见影的效果，两个细胞的胚胎，16日至18日曝光12小时后死亡。与此低氧室设置，曝光下限受制于必要的气氛中交换达到平衡率。 </li><li>一个标签为1.5 mL离心管和一个15毫升锥形管每个实验样本。缺氧的蠕虫更容易坚持管双方在收获。为了防止这种情况，地方100μL1％十二烷基硫酸钠（SDS），在每15毫升锥形管。如果SDS抑制下游应用，牛血清白蛋白（BSA）可以用来严防死守。常规使用的SDS或BSA似乎并不有一个明显的差异。加入50μL2X蛋白样染料（4％SDS，10％2 - 巯基乙醇和溴酚蓝30％甘油（W / V））1.5毫升离心管追踪。有液氮的杜瓦准备。 </li><li>缺氧和记录删除蠕虫后，时间步骤。取下盖子缺氧室，一个样板，并重新密封腔。用蒸馏水洗到尼龙过滤器的蠕虫，然后倒入15毫升锥形管。在台式离心机旋转的蠕虫在1500 XG与制动的15-20秒。 </li><li>使用真空大部分从管中取出上清液，留下的蠕虫病毒颗粒不变。 </li><li>使用吸管，将在50微升至1.5 ml离心管中的蠕虫病毒颗粒。密封管和浸在液态氮。 </li><li>重复，直到所有的样品已被隔​​离。室内空气样品的一致性控制遵循这些程序。样品可存放于-20°C。 </li></ol> 6。代表结果缺氧有机体的影响可以看出通过审查的可行性C 的成年线虫 （ 图2）。室内空气氧浓度（O 2的 21万PPM），奠定了野生型的布里斯托（N2）和HIF-1（ia04）缺失突变体的胚胎全部存活。 氮气蠕虫是能够适应和生存在5000 ppm的直径2至成年，而HIF-1的胚胎是不可行的。这表明，HIF-1是为适应不断变化的环境10氧水平的关键。无论是氮气，也不HIF-1的动物可以生存暴露于1,000 ppm的氧气 。 缺氧蠕虫直接可视化的使用范围和明确的容器（ 图3）解剖上是可行的。直接放置在低氧室的解剖范围，也没有必要从缺氧蠕虫观察有机体反应。的范围可以装有荧光illumation的（ 如图3 </stronG&gt;），进一步扩大可能在缺氧的意见类型。 <img alt="图1" src="/files/ftp_upload/4088/4088fig1.jpg" /> 图1。低氧室。气流方向的例子是用箭头表示。气体存储在定义的O 2的浓度（1）和连接两个阶段调节器（2）压缩气体罐。气体进入流管（3）的底部，顶部的退出在正确的流量。气体，然后流入泡沫瓶（4），保湿气体（确保正确的连接，通过观察气泡泡沫瓶）。水合天然气，然后将其传递到流入阀（5）在低氧室，暴露样品缺氧。最后的气体通过排气口钻室的房间的通风口进入。 <img alt="图2" src="/files/ftp_upload/4088/4088fig2.jpg" /> 图2。胚胎暴露于1000ppm的O 2的 5000 PPMØ可行性 <sub&gt; 2和常氧（〜21万ppm的氧气 ）。胚胎暴露于胚胎每24小时的连续流动氧舱氧条件。 蠕虫被转移到常氧条件下，允许48小时发展到成年，到成年的可行性，然后拿下。 N&gt; 50，n = 5。 <img alt="图3" src="/files/ftp_upload/4088/4088fig3.jpg" /> 图3。 C 的可视化线虫缺氧显微镜。蠕虫暴露对缺氧的使用方法概述。直接置于透明的环境室（构建具有耐热结晶菜和玻璃板）上的解剖范围的阶段。两种观点都表明，一个包括整个气流量设置，只用显微镜舞台上腔。

