作者想感谢里克·帕吉特博士（瓦克斯曼研究所，罗格斯大学，新泽西州）DBL-1基因和注射标记，博士，克里Rongo（瓦克斯曼研究所，罗格斯大学，新泽西州）的礼物。巴特格兰特博士的实验室进行基因枪轰击低拷贝数一体化的GFP标记DBL-1构造。勒内·加西亚实验室提供在创造texIs100技术援助。勒内·加西亚，罗宾Lints，李洪敏秦实验室提供了富有成效的意见。这项工作的启动资金是由TAMHSC从分子和细胞医学系。由部门和医学院院长办公室TAMHSC学院提供的资金购买该化合物的范围和旋转盘共聚焦。

1。筛选菌株建设精心设计的筛选菌株屏幕上的成功是至关重要的，并已在别处3。对于一些研究人员，用应变表示有形的产品，从转基因的实验需要。许多窝藏集成转基因的菌株是从总部大楼或个别研究人员。如果转基因株所需的屏幕上，但不可用，那么它可以使用像轰击紫外/ TMP 5，4，或MOS座子插入6发表的方法产生。为了形象化我们感兴趣的蛋白质，我们在成熟cDNA序列的帧中插入GFP编码序列（我们使用DBL-1序列）。因为这GFP融合蛋白是不可见的解剖范围，我们使用了共注射是可见的标记，并没有影响到我们感兴趣的蛋白质观看（TTX-3P :: RFP </eM&gt;，审查其他几个标准标记，见7）。然后，我们创建了一个整合的转基因染色体外数组。我们发现，转基因轰击产生一个低拷贝数的集成线路，无论是获救的解剖显微镜型，也不产生可见GFP标记的产品水平的（Beifuss和Gumienny，未发表）。紫外/ TMP集成多个拷贝的染色体外最初是由注射8,9阵列产生可见，挽救转基因产品的水平（图3A）。抗GFP抗体免疫印迹证实，转基因（等位基因名字texIs100）表示，正确处理（Beifuss和Gumienny，未发表）。应综合转基因异交五次，删除多余的背景突变，不论其来源。 对于我们的屏幕上，我们希望利益的唯一蛋白转基因标签的形式。因此，我们删除功能的内源性Ğ烯引入损失的功能突变DBL-1等位基因。 最后，我们的应变敏RNA干扰的影响。许多基因，从库RNAi技术将产生更严重的一个基因产物减少，如果动物含有突变的敏感动物的RNAi 10,11的影响组织的利益（S）应考虑选择适当的时RNA干扰敏感的背景11。我们使用规范的RRF-3（pk1426）等位基因，使我们的筛选菌株。快速反应部队-3是一种RNA指导的RNA聚合酶（RDRP）同源，通常体RNA干扰抑制10。可以用在其他的RNAi ERI-1或林ERI-1-15B hypersensitizing基因突变，而增加的RNAi的有效性11-13。 我们做了筛选应变基因型RRF-3（pk1426）; texIs100; DBL-1（NK3）。 2。选择和准备RNA干扰库市售C。线虫基因库代表约55％的预测基因在 C或87％ 线虫单独（维达尔实验室或Ahringer的实验室图书馆，分别）。个人克隆转让（开放式生物系统公司，Geneservice有限公司）。我们选择了由，维达尔实验室（开放Biosystems公司）建造的ORF的RNAi库，因为它的克隆大多是全长cDNA克隆的网关和下游应用（Invitrogen公司，卡尔斯巴德，加利福尼亚州）的准备。原Ahringer实验室的基因组cDNA文库包含不广泛用于下游表征实验14,15有用的cDNA片段。这两个库使用一个向量，它包含两个T7启动子侧翼插入（图1）。生长在菌株HT115异丙基-β-D-硫代半乳糖苷（IPTG）诱导后，表达T7 RNA聚合酶的结构。包含库克隆的细菌，用IPTG诱导。产生双链RNA（见第3.4步）。 在收到重复的整个库，并为所有的实验中使用的重复。正本和副本库应存放在不同的-80℃的冰柜连接电气线路分开。 3。