这项研究部分由美国国立卫生研究院，研究所院内研究老龄问题方案（Z01 AG000746-08）和范可尼贫血研究基金的支持。

1.挖-TMP的制备<ol><li> 混合50毫克（0.18毫摩尔）的4&#39;-氯甲基-4,5&#39; ，8-三甲基补骨脂素和590毫克（2.7毫摩尔）-4,7,10-三氧杂-1,13- tridecanedi胺在干燥的25mL圆在氮气下汉字烧瓶中。添加10mL的甲苯，并回流12个小时。在减压下在旋转蒸发器除去溶剂。 <ol><li>纯化在硅胶残余物通过快速柱色谱法。 （1:1:0.5 9），用氯仿，甲醇和28％氨水溶液洗脱该柱。蒸发在减压下在旋转蒸发器除去溶剂，回收纯4" - [N-（13-氨基-4,7,10- trioxatrideca）]氨甲基-4,5"，8-三甲基补骨脂素，为浅黄色粘性液体。 </li><li>的4混合5.5毫克（0.012毫摩尔）" - [N-（13-氨基-4,7,10- trioxatrideca）]氨甲基-4,5"，8-三甲基补骨脂素与5毫克（0.008毫摩尔）洋地黄毒苷NHS酯的在氮气下干燥的圆底烧瓶中。添加0.5毫升无水二甲基甲酰胺（DMF）的ð3.4μL三甲胺，并搅拌在50℃下18小时。 </li><li>在减压下在旋转蒸发器除去溶剂。 </li><li>溶于二氯甲烷中的最小量的残余物。适用于2μL斑点溶液水平地横跨用硅胶作为固定相制备性薄层板的底部。甲醇:氯仿运行制备型TLC 28％氢氧化铵（8:1:0.1）。 </li><li>屏蔽板除了垂直边缘，并与短波长UV 254nm的灯识别产品的带。从用刮刀将板刮去产品，然后使用氯仿分离纯产物:甲醇:28％氢氧化铵（8:1:0.1）的混合物。 </li><li>在减压下在旋转蒸发器除去溶剂。溶解残留的粒料在1mL 50％乙醇:H 2 O，分配到在1.5mL管中（约20个等分）和干燥的50μL等分试样在离心蒸发器，直到所有的溶剂被蒸发（〜3小时）。 </li><li>在离心蒸发器再次H 2 O和干燥:再溶解在50％EtOH中的200μL每个等分试样。在50％EtOH中的200μL再次溶解每个等分试样:H 2 O，在-20℃下长期储存干在离心蒸发器和存储粒料。 </li></ol></li><li> 为了使用，溶解于50％EtOH中的50μL的丸粒:H 2 O.准备1:100稀释溶解的挖-TMP的在H 2 O和在250nm测量OD。挖-TMP的消光系数是25,000。如果储存在-20℃下将溶液是约一个月稳定。通过在每次使用前在250nm处测量OD验证的浓度。 <ol><li>计算库存浓度:腹部×100×10 6/25000 =浓度（μM）。典型地，所述原液是约3mm。是在该范围内，以尽量减少的EtOH加入到培养基中的量是重要的。 </li></ol></li></ol> 2.激光局部地高辛-TMP的ICL <ol><li> PE与具有SRS氮激光器（337 nm）的共焦显微镜rform激光定位通过染料细胞泵送发射线的365纳米和在10赫兹烧成3个纳秒脉冲，0.7纳瓦的功率。 </li><li>使在35 mm的玻璃侧的垂直标记有底有标记笔细胞培养皿。划伤与金刚石笔培养表面上的玻璃的中心的横使得在盘侧的黑条是在交叉的一个臂的顶部。横标上的玻璃的生长表面，使得它与细胞将在相同的焦平面中。 </li><li>用70％乙醇的漂洗后标记消毒板。电镀细胞之前干燥。 </li><li>板细胞（标准实验室菌株例如HeLa或U2OS​​）在交叉之前一天或两天标记培养皿。小区应积极分裂，并在实验当天50-70％汇合。孵育24小时用1μM5-氯-2-脱氧尿苷（CldU）均匀地标签DNA。 </li><li>加挖-TMP，在50％EtOH中:H 2 O至细胞培养基至20μM的终浓度。使介质至37℃。改变在细胞上，并装在培养箱培养基（37℃，5％CO 2）30分钟，以允许该DIG TMP达到平衡。 </li><li>而将细胞培养，建立的视场，其中所述激光是活性的x / y坐标。按照该激光是由软件涉及蚀刻沿着水平和对角线镜像载玻片制造商的校准程序。两条线限定，其中该激光能够求取目标场的边界。 </li><li>放置板在环境室，在37℃下具有受控的CO 2和湿度，显微镜载物台上，并用在十字交叉的60X油浸物镜聚焦。 </li><li>引导365nm的激光束的ROI，通过在该软件的形状工具研究者成立，以形成3条带＃181; 0.6 MX微米。 ROI的轮廓由光标在位于十字交叉的附近细胞的核放置。调节Z焦平面到目标通过细胞核的激光中途。在350-370 nm范围内使用的激光器。 405nm的激光器不能诱导交联19。 注意:我们定位核仁，这可以从微分干涉对比（DIC）成像核质来区分外核质区域的投资回报率。 </li><li> 通过增加功率设置到足以抹杀一个小区（小区会变暗，然后消失）的电平确认激光的焦点和活性。