<ol>
	<li>Danovaro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+metazoa+living+in+permanently+anoxic+conditions.">The first metazoa living in permanently anoxic conditions.</a> BMC Biology. 8, 30 (2010).</li><li>Birner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Hypoxia-inducible+Factor+1+alpha+Is+a+Marker+for+an+Unfavorable+Prognosis+in+Early-Stage+Invasive+Cervical+Cancer.">Overexpression of Hypoxia-inducible Factor 1 alpha Is a Marker for an Unfavorable Prognosis in Early-Stage Invasive Cervical Cancer.</a> Cancer Research. 60, 4693-4696 (2000).</li><li>Harris, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+-+a+key+regulatory+factor+in+tumour+growth.">Hypoxia - a key regulatory factor in tumour growth.</a> Nat. Rev. Cancer. 2, 38-47 (2002).</li><li>Ramirez-Bergeron, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+affects+mesoderm+and+enhances+hemangioblast+specification+during+early+development.">Hypoxia affects mesoderm and enhances hemangioblast specification during early development.</a> Development. 131, 4623-4634 (2004).</li><li>Staff, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wheel-well+Stowaways+Risk+Lethal+Levels+of+Hypoxia+and+Hypothermia.">Wheel-well Stowaways Risk Lethal Levels of Hypoxia and Hypothermia.</a> Human Factors and Aviation. 44, 1-5 (1997).</li><li>Shen, C., Powell-Coffman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Analysis+of+Hypoxia+Signaling+and+Response+in+C.+elegans.">Genetic Analysis of Hypoxia Signaling and Response in C. elegans.</a> Annals of the New York Academy of Sciences. 995, 191-199 (2003).</li><li>Shen, C., Nettleton, D., Jiang, M., Kim, S. K., Powell-Coffman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+the+HIF-1+Hypoxia-inducible+Factor+during+Hypoxia+Response+in+Caenorhabditis+elegans.">Roles of the HIF-1 Hypoxia-inducible Factor during Hypoxia Response in Caenorhabditis elegans.</a> Journal of Biological Chemistry. 280, 20580-20588 (2005).</li><li>Wang, G. L., Jiang, B. H., Rue, E. A., Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor+1+is+a+basic-helix-loop-helix-PAS+heterodimer+regulated+by+cellular+O2+tension.">Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.</a> Proceedings of the National Academy of Sciences. 92, 5510-5514 (1995).</li><li>Epstein, A. C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+EGL-9+and+Mammalian+Homologs+Define+a+Family+of+Dioxygenases+that+Regulate+HIF+by+Prolyl+Hydroxylation.">C. elegans EGL-9 and Mammalian Homologs Define a Family of Dioxygenases that Regulate HIF by Prolyl Hydroxylation.</a> Cell. 107, 43-54 (2001).</li><li>Nystul, T. G., Goldmark, J. P., Padilla, P. A., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suspended+Animation+in+C.+elegans+Requires+the+Spindle+Checkpoint.">Suspended Animation in C. elegans Requires the Spindle Checkpoint.</a> Science. 302, 1038-1041 (2003).</li><li>Gray, 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=Oxygen+sensation+and+social+feeding+mediated+by+a+C.+elegans+guanylate+cyclase+homologue.">Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue.</a> Nature. 430, 317-322 (2004).</li><li>Miller, D. L., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+Elegans+Are+Protected+from+Lethal+Hypoxia+by+an+Embryonic+Diapause.">C. Elegans Are Protected from Lethal Hypoxia by an Embryonic Diapause.</a> Current Biology. 19, 1233-1237 (2009).</li><li>Padilla, P. A., Nystul, T. G., Zager, R. A., Johnson, A. C. M., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dephosphorylation+of+Cell+Cycle-regulated+Proteins+Correlates+with+Anoxia-induced+Suspended+Animation+in+Caenorhabditis+elegans.">Dephosphorylation of Cell Cycle-regulated Proteins Correlates with Anoxia-induced Suspended Animation in Caenorhabditis elegans.</a> Molecular Biology of the Cell. 13, 1473-1483 (2002).</li><li>Hu, C. -. J., Wang, L. -. Y., Chodosh, L. A., Keith, B., Simon, 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=Differential+Roles+of+Hypoxia-Inducible+Factor+1{alpha}+(HIF-1{alpha})+and+HIF-2{alpha}+in+Hypoxic+Gene+Regulation.">Differential Roles of Hypoxia-Inducible Factor 1{alpha} (HIF-1{alpha}) and HIF-2{alpha} in Hypoxic Gene Regulation.</a> Molecular and Cellular Biology. 23, 9361-9374 (2003).</li><li>Treinin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1+is+required+for+heat+acclimation+in+the+nematode+Caenorhabditis+elegans.">HIF-1 is required for heat acclimation in the nematode Caenorhabditis elegans.</a> Physiological Genomics. 14, 17-24 (2003).</li><li>Miller, D. L., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+sulfide+increases+thermotolerance+and+lifespan+in+Caenorhabditis+elegans.">Hydrogen sulfide increases thermotolerance and lifespan in Caenorhabditis elegans.</a> PNAS. 104, 20618-20622 (2007).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Massie, M. R., Lapoczka, E. M., Boggs, K. D., Stine, K. E., White, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+the+metabolic+inhibitor+sodium+azide+induces+stress+protein+expression+and+thermotolerance+in+the+nematode+Caenorhabditis+elegans.">Exposure to the metabolic inhibitor sodium azide induces stress protein expression and thermotolerance in the nematode Caenorhabditis elegans.</a> Cell Stress Chaperones. 8, 1-7 (2003).</li><li>Salceda, S., Caro, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+Factor+1&#945;+(HIF-1&#945;)+Protein+Is+Rapidly+Degraded+by+the+Ubiquitin-Proteasome+System+under+Normoxic+Conditions.">Hypoxia-inducible Factor 1&#945; (HIF-1&#945;) Protein Is Rapidly Degraded by the Ubiquitin-Proteasome System under Normoxic Conditions.</a> Journal of Biological Chemistry. 272, 22642-22647 (1997).</li><li>Theilacker, J. C., White, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+Gases+in+Air+and+Its+Affect+on+Oxygen+Deficiency+Hazard+Abatement.">Diffusion of Gases in Air and Its Affect on Oxygen Deficiency Hazard Abatement.</a> AIP Conference Proceedings. 823, 305-312 (2006).</li><li>Chua, B., Kao, R. L., Rannels, D. E., Morgan, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+protein+degradation+by+anoxia+and+ischemia+in+perfused+rat+hearts.">Inhibition of protein degradation by anoxia and ischemia in perfused rat hearts.</a> Journal of Biological Chemistry. 254, 6617-6623 (1979).</li><li>Probst, G., Riedinger, H., Martin, P., Engelcke, M., Probst, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Control+of+DNA+Replication+in+Response+to+Hypoxia+and+to+Inhibited+Protein+Synthesis+in+CCRF-CEM+and+HeLa+Cells.">Fast Control of DNA Replication in Response to Hypoxia and to Inhibited Protein Synthesis in CCRF-CEM and HeLa Cells.</a> Biological Chemistry. 380, 1371-1382 (1999).</li><li>Semenza, G. L., Sen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Oxygen Sensing. 381, (2004).</li><li>Chan, K., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoxia-Induced+Suspended+Animation+in+Budding+Yeast+as+an+Experimental+Paradigm+for+Studying+Oxygen-Regulated+Gene+Expression.">Anoxia-Induced Suspended Animation in Budding Yeast as an Experimental Paradigm for Studying Oxygen-Regulated Gene Expression.</a> Eukaryotic Cell. 7, 1795-1808 (2008).</li><li>Nystul, T. G., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+monoxide-induced+suspended+animation+protects+against+hypoxic+damage+in+Caenorhabditis+elegans.">Carbon monoxide-induced suspended animation protects against hypoxic damage in Caenorhabditis elegans.</a> PNAS. 101, 9133-9136 (2004).</li></ol>