细菌库克隆的制备<ol><li> （在两个月内选定喂养克隆库，以及阳性和阴性对照，（我们增长8％100毫米板文化）标记LB-羧/四环素板的连续细菌实验）和孵化，在37°C过夜（ &gt; 16小时）（图1）。对于每24孔板中，我们使用两个控件。 BLI-4（K04F10.4），阳性对照，产生剂量依赖后胚胎pheontype 16,17。阴性对照，C06C3.5，是一个预测的假（ <a href="http://WormBase.org">WormBase.org</a> ）的，导致没有观察到的表型（Beifuss和Gumienny，未发表）。 </li&gt; <li> （实验后两个月内，最好早晚）准备24线虫生长培养基（NGM）板含有25 - 50微克/毫升羧和1mM的IPTG的。保存于4°C。 </li><li> （1天），对于每个克隆，接种2毫升LB培养基含有50微克/毫升羧与从挑染板（S）（步骤3.1）使用标准的无菌技术的单菌落。在37°C过夜（16小时）在振动筛一套220 - 240转（图1）。 16小时后，该解决方案应该是阴天。 </li><li> （2日）第二天早上，诱导双链RNA（dsRNA）的生产，加入IPTG至终浓度为1mm的文化一倍。孵育文化在37°C间，220 - 240转，一个额外的4 - 5小时。这一步，​​确保更加一致和强大的RNAi的基因产物诱导比单纯依靠IPTG的NGM的板（3.5步）的琼脂击倒。 </li><li>现货30μL每个诱导细菌立方米lture成独立的井，24井NGM的RNA干扰盘含有25 - 50微克/毫升羧和1mM IPTG（图1，用于跟踪RNAi实验的模板见图4）。放板，发现，在20分钟的无菌流罩或直至细菌斑点干。不要overdry。过度干燥导致琼脂边缘拉板和动物将是无可挽回的，如果它们产生的缝隙爬进。 </li></ol> 4。线虫的制备启动与人口的线虫上演。举办​​动物之间的控制和实验RNA干扰试验中观察到的差异仅仅是由于在动物发育阶段的差异（不过，如果RNA干扰导致发育迟缓，这应该由筛选（图2）所指出的降低了机会） 。此外，开始与L1幼虫的RNAi实验省却根RNA干扰胚胎致死的潜在的影响因子ES也可起到胚后角色的利益。 分期完成第一漂的混合菌株的筛选，其中只有蛋壳保护胚胎生存阶段人口。这个阶段的动物，在20°C时约12小时，或约18小时16°C间18。要更加紧密地阶段的动物，动物幼虫在第一阶段（L1）的M9培养基中没有食物，让胚胎孵化逮捕。饥饿的L1动物放在粮食恢复性增长，从同一起跑线19岁。 <ol><li> （1天），使用三个100毫米美联储板，干净的筛选菌株，包含妊娠的成人和胚胎的动物。从使用无菌M9的板洗整个板面的液体轻轻pipeting松开动物胚胎的动物。转移到无菌的15 ml管洗螺丝帽。 </li><li>如果洗大于3.5毫升，自旋向下的动物（〜3000转每分钟为30秒），并取出3.5毫升液体。如果洗是lESS超过3.5毫升，加H 2 O或M9管占3.5毫升。 </li><li>处理化学物品时，佩戴适当的防护装备（实验室外套，手套和护目镜）。混入1毫升新鲜的漂白剂（5％次氯酸钠，小于1岁）0.5毫升5 N的氢氧化钠。这种混合物加入3.5毫升线虫洗。启动一个定时器计数。这个过程不应该超过10分钟。如果确实如此，那么胚胎将被威胁，可能会减少产量，或漂白剂是老的，应予以更换。 </li><li>涡管约10秒钟。重复4至10分钟，每两分钟的涡旋。每个涡旋后，观察动物尸体的解剖范围下的解决方案。 </li><li>当胚胎被释放和分散成人（6 - 8分钟），颗粒管通过旋转表离心机（〜3000转，30秒）的胚胎。 </li><li>尽可能多的上清吸不干扰颗粒。要小心，不贪婪。 </li><li>添加无菌M9的〜10毫升。涡简要或摇晃以及悬浮颗粒。 </li><li>旋转并再次上清。如果漂白剂的气味可以在管检测，重复冲洗必要。 </li><li>重悬胚胎在10 - 15毫升无菌M9和转移到一个小的（25毫升）烧瓶（曝气）。握手，在适当的应变和实验温度（15 - 25℃）过夜（&gt; 16小时在20°C）。动物在这段时间内，将所有孵化和在L1滞逮捕。 </li><li> （2日）降速L1的阶段动物15毫升管和重悬在0.5 - 1毫升M9（〜3000转，30秒）。 5μL的溶液放置在一个单独的板块和计数动物的数量。作出调整音量，如果需要的话，产生30蠕虫/ 3 - 10μL的解决方案。 </li><li>甚至帮助他们识别科学约30到细菌的次氯酸钠同步的L1准备的24孔板以及在每个阶段的动物。太多莫重新超过30％以及动物可能导致臻于成熟前的饥饿人口。让干板盖歪。盖上盖子，用橡皮筋固定。翻转板，并放置在适当的温度，它需要进球阶段所需的时间。因为RRF-3（pk1426）是一种温度敏感等位基因，我们筛选应变动物生长在15 - 17°C的三到四天观察成年阶段的动物（图1）L4。 </li></ol> 5。线虫的观察一旦线虫生长所需的阶段（图2A）（5天），确认阳性和阴​​性对照产生预期的表型，用解剖显微镜。我们使用BLI-4控制RNAi的疗效，因为它会产生剂量依赖性后胚胎的缺陷，发育被捕，小幼虫（水泡成人（温和的RNAi型，不显示）的范围从强，电子xpected RNA干扰型）（图2B）。然后观察各实验动物使用解剖显微镜，并注意明显的异常表型诱导RNA干扰（图2C）。被证实后控制总表型注意到，感兴趣的表型筛选RNAi实验。我们使用复式显微镜配备荧光和63X客观观察至少五个每个RNAi实验动物，开始与对照组（图3）。 <ol><li>准备4％的琼脂垫（H 2 O 4％琼脂），幻灯片（标准的75×25毫米）。要确保琼脂垫的厚度均匀，置于洁净的载玻片之间的两个垫片（每间隔两个实验室磁带层上，在显微镜幻灯片）。约100μL熔融的4％到清洁玻片中心琼脂（2至3滴巴斯德玻璃吸管）吸取。形成覆盖很快，但轻轻地放置在顶部的垫片使用一个额外的幻灯片熔化的琼脂培养基垫第二熔化的琼脂。暴露琼脂轻轻滑动载玻片分开，坚持一个幻灯片离开垫垫。 </li><li>将1〜5μL麻醉下降琼脂板上（左旋咪唑或叠氮化钠，例如）。山8 - 12日在麻醉动物。覆盖22×22毫米＃1.5盖玻片（或最适合的复合显微镜的盖玻片厚度）使用复式显微镜观察动物。 </li><li>每个RNAi实验纪录基因，​​观察动物的数量和表型的结果。一个屏幕，制造和使用标准模板（例如，图5）。 </li><li>另外，二次型验证RNAi的生产型，如果可能的话，重复实验。 （例如，例如屏幕显示用GFP标记后海湾干线，1 20作为辅助屏幕作为主屏幕和车身尺寸的本地化改动。）细菌可以从相同的连胜（储存在4°C）使用长达两个月。老年人条纹5月r在液体中过夜培养和穷人的经济增长esult应首先restreaked。 </li><li>至少序列的cDNA插入，以确认插入匹配库的标签（Ahringer库了可靠性分析;的结束<a href="http://biocompute.bmi.ac.cn/CelRNAi/">http://biocompute.bmi.ac.cn/CelRNAi/</a> ）21。推荐的引物（无论从库中插入）退火L4440-派生的矢量和侧面插入部位网站:5&#39;-AGCGAGTCAGTGAGCGAG的3&#39;和5&#39;-GTAAAACGACGGCCAGT 3"（M13f20底漆）。 </li></ol>使用这种方法，一个人可以合理地举办和观察每周两到四个实验台。