这是用于通过显微镜，然后通过盖玻片并进入细胞中的激光不受阻碍光路的一个重要的诊断。 <ol><li>降低功率设置到刚好足以激活补骨脂素和诱导ICL DDR（这必须根据经验来确定对于每个激光/麦克风roscope组合）。使用激光强度设定（这里为1.7％），其不激活DDR在不存在补骨脂素（由γ-H2AX的形成监测）。 注意:这是一个重要的判定，并且如果激光源或在光路中的分量改变所述设置可能需要调整。遵循GFP的积累标记修复蛋白如XPC或FAN1，这两者都是迅速招募（秒）到激光补骨脂条纹，但不ROI暴露于单独的激光器。在DDR的一些蛋白质出现几秒钟内，而几分钟就可以要求别人。这需要进行时间过程确定每个感兴趣的蛋白质。我们还注意到，在没有暴露于激光细胞没有响应蛋白的条纹。 </li></ol></li><li>后在场内的所有单元已经有针对性的举措板以一个新的领域，力求贴近十字交叉。 </li><li>在实验中，旨在determ的INE间病灶距离的分布的细胞暴露于所述激光在20-25字段（4-5个细胞/场）围绕交叉点。为了分析在局部的ICL的附近复制图案孵育与激光器发射后10μM5-碘-2-脱氧尿苷（IDU）细胞。 注:孵化与IDU的时间是有点武断。我们使用短至20分钟，只要60分钟（见下文）。较长的脉冲（几个小时）通常导致多个复制大片聚结，从而失去的机会，以图像单叉的进度。在一些实验中CES都标有CldU 24小时，以在暴露于高辛TMP随后复制大片脉冲标记均匀地标签DNA。 </li></ol> 3.细胞收获和DNA纤维的拉伸<ol><li>从载物台取下板，除去培养基，并用磷酸盐缓冲盐水（PBS）洗涤。除去PBS溶液。将商业胰蛋白酶的10微升下降/上交叉的在玻璃表面的中间的交点EDTA溶液。 </li><li>孵育在室温下（RT）3-4分钟，然后绘制与所述分离的细胞进入吸管尖的胰蛋白酶溶液。没有必要以中和胰蛋白酶。 </li><li>放置胰蛋白酶/ EDTA的与细胞的下降在硅烷化的玻璃显微镜载片的一端。裂解细胞，并通过加入0.5％SDS溶液10μL的释放DNA，并在室温下孵育3-4分钟用吸管尖轻轻混合，使池干燥的周边。 </li><li>倾斜滑动20º，并允许液体下降到结束时运行。的DNA纤维扩展/流动液体的水动力拉伸。让载玻片风干（〜10分钟），并在3固定:1个甲醇/乙酸10分钟。从修复方案，再风干取出幻灯片。在这一点上，他们可以在-20℃下被无限期地贮存在70％EtOH中。从70％的乙醇去除允许在空气中干燥网元之前XT一步。 </li><li>在PBS洗涤玻片，然后在2.5的1M HCl变性在RT 1个小时。这种处理depurinates DNA使掺入卤化核碱基可访问的抗体。 </li><li>中和的0.4M的Tris-HCl，pH 7.4中的幻灯片。在PBS中，每次5分钟洗涤两次/ 0.5％吐温20（PBST）。 </li><li>块滑动，用5％牛血清白蛋白（BSA）和10％山羊血清的PBS中。用封口膜，以在所述滑动均匀地散布在封闭溶液并在室温下孵育1个小时轻轻覆盖滑动。 </li><li>吸取100μL的1:200稀释于阻断大鼠抗溴脱氧尿苷的溶液（特异性检测CldU）初级抗体每张幻灯片。用封口膜，以在所述滑动均匀地散布在稀释和培养在湿润的腔室，在室温1个小时轻轻覆盖滑动。除去石蜡膜和洗载玻片在PBST三次，每次5分钟。 </li><li>吸取100μL的稀释的（1:100）的山羊抗大鼠Alexa氟647在封闭液中，以每张幻灯片。覆盖用封口膜轻轻滑动并在室温下孵育45分钟。洗载玻片在PBST三次，每次5分钟。 </li><li>吸取100μL的稀释（1:40）的小鼠抗溴脱氧尿苷（检测LDU和CldU这种双特异性通过在现有孵育大鼠抗BrdU的必要CldU的阻塞。）的第一抗体和兔抗DIG抗体（1:200 ）在封闭液中，以每张幻灯片。用封口膜轻轻覆盖滑动并在湿润室中在室温下孵育1个小时。洗载玻片在PBST三次，每次5分钟。 </li><li>吸取100微升稀释的在封闭液中，以每个幻灯片的山羊抗兔探针（ 例如 ，量子点655）（1:100）的山羊抗-小鼠Alexa Fluor488的和c（5000 1）。覆盖载玻片轻轻用石蜡膜和在室温下孵育45分钟。洗载玻片在PBST三次，每次5分钟。 </li><li>排出多余的PBST到纸巾上。添加抗褪色封固剂的50μL至每个载玻片，并用盖玻片覆盖。图片或商店在-20℃下不超过48小时。 </li></ol> 4.荧光显微成像纤维<ol><li>通过63X物镜在倒置显微镜上进行图像采集具有附接的全电动滤光轮与FITC，Cy5的和Q点655检测。激励滤光器为425纳米，和发射滤波器是具有在发光峰值20中心20个纳米655纳米。 </li></ol>