没有利益冲突的声明。

这种方法提出了一个缺氧的环境，允许精确的氧气浓度维持在实验室环境建设的战略。这些商会提供了一个简单的方法暴露生物体特定的低浓度的O 2和监测的分子和生理输出。环境试验箱组装实验室，而不是商业购买，因此可以进行修改，以满足实验的需要。 这种方法的一个明显优势是连续流设计。这消除了通常与维护商会O 2的低浓度时，外部的氧?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>管（FEP和PTFE） </td><td>科尔帕默 聚乙烯</td><td> YO-95821-00（1/8“配屋） 06605-27（1/16×1/8“PTFE）”R-3603 </td></tr><tr><td>压缩配件。 </td><td>西雅图流体系统</td><td> 06363-58（M耦合器的1/16“） 06363-62（楼耦合器的1/16“） 06363-60（M耦合器的1/8“） 06363-61（楼耦合器的1/8“） </td></tr><tr><td>流管</td><td>奥尔堡</td><td> PMR3-010073（3输出） PMR1-013520（1输出） </td></tr><tr><td>质量流量控制器</td><td>塞拉利昂仪器</td><td> 810S - L-DR-2-OV1-SK1-V1的S1（大众特拉克） C100L-DD-2-OV1-SV1的PV2-V1 - 的SO-C10的（智能牵引控制系统2） </td></tr><tr><td>压缩气体罐</td><td>公司Airgas </td><td>订做&lt;/ TD&gt; </tr><tr><td>塑料男性鲁尔软管倒钩接头</td><td>科尔帕默</td><td> 45505-41（500系列1/16“） </td></tr><tr><td>投压克力盒</td><td> ellard仪表</td><td>定做</td></tr><tr><td>管接头（黄铜或不锈钢） </td><td>西雅图流体系统</td><td>的B-402-1（1/4“螺母） 架B-200-3（1/8“联盟T恤） B - 400集（1/4“套管） 的B-QM2-B1-200（质量管理机构的QC） 架B-200-1-2（1/8×1/8“男性CONN） </td></tr><tr><td>道康宁真空润滑脂</td><td> Sigma-Aldrich公司</td><td> Z273554 </td></tr><tr><td> AnaeroPack盒</td><td>三菱瓦斯化学公司</td><td> R684004（0.4升） R685025（2.5升） R685070（7.0升） </td></tr><tr><td>耐热气体洗瓶</td><td> Sigma-Aldrich公司</td><td> CLS31770500C（500毫升） CLS31770250C（250毫升） CLS31770125C（125毫升）&lt;/ TD&gt; </tr><tr><td>棕榈酸</td><td> Sigma-Aldrich公司</td><td> P0500 </td></tr><tr><td>羊抗鼠IgG辣根过氧化物酶</td><td>南方生物技术协会</td><td>一零三二年至1005年</td></tr><tr><td>的SuperSignal西微微Chemiluminsecent基板</td><td>皮尔斯化学</td><td> 34077 </td></tr><tr><td> 100×50玻璃结晶菜</td><td> kimax金布尔</td><td> 23000 </td></tr></tbody></table>