例如，动物上演周一和周二可以观察到周四和周五分别，可以再次在周五和周六举行的周一和周二观察。根据表型（S）甄别每一天，一组可能包括一个24孔板复合微SCOPY或表型迅速查明。因此，至少88种不同的RNAi实验可以观察到在一个星期内，考虑到正面和负面的监控和筛查率。解剖范围屏幕可以表现得特别快，而不需要安装上观看幻灯片的动物。多的人也增加惊人的时间表和/使用多种显微镜实验室的吞吐量。种植琼脂薄膜上的动物和转移的琼脂片含有动物直接观看幻灯片的一个替代方法，可以节省时间。这种变化被成功地用于筛选男性在400倍放大倍率22尾异常。 6。代表结果正常和改变本地化的GFP标记的后海湾干线-1的例子如图3所示。正常表达GFP标记后海湾干线-1包括腹神经索的细胞体和一排的punctae（图3A）。孖宝1小号严重衰减，当动物喂食的RNA，防止DBL-1 mRNA的翻译（图3B）。DBL-1（RNAi）技术也产生了小动物，在这个例子中（数据未显示）的辅助屏幕。很容易发现使用此屏幕设计应变（图3C）RNA干扰基因的影响后海湾干线-1定位。 <img src="/files/ftp_upload/3442/3442fig1.jpg" alt="图1" /> 图1。屏幕计划，以确定孖宝-1信号外监管 。一夜之间从进料库的细菌生长，用IPTG诱导，培养一个额外的4个小时，在37°C，使细菌产生双链RNA（dsRNA）的。 30诱导细菌培养液，发现每到24井NGM的（线虫的生长介质），含有25微克/毫升羧和1mM的IPTG的板，并允许在无菌流罩干。我们第一幼虫期（L1）的幼虫阶段让次氯酸钠处理电磁制动达索孵化媒体没有食物，从而产生一个L1滞（被捕的发展）。镀上约30同步L1阶段动物的RNAi库，它允许上演动物恢复性增长和消耗的dsRNA的细菌23创建从每口井含有细菌。在15°C时，年轻的成虫是一个可见的表型筛选后72小时。在这个年龄段，车身尺寸的缺陷是显而易见的荧光明亮从texIs100转基因表达。 <img src="/files/ftp_upload/3442/3442fig2.jpg" alt="图2" /> 图2。筛选前确定表型的例子 。所有板的动物图像，以及在相同的使用解剖显微镜的放大倍率。动物为饥饿的L1幼虫电镀成​​像后约72小时。所有图像处理相同。一）无毛的形态缺陷的基因，使RNA干扰。动物出现野生型。 b）护士艾BLI-4。动物发育停止显示，相比是微小的动物在面板A. C）DPY-13 RNA干扰。动物是在同一发展阶段，作为一个动物在面板，但显示一个"矮胖"的身体形态。 <img src="/files/ftp_upload/3442/3442fig3.jpg" alt="图3" /> 图3。荧光标记的蛋白质的例子和特定基因的RNAi改变本地化模式 。所有图像被旋转盘共聚焦显微镜，5秒630x放大倍率。曝光。比例尺= 10微米。所有图像处理相同。开放箭头表明细胞体。箭头标示punctae线。充满箭头指示GFP的一些aberrantly本地化标签后海湾干线，1。一）RNAi的喂养，假C06C3.5（"野生型"）。二）DBL-1（RNAi）的控制。 c）需要一个正常的本地化的GFP标记的后海湾干线-1基因的RNA干扰。 <img src="/files/ftp_upload/3442/3442fig4.jpg" alt="图4"/&gt; 图4。 RNAi实验（库克隆）在24孔板进行跟踪模板的例子 。此模板可用于创建一个永久记录的实验，不像直接标注板。 <img src="/files/ftp_upload/3442/3442fig5.jpg" alt="图5" /> 图5。为录制的RNAi表型的模板示例 。按需扩展。