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Cell. 40, 179-204 (2010).</li><li>Mori, T., Matsunaga, T., Hirose, T., Nikaido, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+monoclonal+antibody+recognizing+ultraviolet+light-induced+(6-4)+photoproducts.">Establishment of a monoclonal antibody recognizing ultraviolet light-induced (6-4) photoproducts.</a> Mutat. Res. 194, 263-270 (1988).</li><li>Poirier, 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=Antisera+specific+for+carcinogen-DNA+adducts+and+carcinogen-modified+DNA:+applications+for+detection+of+xenobiotics+in+biological+samples.">Antisera specific for carcinogen-DNA adducts and carcinogen-modified DNA: applications for detection of xenobiotics in biological samples.</a> Mutat. Res. 288, 31-38 (1993).</li><li>Henderson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+G-quadruplex+DNA+in+mammalian+cells.">Detection of G-quadruplex DNA in mammalian cells.</a> Nucleic Acids Res. 42, 860-869 (2014).</li></ol>

作者什么都没有透露。

激光定位技术要求与在明亮的视野显微镜可见细胞核使用贴壁细胞。我们已经试图附加非贴壁细胞，如初级淋巴细胞，或松散粘连培养细胞如AD293，与细胞粘附制剂，如聚赖氨酸或胶原，或更复杂的混合物在玻璃表面。虽然这些治疗方法可以将细胞结合到表面，我们发现，他们一般留圆角使其很难集中到细胞核。此外，细胞通常不能存活除去生长培养基后的初始洗涤。然而，对于贴壁细胞的要求内，我们已经成功地使用宽范围的标准细胞系以及原代细胞用于这些研究。它是测试支原体污染的细胞是有用的。我们发现，细胞反应是由这些感染改变。 激光的焦点平面是非常重要的。我们的目标是放置的ICL在细胞核内。过高和光活化将成为核的上方;太低了，这将是在玻璃上。我们会发现它有用在细胞质和细胞核的交集锐化图像DIC的焦点。 纤维检测是不难，但需要一些练习解释字段被回收之前。正确地实现这种技术是非常重要的。从激光实验中回收的细胞的数量是低，并且每个样品在其全部使用在单个幻灯片。因此有必要使从各实验产量，作为样品不能被分割，保存用于后续分析等分试样。然而，数以百计的事件可以从单个样品进行回收。该过程的两个功能是至关重要的。一个是细胞裂解液倒滑的流速。如果速度过快，大部分的DNA会流失的幻灯片。玻片的质量也很重要。我们偶尔会RECE香港专业教育学院很多是无效的。因此每一批新必须在使用前进行测试。 对于所有的实验室操作是真正的商业试剂，抗体，immunoquantum点等的可靠性，始终是一个问题。这些并不总是稳定的，实验失败通常追踪到一个或另一种试剂的问题。的immunoquantum点是特别有问题的，超过一个许多可能需要以识别一个能够接受的被测试。无效批次由高背景信号标。这些不与纤维相关联，并且在不具有DNA滑动的区域可以看到。 因为它是由邦纳组4介绍了使用激光器以引入本地DNA损伤已经成为非常流行。在那些，最后续实验的激光功率设置被设置，以便直接诱导DDR。我们已经使用了功率设置足够低SUCh上的DDR的活化依赖于激光和补骨脂素。如上所讨论的，补骨脂素加合物之间的分离认为，我们正在寻找在响应于各个事件。虽然目前还不知道由内源性的交联剂形成的ICL的数量，我们注意到，补骨脂素加合物的定位不杀死细胞，因为它们能够内6-8小时（未公布的数据）移除补骨脂加合物和存在健康24个小时后。因此，这种方法是将细胞比需要暴露于引入整个核和线粒体DNA损伤剂协议少得多的毒性。 激光/补骨脂素组合引入螺旋扭曲结构，其激活DDR。在这点上，从该方法获得的结果是适用于一系列螺旋扭曲病变，给出的DDR的级联性质中，ATM / DNAPK / ATR的活化激酶以下22。在这方面本文所描述的DNA纤维的方法可以扩展到能够由可通过荧光来显示免疫或化学或生物化学手段检测的任何DNA结构。这些可以包括UV光产物23，笨重烷化剂24，G四链25，和氧化损伤5。