creating defined gaseous environments to study the effects of hypoxia on c. elegans

氧气是必不可少的，所有的多细胞动物的生存与一个已知的例外1。降低O 2的可用性（缺氧）中出现的疾病，发育正常或在2-5环境条件的变化状态。了解细胞信号途径参与缺氧反应可以分为治疗策略提供新的见解，对不同的人类疾病，中风，癌症。已阻碍了这一目标，至少有一部分，控制低氧暴露在模式生物基因服从相关的技术困难。 线虫作为模式生物缺氧反应的研究，因为它非常适合容易培养和基因操纵。此外，它可能没有因为C.影响因子的细胞反应到具体缺氧O 2的浓度研究线虫获得O 2的 （和其他气体扩散），而不是一个促进呼吸道系统6。 在 C已知缺氧反应涉及的因素是保守线虫 。缺氧的实际响应依赖于特定的O 2浓度可用。 在 C 线虫 ，中度缺氧暴露引出一个主要介导的HIF-1的转录反应，高度保守的缺氧诱导转录因子6-9，C。线虫胚胎需要HIF-1在5,000-20,000 ppm的生存Ø2 7,10 。缺氧是一个“O 2的比正常少”的总称。常氧（O 2的正常），也可以是很难界定。我们普遍认为室内空气，这是21万ppm的O 2的是常氧。然而，它已被证明是C。线虫有行为偏好O 2的浓度从5-12％（50,000-120,000 ppm的氧 ）11。在Larvae和成年人，HIF-1的行为，以防止缺氧诱导滞育的5000 PPMØ2 12。然而，HIF-1不玩作用，在低浓度氧 （缺氧，&lt;10 ppm的O 2的操作性定义）13。在缺氧，C。线虫进入一种假死的可逆状态，在所有显微镜下观察到的活动停止10。事实上，出现不同的生理反应，强调在不同条件下的缺氧O 2的浓度在实验控制的重要性。 在这里，我们提出了一个可靠和可重复性缺氧条件下产生的O 2浓度定义的环境商会的建设和实施方法。在不断流的方法，确保迅速达到平衡室，并增加了系统的稳定性。此外，透明度和无障碍商会让动物受到缺氧的直接可视化。我们进一步证明有效的方法收获C.线虫样本迅速缺氧暴露后，这是必须遵守许多的迅速反转的变化发生在缺氧10,14。这种方法提供了一个基本的基础，可以很容易地修改个别实验室的需求，包括不同的模型系统和各种气体。

Watch this Scientific Journal Video about Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans at JoVE.com

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

本文详细介绍了如何使用连续流缺氧商会产生与定义的O浓度的大气<sub&gt; 2</sub&gt;了解生物反应下降Ø<sub&gt; 2</sub&gt;。该系统易于安装和维护，并具有足够的灵活性，以适应广泛的O<sub&gt; 2</sub&gt;浓度和模型系统

创建定义的气体环境研究缺氧对<em&gt; C。线虫</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

creating-defined-gaseous-environments-to-study-effects-hypoxia-on-c

Research

JoVE Journal

Biology

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

12.0K 次观看.  University of Washington. 本文详细介绍了如何使用连续流缺氧商会产生与定义的O浓度的大气 2了解生物反应下降Ø 2。该系统易于安装和维护，并具有足够的灵活性，以适应广泛的O 2浓度和模型系统

视频: Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

线虫神经肌肉交界处的解剖和录制

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

生产 C。线虫</em通过重组工程与转基因 galK选择标记

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

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 ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...