Funding was provided by the Deutsche Forschungsgemeinschaft (SPP1230, grant of the Tumor Center Heidelberg/Mannheim), by the Bundesministerium f&#252;r Bildung und Forschung (iGene), by the VIth + VIIth Framework Programs of the European Commission (CONSERT, CLINIGENE and PERSIST). We thank Ina Kutschera for demonstrating the protocol technique in the video.

链接器的磁带1。准备（LC） <ol><li>混合40微升LC1寡核苷酸（ 表1），40微升LC2寡核苷酸（ 表1中 ，用适当的限制性内切酶悬），110微升Tris-盐酸（100毫米，pH值7.5），和10微升250毫摩尔MgCl 2组成。 </li><li>孵育在95℃下5分钟，让反应冷却下来慢慢至室温。加入300微升H 2 O和集中dsLinker-DNA在离心过滤器。加入80微升H 2 O至洗出液和分装10微升的准备盒式连接器在0.2 PCR管。 </li></ol>载体基因组结2。前置放大<ol><li>对于每个待分析样品制备50μl的PCR反应。 <ol><li>确定DNA样品的浓度。移液器x微升DNA插入0.2 ml的PCR管（1-1000纳克的LAM/100-1，000毫微克的nrLAM）。的DNA的量应该等于在每个样品，并在然GE的0.5至25微升。 </li><li>如表2中所述制备PCR主混合物混合（50 - x）的微升主混合物用在0.2ml PCR管中各DNA样品。 </li></ol></li><li>使用例示于表2中的PCR条件。PCR完成后Preamplify载体基因组结微升Taq酶添加0.5到每个PCR管中，然后重新运行PCR程序。 PCR产物可被储存在4℃下4天或长期于-20℃。 </li></ol> PCR产物的3。磁选<ol><li>磁珠的制备<ol><li>移液管20微升（200微克）的链亲和素包被的磁珠到1.5ml试管并暴露，以磁性颗粒分离器（MPS），在室温下放置1分钟。弃上清液。 </li><li>从MPS除去管，重悬磁珠在40微升PSB / 0.1％BSA（pH 7.5）中。暴露在MPS 1分钟，弃上清。一旦重复此步骤。 </li><li>洗珠无线第20微升的3M氯化锂溶液（3M氯化锂，10毫米的Tris-HCl，1 mM EDTA）中暴露于MPS 1分钟，弃上清。重悬珠在50μl6M的氯化锂溶液（6M的LiCl，10mM的Tris-盐酸，1mM EDTA）中。 </li></ol></li><li>混合来自步骤2.2与50微升制备的磁珠整个PCR反应。孵育在水平振荡器上（转速300rpm）至少2​​小时，在室温下进行。这个步骤可让生物素化的PCR产物链霉包被的磁珠（DNA-珠复合物）的结合。 DNA的磁珠复合物可以在摇床上过夜孵育，或储存在4℃下4天。 </li><li>暴露的DNA-珠复合体的MPS，在室温下放置1分钟，除去上清，重悬DNA-珠复合于100μlH 2 O。立即着手与LAM或nrLAM第5步第4步。 </li></ol> 4，LAM-手续<ol><li>双链DNA（dsDNA）的合成（林只） <ol><li>暴露的DNA-珠复杂步骤3.3至总薪级第1分钟和DIS卡上清液。加入8.25微升H 2 O，1微升10X六核苷酸缓冲液，0.25μL的dNTP（10毫米）和0.5微升（2学分）Klenow聚合酶。在37℃下1小时。 </li><li>加入90微升H 2 O和暴露在MPS 1分钟。弃上清，重悬DNA-珠复杂的100微升H 2 O </li></ol></li><li>限制文摘<ol><li>暴露的DNA-珠复杂，MPS 1分钟，弃上清。加入8.5微升H 2 O，1微升10X限制性内切酶缓冲液和0.5μL限制性内切酶和孵化1小时反应。重复步骤4.1.2。 注意：孵育反应物在所建议的限制酶的制造商的温度。确保有内没有限制性位点存在，或在感兴趣的DNA用于预扩增的引物结合位点的下游。对于所选择的合适的限制性内切酶/限制性内切酶的组合是指9。 </li></ol></li><li> DS结扎链接器（LK） <ol><li>暴露的DNA-珠复杂，MPS 1分钟，弃上清。加入5微升H 2 O，1微升10X FastLink缓冲液，1微升ATP（10毫米），从步骤1.2 2微升连接器卡带，和1微升快速连接DNA连接酶（2 U /μL）。在室温下孵育5分钟。重复步骤4.1.2。 </li></ol></li><li>合成的双链DNA变性<ol><li>暴露的DNA-珠复杂，MPS 1分钟，弃上清。重悬DNA-珠复杂的5微升0.1N NaOH中。孵育5分钟，在室温下在水平摇动器。 </li><li>暴露的DNA-珠复合体的MPS为1分钟，并收集上清液含于新的1.5毫升管预扩增载体基因组的交界处。立即在-20°C进行第6步或存储上清液</li></ol></li></ol> 5，nrLAM-手续<ol><li>单链连接器的连接（SSLC）（nrLAM只） <ol><li>暴露的DNA-珠复杂步骤3.3至公安部1分钟，弃上清。加入6.5微升H 2 O，1微升CircLigase 10X反应缓冲液，0.5μL的MnCl 2（50毫米），0.5微升ATP（1毫米），1微升ssLinker寡核苷酸和0.5微升CircLigase（100 U /μL）。孵育在60℃下1小时。 </li><li>加入90微升H 2 O和暴露在MPS 1分钟。弃上清，洗DNA-珠复杂的100微升H 2 O再次暴露在MPS 1分钟，弃上清，重悬DNA-珠复杂的10微升H 2 O </li></ol></li></ol> 6，指数扩增我<ol><li>对于每个待分析样品制备50μl的PCR反应。 <ol><li>吸取2μL模板DNA（从步骤4.4.2（LAM）或5.1.2（nrLAM））到0.2 ml的PCR管。 </li><li>制备PCR主混合物如表3所述。加入48μl反应混合液到每个样品从步骤6.1.1和通过PCR扩增的天气下载体基因组结纳秒列举于表3。PCR产物可以被储存在4℃下4天或长期于-20℃。 </li></ol></li></ol> PCR产物7。磁分离<ol><li>制备磁珠所例示的步骤3.1.1 - 3.1.3。重悬珠在20μl（对于LAM）或6米的LiCl溶液（6M的LiCl，10mM的Tris-盐酸，1mM EDTA）中加入50μl（对于nrLAM）。混合20微升（LAM）或从步骤6.1.2 PCR反应以制备的磁性小珠（步骤7.1）的50微升（nrLAM）孵育在水平振荡器上（转速300rpm）搅拌2小时，在室温下。 DNA的磁珠复合物可以在摇床上过夜孵育，或储存在4℃下4天。 </li><li>暴露的DNA-珠复合体的MPS为1分钟，除去上清，重悬DNA-珠复合于100μlH 2 O。暴露的DNA-珠复杂，MPS 1分钟，弃上清。 </li><li>重悬DNA-珠复杂的20微升（LAM）或5微升（nrLAM）0.1N氢氧化钠。培育大怒，在室温下在水平摇床10分钟，暴露在MPS 1分钟，并收集在新的1.5 ml管扩增含DNA的上清液。立即在-20°C继续执行步骤8.1或存储上清液</li></ol> 8，指数扩增二<ol><li>对于每个待分析样品制备50μl的PCR反应。 <ol><li>移液管加入2μl模板DNA（来自步骤7.3）到0.2 ml的PCR管中。 </li><li>制备PCR主混合物，如表4所述。加入48μl反应混合液到每个样品从步骤8.1.1并通过举例说明在表4中的PCR条件扩增载体基因组结点。PCR产物可以被储存在4℃下进行长达4在-20℃天或长期</li></ol></li><li>可视化（NR）LAM-PCR产物，负载10微升的PCR产物从2％琼脂糖凝胶步8.1.2。如果带不可见，分析10微升的LAM-PCR产物的高分辨率凝胶。注意：逸hidium溴化物是致突变物。工作很认真，总是穿戴适当的手套。 </li></ol> 9。用于制备高通量测序<ol><li>的（NR）LAM-PCR产物纯化<ol><li>从步骤8.1.2用44μL室温AMPure XP的磁珠混合40微升的PCR产物。孵育5分钟，在室温下暴露于MPS额外的2分钟。 </li><li>弃上清，用200μl70％乙醇洗两次在MPS。 </li><li>弃上清，重悬DNA-珠复杂的30微升H 2 O孵育1分钟，上清液转移至新鲜0.2毫升管。确定纯化的DNA的浓度。 </li></ol></li><li> Fusionprimer PCR加测序专用适配器。 <ol><li>对于每个待分析样品制备50μl的PCR反应。 </li><li>移液器x微升（40毫微克）的DNA为0.2 ml的PCR管。的DNA的量应该等于在每个样品和在0.5至25微升的范围内。 </li><li>制备PCR主混合物，如表5中所述添加（50 - x）的μl反应混合液到每个样品从步骤9.2.2和通过列举在表5中的PCR产物的PCR条件引入测序适配器（NR）LAM-PCR产物。可以被保存在4℃下4天或长期在-20℃下</li><li>形象化Fusionprimer-PCR产物负载10微升的PCR产物从2％琼脂糖凝胶步骤9.2.3。 9.1.3 - 按照步骤9.1.1中所述净化剩余的PCR产物。从自动高分辨率电泳设备准确量化的Fusionprimer-PCR产物的浓度和片段大小的步骤9.4分析1微升纯化PCR产物。 </li></ol></li></ol>

