Mutant worm strains were kindly provided by the Caenorhabditis Genetics Center. This research was supported by a grant of the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (grant number: HI14C1277).

1.标记探针与地高辛的dUTP通过PCR <ol><li>用含地高辛的dUTP 10X dNTP混合物，如前所述13进行PCR标记。 </li><li>根据制造商的指令用旋转柱纯化纯化PCR产物。 <ol><li>如果探针是短于200碱基对，除去游离的地高辛的dUTP从反应混合物自旋柱色谱法，而不是旋转柱纯化。 </li></ol></li></ol> 2.准备聚赖氨酸涂层幻灯片注意:整个过程大约需要2小时。大多数的步骤都在室温下，除了在干燥步骤完成。 <ol><li>清洁幻灯片<ol><li>放置载玻片在一个塑料容器和漂洗载玻片简要地用蒸馏水​​（DW）。除去水和用含有1％玻璃清洁剂DW填充容器。 </li><li>搅拌15分钟载玻片在室温下（RT）50的转速。用DW洗涤3幻灯片次在RT每次5分钟。 </li><li>洗用70％乙醇的幻灯片搅拌15分钟。丢弃70％的乙醇，然后将玻片65℃的干燥块和风干15分钟上。 </li></ol></li><li>多井载玻片的聚赖氨酸涂层 注:玻璃片聚赖氨酸涂层是重要的一步，因为它提供了在整个染色过程中样品的附着力。差的涂覆的滑动将导致样品的损失。 <ol><li>稀释聚赖氨酸原液〜0.01％（重量/体积）在蒸馏水。添加的稀释0.01％20微升（重量/体积）聚赖氨酸到干净的载玻片上的孔中。 </li><li>在室温下孵育5分钟载玻片。 </li><li>放置在幻灯片的65℃的干体和空气干燥1小时上。 </li><li>存放在无尘箱聚赖氨酸幻灯片。 </li></ol></li></ol> 3.在载玻片上蠕虫固定（图1） <ol><li>准备胚胎FISH 注:嘉实蠕虫所有T之前他细菌食物被看显微镜下生长培养基消耗。饥饿减少产蛋成虫并增加卵孵化。详细方法在14,15进行说明。 <ol><li>根据标准方法14生长的蠕虫在50mM培养皿。 </li><li>所有的细菌食物消耗后，切琼脂培养基在季度锅铲。切割，以防止污染前消毒锅铲。 </li><li>把所有的一块琼脂上100mm的线虫生长介质（NGM）板。打开琼脂片倒挂的蠕虫到达新鲜食品的细菌。 </li><li>经过48〜72小时，收集与M9的缓冲区蠕虫。加入3 - 上NGM板5毫升M9的缓冲区中。 NGM的表面上吸移管的M9缓冲洗蠕虫。 </li><li>收集蠕虫的液体，并添加到15毫升管。 注意:如果在30％的蔗糖溶液的收获，离心后的蠕虫琼脂碎片。虽然碎片成团，虫子浮在的表面上。 </li><li>添加M9的缓冲区，以弥补体积到15ml。沉淀通过离心蠕虫在300×g离心3分钟，并除去大部分的M9缓冲液中。重复此步骤2次以上。 注意:如果蠕虫离心后仍然漂浮，设置离心机的制动电平为值= 1。 </li><li>吸M9的缓冲区。添加漂白解决蠕虫。每0.5ml蠕虫，添加6.5毫升的DW，将2ml次氯酸盐，加入1ml的5M的KOH。 </li><li>在50rpm在RT摇动3分钟孵育蠕虫。 15秒涡虫以机械剪切蠕虫和暴露的蛋。 </li><li>观察漂白过程中解剖显微镜下的管中。确保蠕虫切成两半和鸡蛋被释放。当最成年体的溶解，添加的M9缓冲液补足体积至15ml。 </li><li>离心在300×g离心3分钟。吸大部分的M9和补充新鲜M9的缓冲区，以弥补体积到15ml。重复洗涤步骤3次。 注意:避免使用蠕虫过量，因为它们妨碍漂白过程。保持整个反应时间不超过8分钟，直到洗涤。过度漂白鸡蛋产生强烈的自体荧光。 </li><li>添加具有聚山梨醇酯20（PBST）的磷酸缓冲盐水高达200微升和200微升4％低聚甲醛（PFA），以使2％的PFA。 注意:由于PFA是致癌物质，穿防护服，手套和眼罩使用PFA之前。 </li><li>添加40微升在2％PFA中的鸡蛋到聚赖氨酸涂覆的滑动的良好。 </li><li>放置载玻片在潮湿室中，并在室温下孵育15分钟。关闭潮湿室的幻灯片被放在里面之后。 注意:鸡蛋沉降到滑动件的底部而被固定。 </li></ol></li><li>准备FISH解剖性腺<ol><li>嘉实成虫吹打1毫升M9缓冲生长在50毫米NGM板。细菌性食物前收获蠕虫被耗尽。 </li><li>从任何细菌机智洗净虫体^ h M9的缓冲区，日2次。 注:残余细菌可能粘附到聚赖氨酸包被的载玻片与解剖性腺干扰。 </li><li>颗粒离心蠕虫在300×g的。删除M9和微量转移蠕虫空NGM板。 </li><li>加入30微升含有聚赖氨酸上的处理幻灯片的好2毫米左旋咪唑M9缓冲。 </li><li>加入500微升的M9缓冲液中至1毫升管中。使用该缓冲区口吸管转移的蠕虫。 </li><li>通过将毛细管在含有M9的缓冲区1毫升管填充M9缓冲嘴吸管尖。 </li><li>在解剖显微镜，把口吸管尖刚刚在成虫并拖动口吸管的头前，使蠕虫的头进入口腔吸管。一旦蠕虫的头部进入尖端，蠕虫的整个主体将被吸引到吸头。 </li><li>转移蠕虫使用吸管口聚赖氨酸涂层幻灯片。 </li><li>使用剃刀，切F中的头部或幻灯片上蠕虫的尾部的尖端。当蠕虫被切断，性腺就会弹出。性腺会坚持到幻灯片。 <ol><li>在一口井至少准备30蠕虫。多个孔可以用于另外30蠕虫。 </li></ol></li><li>把幻灯片在潮湿的室内和吸掉用口吸液管M9的缓冲区。 </li><li>通过在湿润室中在室温下加入20微升2％的PFA的15分钟固定的样品。 </li></ol></li></ol> 4.固定和透<ol><li>放置在干冰上的铝块，并将其存储在深冷冻机（-80℃）。商店甲醇和丙酮在-20℃。 </li><li> PFA固定步骤3.1.15或3.2.11后，用微量留下〜5微升固定液除去固定液。 </li><li>放在样品载玻片另一聚赖氨酸涂层幻灯片。除去用纸巾固定液如果溶液是过分的。一旦它们粘在一起，不要移动幻灯片。 </li><li>冻结幻灯片在铝块至少15分钟。 注:样品可以贮存至少2 - 3天。 </li><li>虽然被冻结的幻灯片，放冷含有甲醇和丙酮冰的罐子。 </li><li>就拿滑出并扭转他们冻结破解样本。丢弃上滑动。立即浸泡滑入冰冷甲醇5分钟。 </li><li>转移的幻灯片到冰冷的丙酮5分钟。 </li><li>洗玻片用PBST 3次，每次5分钟，以去除残留的固定剂。继续进行下一步骤，或在100％乙醇的幻灯片储存于4℃。 注:样品可以贮存至少2 - 3天。 </li></ol> 5.修正了杂交细胞<ol><li>添加的RNA酶溶液20微升（含10微克/毫升RNA酶A PBST）。