DNA是从下外源性试剂如辐射，紫外线，环境毒素，燃烧产物，等。此外，还通过氧化代谢产生的内源性自由基物种攻击恒定攻击。所有这些必须化学或物理破坏DNA的完整性1的潜力。在基因组扰动可以激活DNA损伤反应（DDR），招聘和翻译后修饰级联数百，甚至数千名参与损伤修复蛋白和微RNA，细胞周期，细胞凋亡，衰老和炎症通路的调控2。 大多数的我们对DDR的信息来自于与DNA双链断裂的研究。这在很大程度上是由于技术在活细胞中导入3断裂，包括序列特异性断裂，在基因组DNA的可用性。此外，propensit断裂的y以诱导蛋白质DDR，这可以通过免疫荧光来显示的病灶，已经非常有帮助，用于识别的动力学和响应蛋白质的要求。一个用于研究DDR的关键技术是由邦纳和他的同事，谁在活细胞4的核使用激光束直接DNA双链断裂的条纹在"利益区域"（ROI）推出。实际上，他们创造了一个漫长的焦点，其中DDR的蛋白质可以通过免疫识别。这是由它们的磷酸化组蛋白H2AX的激光暴露的细胞的强条带（γ-H2AX）的示范说明。此后，激光方法已通过DNA双链断裂引起的DDR的众多研究中。虽然功能强大和流行，和戏剧性的免疫荧光图像的来源，应该指出的是，在大多数实验的激光强度进行调整，从而产生可观察到的结果，而对病变身份的关注，密度，或间距。事实上，它可能很难做这类估算。因此，它们在很大程度上忽略了，尽管由激光器5引入DNA损伤的多重性。这有助于在文献6的许多矛盾。 与此相反，以双链断裂，DNA的大多数化学修饰不刺激DDR蛋白质的分离的焦点的形成。这是我们目前的病变频率的理解之光重要的。据估计，在培养的人细胞招致每个细胞周期多达50双链断裂，在S期7，8，9很大程度上形成。形成在非增殖细胞更少。与此相反，核碱基丢失或修改事件，这是在数万每细胞/ 1天，10的数目。因此，我们最大约知道通过比较少见，而那些通过螺旋扭曲病变，合共更为常见诱发少得多的事件引起的DDR。 为了解决有关的细胞反应共价基因组DNA的修改问题，我们希望具有螺旋扭曲的DNA加合物是有内在的DDR诱导活性的工作。此外，为了方便实验设计和解释，我们感兴趣的是其引入可能对于控制时间，是适合于可视化的结构。因此，我们开发了基于补骨脂素的战略。补骨脂素被很好地表征光敏DNA嵌入有利于5&#39; TA:AT网站。不像其他的交联剂如氮芥和丝裂霉素C（MMC）它们不是DNA的反应性，除非暴露于长波紫外光（UVA）光。插层的分子与相反链上，以产生螺旋扭曲间交联胸腺嘧啶碱基（的ICL反应）11。在我们的实验中使用的三甲基补骨脂素大多数产品都是的ICL，相对少的单加成物产生（低于10％）12，以及在一条链上相邻碱基之间的内交联不形成。因为他们是强大的块复制和转录，补骨脂等交联剂，如顺铂和MMC，通常用于化疗。因此补骨脂启用研究，通过一个螺旋扭曲结构跟着DDR的激活，并且还提供了洞察到与临床重要性的化合物的细胞应答。 我们合成，其中三甲基补骨脂素是有联系的地高辛（DIG）的试剂，植物甾醇未在哺乳动物细胞中发现的，并经常用作immunotag。用于光活化的要求在活细胞中的细胞核定位允许通过补骨脂素的ICL在定义的ROI的激光（365纳米）。这些可以通过IMM显示unofluorescence反对挖标签。 DNA修复和DDR蛋白出现在激光的局部的ICL 13,14的条纹。 通过用于产生双链断裂的高激光强度激活DDR可能是由于分离的或聚集损伤15,16。因此，从这些实验结果的相关性，以天然存在的病变，本在低得多的浓度，是不确定的。为了解决有关的DNA加合物补骨脂频率和间距类似的问题，我们利用DNA纤维技术17和immunoquantum点。量子点是比荧光染料更明亮，没有被曝光漂白光。因此，它们经常用于单分子成像18，一个应用程序，其为荧光染料是不够明亮。个人DNA纤维可以上克被拉伸小姑娘滑动，并且可以通过对在细胞收获前的温育过程中掺入的核苷类似物免疫荧光来显示。我们处理的细胞与地高辛补骨脂素和露出的ROI，以激光微照射。纤维从细胞和个体辛补骨脂素加合物准备可以与immunoquantum点进行可视化。将细胞暴露于核苷类似物为相对短的时间（20-60分钟）允许复制大片在激光局部的ICL的附近的显示。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Digoxigenin NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>11333054001</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloro-psoralen</td>
			<td>Berry and Associates</td>
			<td>PS 5000</td>
			<td></td>
		</tr>
		<tr>
			<td>diaminoglycol</td>
			<td>Sigma-Aldrich</td>
			<td>369519</td>
			<td>4,7,10-Trioxa-1,13-tridecanediamine</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Acros Organics</td>
			<td>423550040</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A4524</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium solution</td>