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Mol. Life. Sci. , (2011).</li><li>Berkowitz, L. A., Knight, A. L., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Stable+Transgenic+C.+elegans+Using+Microinjection.">Generation of Stable Transgenic C. elegans Using Microinjection.</a> J. Vis. Exp. (18), e833 (2008).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods in Cell Biology. 48, 451-482 (1995).</li><li>Simmer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+the+putative+RNA-directed+RNA+polymerase+RRF-3+makes+C.+elegans+hypersensitive+to+RNAi.">Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi.</a> Curr. Biol. 12, 1317-1319 (2002).</li><li>Zhuang, J. J., Hunter, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specificity+of+Caenorhabditis+elegans+enhanced+RNAi+mutants.">Tissue-specificity of Caenorhabditis elegans enhanced RNAi mutants.</a> Genetics. , (2011).</li><li>Samuelson, A. V., Klimczak, R. R., Thompson, D. B., Carr, C. E., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Caenorhabditis+elegans+genes+regulating+longevity+using+enhanced+RNAi-sensitive+strains.">Identification of Caenorhabditis elegans genes regulating longevity using enhanced RNAi-sensitive strains.</a> Cold Spring Harbor Symposia on Quantitative Biology. 72, 489-497 (2007).</li><li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+misexpression+of+germline+P+granules+and+enhanced+RNA+interference+in+retinoblastoma+pathway+mutants.">Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants.</a> Nature. 436, 593-597 (2005).</li><li>Fraser, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+analysis+of+C.+elegans+chromosome+I+by+systematic+RNA+interference.">Functional genomic analysis of C. elegans chromosome I by systematic RNA interference.</a> Nature. 408, 325-330 (2000).</li><li>Kamath, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+functional+analysis+of+the+Caenorhabditis+elegans+genome+using+RNAi.">Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.</a> Nature. 421, 231-237 (2003).</li><li>Peters, K., McDowall, J., Rose, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+bli-4+(I)+locus+of+Caenorhabditis+elegans+disrupt+both+adult+cuticle+and+early+larval+development.">Mutations in the bli-4 (I) locus of Caenorhabditis elegans disrupt both adult cuticle and early larval development.</a> Genetics. , 129-195 (1991).</li><li>Thacker, C., Peters, K., Srayko, M., Rose, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bli-4+locus+of+Caenorhabditis+elegans+encodes+structurally+distinct+kex2/subtilisin-like+endoproteases+essential+for+early+development+and+adult+morphology.">The bli-4 locus of Caenorhabditis elegans encodes structurally distinct kex2/subtilisin-like endoproteases essential for early development and adult morphology.</a> Genes &#38; Development. 9, 956-971 (1995).</li><li>Byerly, L., Cassada, R. C., Russell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+cycle+of+the+nematode+Caenorhabditis+elegans.+I.+Wild-type+growth+and+reproduction.">The life cycle of the nematode Caenorhabditis elegans. I. Wild-type growth and reproduction.</a> Dev. Biol. 51, 23-33 (1976).</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>Savage-Dunn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screen+for+small+body+size+mutants+in+C.+elegans+reveals+many+TGFbeta+pathway+components.">Genetic screen for small body size mutants in C. elegans reveals many TGFbeta pathway components.</a> Genesis. 35, 239-247 (2003).</li><li>Qu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+analysis+of+the+Ahringer+Caenorhabditis+elegans+RNAi+feeding+library:+a+guide+for+genome-wide+screens.">Reliability analysis of the Ahringer Caenorhabditis elegans RNAi feeding library: a guide for genome-wide screens.</a> BMC Genomics. 12, 1471-2164 (2011).</li><li>Nelson, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Bow-Tie+Genetic+Architecture+for+Morphogenesis+Suggested+by+a+Genome-Wide+RNAi+Screen+in+Caenorhabditis+elegans.">A Bow-Tie Genetic Architecture for Morphogenesis Suggested by a Genome-Wide RNAi Screen in Caenorhabditis elegans.</a> PLoS Genetics. 7, e1002010 (2011).</li><li>Timmons, L., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+interference+by+ingested+dsRNA.">Specific interference by ingested dsRNA.</a> Nature. 395, 854 (1998).</li><li>Sarin, S., Prabhu, S., O'Meara, M. M., Pe'er, I., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+mutant+allele+identification+by+whole-genome+sequencing.">Caenorhabditis elegans mutant allele identification by whole-genome sequencing.</a> Nature Methods. 5, 865-867 (2008).</li><li>Lewis, J. A., Fleming, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+culture+methods.">Basic culture methods.</a> Methods in Cell Biology. 48, 3-29 (1995).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li></ol>