孵育在潮湿室中的滑动，在37℃1小时。 </li><li>在2×盐水和柠檬酸钠与聚山梨酯20（2×SSCT），每次15分钟，洗涤载玻片两次。 </li><li>添加20杂交液微升，把湿热试验箱在37℃培养箱。 1小时后，除去通过移液杂交溶液。 </li><li>去除杂交液之前，准备探头。如果探针双链DNA，通过加热在95℃的干块上5分钟变性探针。加热后，在冰上冷却简要探头。 </li><li>加入10微升含探针与样品的杂交溶液。对于PNA探针，使用浓度以1:1的比例:2,000和DIG标记的探针，使用浓度以1:200的比例。盖上盖玻片样品。 </li><li>放与水的热块（80℃）上浸湿的纸巾。把热块的塑料盒盖以保持湿度和温度。 </li><li>加热块的温度稳定（80℃）后，放置在加热的纸巾样品载玻片，盖上塑料盒盖的样品。变性样品3分钟。 </li><li>公司乌瓦特在潮湿室中的载玻片在37℃下过夜。 </li></ol> 6.清洗和免疫<ol><li>预热杂交洗涤溶液（2×SSC，50％甲酰胺）至37℃。 </li><li>在RT洗在PBST样品两次5分钟。取下玻璃盖。 </li><li>在37℃下洗杂交洗涤溶液中的样品30分钟。 </li><li>洗在PBST中的样品玻片3次在RT。注:在室温下进行的湿热试验箱所有的后续步骤。 </li><li>加入20μl阻断溶液并在室温下孵育在潮湿的腔室1小时。 </li><li>除去封闭溶液，加入FITC缀合的抗洋地黄毒苷抗体溶液（1:200），3小时在RT或过夜，在4℃。 </li></ol> 7.安装和观测<ol><li>用PBST洗样品玻片2次，每次15分钟。 </li><li>加10μl用DAPI安装的解决方案。把玻璃盖，并轻轻按下。删除多余的解决方案用纸巾。 </li><li>为了防止安装溶液的蒸发，密封玻璃盖指甲油的边缘。 </li><li>共聚焦显微镜下观察。排除与高背景胚胎。注重与4场 - 20核。 </li><li>根据制造商的使用100X物镜指令拍摄图像。 注:激发样品与405 nm激光为DAPI，555纳米激光的Cy3，与488纳米激光FITC。 </li></ol> 8.端粒信号的量化注意:定量分析如先前16描述的进行。所有要被比较的图像，也可以采取包括曝光时间和光源相同的设置。 <ol><li>在.TIF格式导出图像。 </li><li>下载并安装图像分析软件。 </li><li>执行图像分析软件，点击同意按钮。 </li><li>单击打开按钮。通过双击图像文件打开端粒FISH图像。 </li><li>点击[编辑] - [选择加工区域]，通过左键单击并拖动选择感兴趣的区域。排除所有的非特异性染色。 </li><li>点击[措施] - [现货光密度]，选择端​​粒信号通道，输入文件名保存在.txt文件的结果。 注:结果列在顺序如下:现货荧光，现货和现货领域的背景强度。 </li><li>复制值和斑点的荧光斑点减去背景强度。值现在可以进行统计学分析。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fluorescence+in+situ+hybridization+(Q-FISH).">Quantitative fluorescence in situ hybridization (Q-FISH).</a> Curr Protoc Cell Biol. 18, 14 (2001).</li></ol>