			<td>Sigma-Aldrich</td>
			<td>5002</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC plates</td>
			<td>Analtech, Inc.</td>
			<td>P02511</td>
			<td></td>
		</tr>
		<tr>
			<td>Flass glass column 24/40, 100ml</td>
			<td>Chemglass Life Sciences</td>
			<td>CG-1196-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon T2000_E2 spinning disk confocal microscope, equipped with automated stage and environmental control chamber and plate holder</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>With Volocity Software</td>
		</tr>
		<tr>
			<td>Micropoint Galvo&#160;</td>
			<td>Andor Technologies</td>
			<td></td>
			<td>with a Nitrogen pulsed laser&#160;</td>
		</tr>
		<tr>
			<td>dye cell</td>
			<td>Andor Technologies</td>
			<td>MP-2250-2-365</td>
			<td></td>
		</tr>
		<tr>
			<td>365 dye</td>
			<td>Andor Technologies</td>
			<td>MP-27-365-DYE</td>
			<td></td>
		</tr>
		<tr>
			<td>IdU&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>17125</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm glass botomm plates 1.5 coverslip, 10mm glass diameter, uncoated</td>
			<td>Matek</td>
			<td>P35G-1.5-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>New Comer Supply</td>
			<td>Part # 5070</td>
			<td>New Silane Slides</td>
		</tr>
		<tr>
			<td>Mouse anti BrdU antibody (IdU)</td>
			<td>BD Biosciences</td>
			<td>347580</td>
			<td>1 in 40</td>
		</tr>
		<tr>
			<td>Rat anti BrdU Antibody (CldU)</td>
			<td>Abcam</td>
			<td>ab6326</td>
			<td>1 in 200&#160;</td>
		</tr>
		<tr>
			<td>Rabbit anti Dig antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>710019</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>Q-dot 655 goat anti Rabbit IgG</td>
			<td>ThermoFisher Scientific</td>
			<td>Q-11421MP</td>
			<td>1 in 5000</td>
		</tr>
		<tr>
			<td>AF647- goat anti Rat IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>112-605-167</td>
			<td>1 in 100</td>
		</tr>
		<tr>
			<td>AF488-goat anti mouse IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>115-545-166</td>
			<td>1 in 100</td>
		</tr>
		<tr>
			<td>Zeiss epifluorescent microscope A200</td>
			<td>Zeiss</td>
			<td></td>
			<td>&#160;with Axiovision software</td>
		</tr>
		<tr>
			<td>Q-dot 655 filter</td>
			<td>Chroma</td>
			<td>39107</td>
			<td></td>
		</tr>
	</tbody>
</table>