我们什么都没有透露。

这里提出的RNAi筛选方法使胚后的表型正常（或基因）所需要的基因产物的敏感和快速分析。所示的例子是屏幕中的荧光标记的蛋白质的亚细胞定位有关的基因。然而，这项协议可以进行修改，以确定影响其他感兴趣的胚后表型的基因。 此方法采用候选基因的方法，通过使用RNA干扰库的优势。利用诱变技术正向遗传筛选确定随机引起的新的等位基因，这些等位基因需要大量时?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> NGM的琼脂</td><td>线虫的生长介质</td><td> IPM的科技，公司</td><td>可以准备NGM的琼脂协议25 </td></tr><tr><td> M9培养基</td><td> 22MM KH 2 PO 4计 ， 42MM的Na 2 HPO 4， 86mm的氯化钠， 1毫米硫酸镁4 </td><td></td><td> 26 </td></tr><tr><td>琼脂</td><td>电解二氧化锰化学品公司</td><td> 1.01614.1000 </td><td> NGM的板在水中的2％。 4％显微镜幻灯片垫（最初釜和微波炉融化后）水。 </td></tr><tr><td>细菌用胰蛋白胨</td><td>碧迪 - Difco公司的CP </td><td> 211677 </td><td> 0.25％ </TD&gt; </tr><tr><td>经IPTG </td><td>研究产品国际股份有限公司</td><td> I56000-5.0 </td><td> 1毫米的最终浓度</td></tr><tr><td>羧</td><td>研究产品国际股份有限公司</td><td> C46000-5.0 </td><td> 50μg/ ml的工作液</td></tr><tr><td> LB培养基伦诺克斯</td><td>碧迪 - Difco公司的CP </td><td> 240230 </td><td> 20克/升</td></tr><tr><td>四环素</td><td>西格玛</td><td> 268054 </td><td> 12.5μg/ ml的工作液</td></tr><tr><td>次氯酸钠</td><td>任何品牌</td><td> 5％的家用漂白水</td><td>使用新鲜的漂白剂。 </td></tr><tr><td>氢氧化钠</td><td>任何品牌</td><td>中科院1310-73-2 </td><td> 5 N股</td></tr><tr><td> M9培养基</td><td> wormlab配方簿</td><td> <a href="http://130.15.90.245/wormlab_recipe_book.htm#Commonlab">http://1</a>30.15.90.245/wormlab_recipe_book.htm＃Commonlab </td><td> 26 </td></tr><tr><td>左旋咪唑</td><td>西格玛</td><td> 31742 </td><td> 100微米 - 1毫米的工作稀释</td></tr><tr><td>叠氮化钠</td><td> Fisher Scientific则</td><td> S227好</td><td> 10毫米M9的工作稀释</td></tr><tr><td> 24孔板</td><td>格雷纳生物一</td><td> 662160 </td><td>厂商VWR分销商</td></tr><tr><td>显微镜载玻片</td><td>任何品牌</td><td> 75×25×1毫米</td><td></td></tr><tr><td>镜盖单</td><td>任何品牌</td><td> 22×22毫米No.1.5 </td><td>使用显微镜制造商建议的厚度。 </td></tr><tr><td>复式显微镜</td><td>卡尔蔡司公司</td><td> A1M </td><td>使用目标和过滤器，以满足实验的需要。 </td></tr><tr><td>媒体泵</td><td>稳压器Varistaltic泵</td><td>凯特 模型＃72-620-000 </td><td>使用油管和设置适当的机器</td></tr></tbody></table>

rnai screening to identify postembryonic phenotypes in c. elegans

线虫已被证明是一个有价值的发现和功能分析很多基因和基因途径1模型系统。在这个系统中的研究更先进的工具和资源，促进具有更微妙的表型或角色的基因的不断发现。 在这里，我们提出了一个广义的协议，我们确定C.适应利用RNAi的利益胚后表型线虫基因。这个过程很容易地修改，以检测表型的选择，无论光或荧光光学显微镜解剖或复合。此场次协议利用生物体的有形资产和C.分子工具线虫研究社会生产。作为一个例子，我们展示了一个综合的转基因表达的RNAi屏幕荧光产品的使用，以确定这是正常的本地化所需的基因产品在后期幼虫和成虫。首先，我们用全长cDNA插入市售的RNA干扰基因库。这个库有助于减少候选基因产品通过RNAi多个考生的快速识别。第二，我们产生了一个综合的利益我们fluorecently标签蛋白基因的表达RNAi敏感的背景。第三，暴露孵出的动物RNA干扰，这个屏幕允许有一个重要的胚胎的作用，否则屏蔽后胚胎的作用，在调节蛋白基因的产品鉴定。最后，该屏幕采用单细胞分辨率配备一个复合显微镜。

Watch this Scientific Journal Video about RNAi Screening to Identify Postembryonic Phenotypes in C. elegans at JoVE.com

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

我们描述了致敏的方法来确定胚后调节蛋白表达和定位<em&gt; C。线虫</em&gt;使用RNAi技术为基础的基因组的屏幕和一个综合的功能，荧光标记的蛋白质基因的表达。

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 ...

rnai-screening-to-identify-postembryonic-phenotypes-in-c-elegans

Research

JoVE Journal

Biology

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

17.0K 次观看.  Texas A&M University System Health Science Center. 我们描述了致敏的方法来确定胚后调节蛋白表达和定位 C。线虫使用RNAi技术为基础的基因组的屏幕和一个综合的功能，荧光标记的蛋白质基因的表达。

视频: RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

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 ...

科研

生物学

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

定量和自动化高通量的全基因组RNAi屏幕 C。线虫</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

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

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

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

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

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, ...

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 ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using "Freeze-Cracking"

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体在染色<em&gt; C。线虫</em&gt;使用"冻结开裂"

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

在大规模的基因敲除<em&gt; C。线虫</em&gt;使用双链RNA料库产生失去功能的鲁棒表型

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...