The authors have nothing to disclose.

我们的协议的主要优点是过程的，但无细胞结构的形态明显损坏的简单性。几个步骤C.进行了优化线虫 FISH在此协议。成功鱼的关键步骤包括探针标记，胚胎和渗透固定。地高辛的dUTP标记方法提供了通过PCR或缺口转译一个易于使用的标记方法。要标记长的靶序列，缺口平移是首选。在这种情况下，探头应用适当的限制性酶消化，以促进探针的渗透。不推荐生物素-dUTP标记，因为生物素标记的探针产生从细胞质背景信号的过量。虽然内源性生物素封闭试剂是可商购的，它不企图。 该协议使用分离的胚胎，以增加效率的定量细胞的密度。 的C肠核。线虫是体积大，是多倍体，这有助于过多的数量和端粒点的强度相比，体细胞核的其余部分。出于这个原因，整个蜗杆不适合在C端粒量化线虫 。与此相反，因为它们提供了均匀的细胞而不多倍体的效果胚胎适合端粒长度的评估。 该协议使用2％PFA固色剂的正常工作端粒鱼。尽管戊二醛被报告给导致较低的背景信号，并在RNA原位杂交更难固定，戊二醛显著18增加自体荧光。这种过量背景治疗硼氢化钠，从而降低未反应的醛基后未废除。出于这个原因，不使用戊二醛。如果信噪比低，预杂交的时间可以延长到几个小时，以阻断非特异性结合位点。在此外，可以提高严格洗涤的时间，以降低背景水平。 在染色技术 线虫可以是适当的透治疗是一个挑战。C.线虫包含厚角质外骨骼抑制抗体和探头的渗透。传统的抗体染色方法涉及胶原酶的治疗，这需要大量的时间和优化过程。酶渗透的方法也损害了蠕虫形态的渗透效率的交流。冷冻裂纹和甲醇 - 丙酮处理被用来促进探针穿透。冷冻裂缝相比，几丁质酶或yatalase治疗19简便，快速。脱水或甲醇系列补液似乎并没有影响到鱼的质量。这些步骤在这个协议进行了简化。虽然有人报道这是必需的RNA原位杂交的蛋白酶K消化端粒的FISH结果与无蛋白酶K处理之间没有观察到19，明显的区别。此外，冷冻裂化提供用于在单个焦平面同时可视化许多细胞对于大定量分析一个易于使用的方法的胚胎。 通过使用PNA探针，端粒信号与同一个DNA探针得到的相比有显著增加。这可能是由于对于它的互补靶和其较小的尺寸（3重复对比4重复）更高的结合亲和力。荧光标记的PNA探针也直接结合到它的目标，减少后续步骤。保持在以下免疫步骤很强的亲和力，使后固定不必要，可避免背景噪声。 然而，一些限制在该技术中存在。其中之一是，在透稍微损害在高效探针穿透的成本的步骤的形态。性腺治疗S和用甲醇和丙酮的胚胎扭曲晶核圆形相比未处理对照。对于需要保存完好的形态实验，不同的通透方法应该尝试。另一个原因是，端粒信号被以任意单位定量。这主要是由于独立的实验中的变化。核糖体DNA可以被认为是内部对照正常化每个样品。更可以在参考20中找到有用的方法。 一个简单的端粒FISH协议这里描述，这需要最基本的步骤。许多步骤，减少了大量胚胎的分析。但是，进一步的修饰可为较强的信号进行，例如几丁质酶治疗的渗透，和其他创新试验。在细胞培养法的组合，超分辨率成像是可能的。该协议可能有助于发现在C.小说端粒维持机制线虫 。

端粒可以防止异常融合和降解染色体末端。哺乳动物端粒是由G-丰富的六聚体重复，TTAGGG和shelterin复合物。线虫的端粒重复序列是相似的哺乳动物（TTAGGC）的。大多数真核生物利用端粒酶端粒重复添加到他们的染色体末端。然而，10 -癌细胞的15％利用端粒酶独立的机制，被称为端粒（ALT）3的替代延长。此前，我们报道了端粒重复序列及其相关序列，命名为TALT，端粒酶的突变系存活的关键不育2端粒扩增。 端粒长度通过定量PCR或通过Southern印迹，其中提供总端粒-4,5,6,7-的平均长度测量。在全基因组测序数据端粒重复的读取次数也总端粒8含量的指标。虽然仙GLE端粒长度分析（STELA）可以提供一个单一端粒的长度，它不能提供端粒9的空间信息。而POT-1 :: mCherry报告蛋白提供在体内的端粒的空间信息，它不能代表双链端粒长度为POT-1是一个单链端粒结合蛋白10。 而上述的方法提供了重复序列的平均信息，原位杂交（FISH）荧光允许观察染色体规模的量和感兴趣的个体的序列的空间图案。代替的DNA的纯化，组织或细胞被固定到保持在FISH本地空间信息。因此，鱼为个别重复序列，如端粒重复序列的观察定量和定性的工具。 这个协议提供了两个TELO同时检测的有效方法基于从先前描述的方法11,12的改进仅仅是与其它重复。C.线虫幼虫或成虫多细胞生物体具有高度分化的细胞。细胞的异质性阻碍对大量端粒斑点的定量分析。为了最大限度地提高了分析的细胞数，胚胎分离并散布在用于FISH聚赖氨酸包被的载玻片。此外，该协议也可以与免疫结合。 作为该协议的工作证明，我们表明，它是可能的观察和量化两个不同的重复序列。针对TALT1 DNA探针，用简单的PCR结合地高辛的dUTP产生。那么这个TALT1探针和荧光标记的端粒PNA探针杂交同时进行。随后，地高辛被规范的免疫荧光法检测。我们在座的地方TALT1在TRT-1共定位与端粒的代表图像</eM&gt;幸存者。