single molecule analysis of laser localized psoralen adducts

DNA损伤反应（DDR）已经被广泛表征在活细胞中诱导激光微束照射的双链断裂（DSB的）的研究。该DDR到螺旋扭曲共价DNA修饰，包括链间交联的DNA（的ICL），没有作为很好的界定。我们研究通过的ICL刺激DDR，通过immunotagged补骨脂素的光敏激光定位，在活细胞中的细胞核。为了解决约加成分布和复制叉遇到的基本问题，我们结合与其他两种技术的激光定位。 DNA纤维经常用于显示通过在短的脉冲掺入核苷类似物的免疫荧光的复制叉的进展。 Immunoquantum点已被广泛用于单分子成像。在新方法中，从携带激光局部的ICL细胞DNA的纤维铺在显微镜载玻片上。该标记的ICL显示有immunoquantum点和日电子病变间距离来确定。复制叉的碰撞与的ICL可以可视化和不同遭遇的图案识别和定量。

激光局部地高辛TMP（ 图1A）的ICL可以通过对连接到补骨脂该DIG标记的免疫荧光来显示。尽管激光可被引导在任意轮廓的区域撞击，条纹不在细胞"自然"形状，并且合法信号可以从假象由于非特异性结合由一次或二次抗体容易区分。使用不到理想的特异性抗体时，此功能非常有用。的DDR，γ-H2AX的公知的标记物，在挖-TMP的ICL的条纹的一个例子示于图1B。 该挖的免疫荧光标记的病变在激光条纹不允许任何结论，加成物的频率和间隔。为了做出该确定细胞用CldU引入的ICL之前温育24小时。对日染色ËCldU允许挖的可视化标记的病灶上的长纤维，并产生更清晰的图案纤维比直接污渍如DAPI（4&#39;，6-二脒基-2-苯基吲哚）或YOYO {1,1&#39; - （-4,4,8- ，8-四甲基-4,8-​​ diazaundecamethylene）双[4 - [（3-甲基 - 苯并-1,3-恶唑-2-基）亚甲基] -1,4- dihydroquinolinium]}四碘化。通过激光光敏引进的ICL后，纤维从靶细胞扩散。在纤维上，如在图2中示出的那些的挖信号的分析表明，该帧间ICL从距离小于10kb的范围为大于160 kb的，如在我们的最近的出版物21中所述。有聚簇合物的证据。因此，挖标签，并在条带中的蛋白质的DDR的积累的条纹，反射良好分离的损伤的存在，至少沿延伸的DNA测定。认为如果形成在核小体阵列的ICL细胞染色质的方面，具有6折延伸的DNA长度的压缩，它们仍然会以大约1.6的DNA kb的等效分离。 实验设有激光诱导的DNA损伤一般遵循DDR的诱导。这些实验很少与有关DNA复制的问题有关。在另一方面，DNA纤维测定法通常用于复制的研究。细胞暴露于在其掺入DNA卤代核苷类似物结果短脉冲。 DNA纤维的免疫荧光分析显示表示最近DNA合成掺入类似物的大片。这是感兴趣，询问是否发生在激光局部ICL条纹DNA复制，如果是这样，这将是复制中的ICL附近的模式？将细胞温育与CldU 24个小时，以促进长纤维的显示。激光局部的ICL被引入到细胞中，随后IDU的1个H脉冲。 DNA˚F<html> IBERS从靶细胞制备和挖标记的ICL并显示复制大片。结果表明，补骨脂素的激光激活不会关闭复制或影响的复制模式的整体频率。复制发生在一侧上，并且还对的ICL的两侧上，与在一个4双面图案:1多数因为我们最近21所示。 <img alt="图1" src="/files/ftp_upload/55541/55541fig1.jpg" /> 图1:激光本地化挖-TMP的ICL的生成。 （A）高辛三甲基补骨脂素的结构。在ROIγ-H2AX（绿色）的（B）含有累积激光局部的ICL（挖，红色）。细胞核被用DAPI染色（蓝色）。明场的照片显示了一个细胞部分地由金刚石笔所完成的标志。注意核仁，和他们外面的ROI的位置。csource.jove.com/files/ftp_upload/55541/55541fig1large.jpg"目标=‘_空白’&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/55541/55541fig2.jpg" /> 图2:DNA纤维用激光局部地高辛TMP信号。 （A）将细胞温育与CldU 24个小时来标记DNA。然后激光局部的ICL进行了介绍和（B）收获细胞和纤维传播。 （C，D）的挖信号用一个immunoquantum点（红色）和免疫荧光（绿色）CldU显示。 <a href="http://ecsource.jove.com/files/ftp_upload/55541/55541fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Single Molecule Analysis of Laser Localized Psoralen Adducts at JoVE.com

Single Molecule Analysis of Laser Localized Psoralen Adducts

激光器在对DNA损伤的细胞应答的研究经常使用的。然而，它们产生的病变，其间隔，频率，和碰撞与复制叉很少表征。在这里，我们描述了一种方法，使这些参数与激光局部间交联的决心。

激光局部补骨脂素加合物的单分子分析

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

single-molecule-analysis-of-laser-localized-psoralen-adducts

Nanyang Technological University

Research

JoVE Journal

Genetics

6.6K 次观看.  National Institute on Aging, National Institutes of Health. 激光器在对DNA损伤的细胞应答的研究经常使用的。然而，它们产生的病变，其间隔，频率，和碰撞与复制叉很少表征。在这里，我们描述了一种方法，使这些参数与激光局部间交联的决心。 

视频: Single Molecule Analysis of Laser Localized Psoralen Adducts

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

激光辅助畜禽受精卵细胞质显微注射

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

科研

遗传学

Metagenomic Analysis of Silage

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of the microbial communities present within them. Two main metagenomic approaches exist; sequencing the 16S rRNA gene coding region, which exhibits sufficient variation between taxa for identification, and shotgun sequencing, in which genomes of the organisms that are present in the sample are analyzed and ascribed to "operational taxonomic units"; species, genera or families depending on the extent of sequencing coverage. 
In this study, shotgun sequencing was used to analyze the microbial community present in cattle silage and, coupled with a range of bioinformatics tools to quality check and filter the DNA sequence reads, perform taxonomic classification of the microbial populations present within the sampled silage, and achieve functional annotation of the sequences. These methods were employed to identify potentially harmful bacteria that existed within the silage, an indication of silage spoilage. If spoiled silage is not remediated, then upon ingestion it could be potentially fatal to the livestock.

Metagenomics was used to investigate the microbiome of silage cattle feed. Analysis was performed by shotgun sequencing. This approach was used to characterize the composition of the microbial community within the cattle feed.

青贮的宏基因组学分析

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

405 Nm 激光微辐照对 DNA 损伤的蛋白质招募研究

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.