<table><tbody><tr><td>PNA probe</td><td>PANAGENE</td><td>custom order</td><td></td></tr><tr><td>Anti-Digoxigenin-Fluorescein, Fab fragments</td><td>Roche</td><td>11207741910</td><td>use 1:200 diluted in PBST</td></tr><tr><td>Digoxigenin-dUTP</td><td>Roche</td><td>11573152910</td><td></td></tr><tr><td>Bovine serum albumin</td><td>SIGMA-ALDRICH</td><td>A-7906</td><td></td></tr><tr><td>Paraformaldehyde</td><td>SIGMA-ALDRICH</td><td>P-6148</td><td>prepare 4% paraformaldehyde by heating in DW with few drops of NaOH. add 0.1 volume of 10x PBS.</td></tr><tr><td>Vectashield</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>Hybridizaiton solution</td><td></td><td></td><td>3x SSC, 50% formamide, 10% (w/v) dextran sulfate, 50 &amp;#956;g/ml heparin, 100 &amp;#956;g/ml yeast tRNA , 100 &amp;#956;g/ml sonicated salmon sperm DNA</td></tr><tr><td>Hybridizaiton wash solution</td><td></td><td></td><td>2x SSC, 50% formamide</td></tr><tr><td>Formamide</td><td>BIONEER</td><td>C-9012</td><td>toxic</td></tr><tr><td>Methanol</td><td>Carlo Erba</td><td></td><td></td></tr><tr><td>Acetone</td><td>Carlo Erba</td><td></td><td></td></tr><tr><td>Heparin</td><td>SIGMA-ALDRICH</td><td>H3393</td><td>make 10 mg/ml for stock solution</td></tr><tr><td>Dextran sulfate</td><td>SIGMA-ALDRICH</td><td>67578</td><td></td></tr><tr><td>10x PBS</td><td></td><td></td><td>For 1 L DW : 80 g NaCl, 2.0 g KCl, 27 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O, 2.4 g KH&lt;sub&gt;2&lt;/sub&gt;PO</td></tr><tr><td>PBST</td><td></td><td></td><td>1x PBS, 0.1% tween-20</td></tr><tr><td>Polysorbate 20</td><td>SIGMA-ALDRICH</td><td>P-2287</td><td>Commercial name is Tween-20</td></tr><tr><td>Poly-L-Lysine solution (0.1% w/v)</td><td>SIGMA-ALDRICH</td><td>P-8920</td><td>prepare fresh 0.01% w/v solution before use</td></tr><tr><td>M9</td><td></td><td></td><td>3 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 6 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 5 g NaCl, 1 ml 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;, H&lt;sub&gt;2&lt;/sub&gt;O to 1 L</td></tr><tr><td>Bleaching solution</td><td></td><td></td><td>20% sodium hypochlorite, 0.5 M KOH</td></tr><tr><td>Antibody buffer</td><td></td><td></td><td>1x PBST, 1 mM EDTA, 0.1% BSA, 0.05% Sodium azide (toxic)</td></tr><tr><td>Blocking solution</td><td></td><td></td><td>Antibody buffer with 5% bovine serum albumin (BSA)</td></tr><tr><td>illustra Microspin G-50</td><td>GE healthcare</td><td>27-53310-01</td><td></td></tr><tr><td>20x SSC</td><td></td><td></td><td>To make 1 L, 175.3 g of NaCl, 88.2 g of sodium citrate, H&lt;sub&gt;2&lt;/sub&gt;O to 1 L, adjust pH to 7.0</td></tr><tr><td>2x SSCT</td><td></td><td></td><td>2x SSC, 0.1% tween-20</td></tr><tr><td>10x digoxigenin-dUTP mix</td><td></td><td></td><td>1 mM dATP, 1 mM dGTP, 1 mM dCTP, 0.65 mM dTTP, 0.35 mM DIG-11-dUTP</td></tr><tr><td>PCR purification columns</td><td>Cosmo genetech</td><td>CMR0112</td><td></td></tr><tr><td>Glass cleaner / ULTRA CLEAN</td><td>Dukssan pure chemicals</td><td>8AV721</td><td></td></tr><tr><td>Multi-well glass slide</td><td>MP biomedicals</td><td>96041205</td><td></td></tr><tr><td>Nematode growth media</td><td></td><td></td><td>To make 1 L, 3 g of NaCl, 17 g of agar, 2.5 g of peptone, H&lt;sub&gt;2&lt;/sub&gt;O to 974 ml. Autoclave and cool the flask. Add 1 ml of 1 M CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 ml of 4 mg/ml cholesterol in ethanol, 1 ml of 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;, 25 ml of 1 M KPO&lt;sub&gt;4&lt;/sub&gt;.</td></tr><tr><td>Levamisole</td><td>SIGMA-ALDRICH</td><td>196142</td><td></td></tr><tr><td>Razor</td><td>Feather</td><td>blade No. 11</td><td></td></tr><tr><td>Rnase A</td><td>Enzynomics</td><td></td><td></td></tr><tr><td>BSA</td><td>SIGMA-ALDRICH</td><td>A7906</td><td></td></tr><tr><td>Confocal microsope</td><td>Zeiss</td><td>LSM 510</td><td>EC Plan-Neofluar 100X was used as objective lens.</td></tr><tr><td>Humid chamber</td><td></td><td></td><td>Plastic box filled with paper towel soaked in DW</td></tr><tr><td>Image Analysis Software&amp;nbsp;</td><td>Dr. Peter Landsdorp</td><td>TFL-telo</td><td>&lt;a href=&quot;http://www.flintbox.com/public/project/502&quot;&gt;http://www.flintbox.com/public/project/502&lt;/a&gt;</td></tr></tbody></table>

observation and quantification of telomere and repetitive sequences using fluorescence in situ hybridization (fish) with pna probes in caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

据报道，此前ALT幸存者可以从端粒酶缺陷的突变通过复制内部本地化&#39;ALT的模板"（TALT）的端粒维持2序列出现，TRT-1（ok410），在低频。使用PNA探针，我们能够想象在解剖性腺（ 图2A）的端粒。微弱的信号端粒检测无论是在TRT-1（ok410）和ALT幸存者。模糊信号被重叠只用DAPI，这表明它们可能不是自发荧光。间质性端粒样重复（ITR）的TRF分析一直被观察到C的研究线虫端粒4,10。考虑PNA探针的高特异性，他们很可能要在ITR分散在整个基因组中。 端粒点的数量约为9％粗线核在TRT-1（ok410）。在以往的研究中，被POT-1 :: mCherry蛋白，结合单链DNA端粒观察10 12重点。在野生型胚胎17中观察到的每核24灶最大。结果表明，mCherry记者方法为，其中端粒的数量应该计数的实验更好。然而PNA FISH能够检测与端粒长度比例双链端粒DNA以及单链端粒DNA。与此相反，端粒斑点的数目约为7中的ALT幸存者，其中融合了染色体（N = 3）2。这一结果与预测相符，端粒斑点将是6中的ALT幸存者。我们的结论是，ALT幸存者的信号强度足以被观察到。 端粒信号是在ALT幸存者与TALT1共定位，表明TALT1用作模板复制​​端粒在没有TELO的聚合酶（ 图3）。 ALT幸存者端粒信号增加相比，亲TRT-1（ok410）突变体，这表明端粒是鲁棒保持在ALT幸存者没有端粒酶（ 图4）。 PNA探针的信号比地高辛标记的探针（ 图4）的大。与PNA寡聚物设计的探头可能会导致地高辛比标签更强的信号。 <img alt="图1" src="/files/ftp_upload/54224/54224fig1.jpg" /> 图1:FISH实验卵概述由漂白成虫收获并固定在2％的PFA在聚赖氨酸包被的载玻片。样品冷冻裂化并用甲醇和丙酮用于探针穿透透。探针加入到样品中，并在37℃下杂交过夜。地高辛标记探针是由免疫荧光法检测。将样品成像，然后quantifie通过图像分析软件ð。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54224/54224fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54224/54224fig2.jpg" /> 图 2: 端粒FISH在解剖性腺 （A）的端粒（红）用的Cy3 PNA-（TTAGGC）3在性腺的远侧末端（箭头）进行检测。 ALT幸存者的强度比TRT-1（ok410）更大。 Z堆栈图像最大投影呈现。由白色箭头指示核吹式的右上角。比例尺，10微米。 （B）的每核端粒斑点在粗线期数量通过目测测量。 N = 50。误差棒，SEM， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54224/54224fig2large.jpg" target="_blank">请点击这里查看</a>一个更大的版本这个数字。 <img alt="图3" src="/files/ftp_upload/54224/54224fig3.jpg" /> 图 3: 端粒和TALT1鱼在胚胎端粒（红色）和TALT1（绿色）鱼的形象代表。端粒和TALT1探针杂交同时胚胎。 DNA用DAPI（蓝色）复染色。比例尺，10微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54224/54224fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54224/54224fig4.jpg" /> 图4:FISH数据端粒的定量和从图3 TALT1强度在图像分析软件（距离彼得Lansdorp博士馈赠）进行定量。每个点与门限电平OV量化器15，以排除非特异性背景。用于评估统计显着性T-检验。 （* p &lt;0.001）。误差线，扫描电镜。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54224/54224fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans at

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

观察与端粒的定量和重复序列用荧光<em&gt;原位</em&gt;杂交（FISH）与PNA探针<em&gt;秀丽隐杆线虫</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

observation-quantification-telomere-repetitive-sequences-using

Nanyang Technological University

Research

JoVE Journal

Biology

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

10.3K 次观看.  Seoul National University. We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT). 

视频: Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

在秀丽线虫中对倍体的操控

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

科研

遗传学

秀丽隐杆线虫的表观遗传遗传和 ChIP

Analysis of Transgenerational Epigenetic Inheritance in C. elegans Using a Fluorescent Reporter and Chromatin Immunoprecipitation (ChIP)

Transgenerational epigenetic inheritance (TEI) allows the transmission of information through the germline without changing the genome sequence, through factors such as non-coding RNAs and chromatin modifications. The phenomenon of RNA interference (RNAi) inheritance in the nematode Caenorhabditis elegans is an effective model to investigate TEI that takes advantage of this model organism's short life cycle, self-propagation, and transparency. In RNAi inheritance, exposure of animals to RNAi leads to gene silencing and altered chromatin signatures at the target locus that persist for multiple generations in the absence of the initial trigger. This protocol describes the analysis of RNAi inheritance in C. elegans using a germline-expressed nuclear green fluorescent protein (GFP) reporter. Reporter silencing is initiated by feeding the animals bacteria expressing double-stranded RNA targeting GFP. At each generation, animals are passaged to maintain synchronized development, and reporter gene silencing is determined by microscopy. At select generations, populations are collected and processed for chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) to measure histone modification enrichment at the GFP reporter locus. This protocol for studying RNAi inheritance can be easily modified and combined with other analyses to further investigate TEI factors in small RNA and chromatin pathways.

作者聚焦:秀丽隐杆线虫的 RNAi 遗传和 ChIP 

This protocol describes an RNA interference and ChIP assay to study the epigenetic inheritance of RNAi-induced silencing and associated chromatin modifications in C. elegans.

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析 秀丽隐杆线虫 的跨代表观遗传

Transgenerational epigenetic inheritance (TEI) allows the transmission of information through the germline without changing the genome sequence, through ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

使用接近连接测定在C.elegans Germline中对核糖蛋白复合体装配的实地检测

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

发育生物学

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. ...

生物学

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

端粒长度和端粒酶活性;阴阳细胞衰老

Telomeres  are repeating DNA sequences at the tip ends of the chromosomes that are diverse  in length and in humans can reach a length of 15,000 base ...

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

免疫与感染

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

观察与端粒的定量和重复序列用荧光

摘要

探索更多视频

在秀丽线虫中对倍体的操控

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析秀丽隐杆线虫的跨代表观遗传

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析秀丽隐杆线虫的跨代表观遗传

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析秀丽隐杆线虫的跨代表观遗传

使用接近连接测定在C.elegans Germline中对核糖蛋白复合体装配的实地检测

定量和自动化高通量的全基因组RNAi屏幕 C。线虫

基本的

端粒长度和端粒酶活性;阴阳细胞衰老

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

观察与端粒的定量和重复序列用荧光

摘要

探索更多视频

在秀丽线虫中对倍体的操控

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析 秀丽隐杆线虫 的跨代表观遗传

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析 秀丽隐杆线虫 的跨代表观遗传

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析 秀丽隐杆线虫 的跨代表观遗传

使用接近连接测定在C.elegans Germline中对核糖蛋白复合体装配的实地检测

定量和自动化高通量的全基因组RNAi屏幕 C。线虫

基本的

端粒长度和端粒酶活性;阴阳细胞衰老

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析秀丽隐杆线虫的跨代表观遗传

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析秀丽隐杆线虫的跨代表观遗传

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析秀丽隐杆线虫的跨代表观遗传

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染