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The authors have nothing to disclose.

在LAM-PCR技术允许识别侧翼已知的DNA区域未知的DNA序列。因为高灵敏度从具有特异性的引物杂交的已知DNA序列中的结的预扩增得到的，能够扩增并检测甚至罕见路口下至单个细胞水平。相反，在多克隆情况LAM-PCR能够扩增数千种不同的结在一个单一的反应。 然而，由于使用了限制性内切酶的integrome只有一个子部分可以由LAM-PCR检测结与每一个特定的限制性内切酶的存在下进行分?...

线性扩增介导的PCR（LAM-PCR）技术可识别和表征邻近任何来源的已知的DNA未知侧翼的DNA。更具体地，LAM-PCR方法已经开发出来，定位病毒载体整合位点（IS）宿主基因组1,2内。像逆转录病毒或转座子的遗传元件整合自己的基因组入在一个（半）随机方式3-6宿主基因组中。在许多情况下，它是决定性的确切地知道这些矢量一体化的立场。 LAM-PCR方法已被证明是优于像连接介导的PCR 7和它的变体或反向PCR 8的替代技术。该方法的灵敏度和鲁棒性源自载体基因组结和磁性选择的扩增的PCR产物的初始预扩增。像提到的替代方法，LAM-PCR依赖于使用限制性内切酶，引入偏差为9-11的检索能力。因此，可以在一个反应中检测出的IS剧目（在integrome）的一个子集。这种偏差是通过使用限制性内切酶9的合适组合的给定样品的平行分析最小化。最近，该技术的一个变种称为非限制性的LAM-PCR（nrLAM-PCR）已经开发出来，绕过使用限制性内切酶，并允许在单个反应9,12的样品的无偏的全基因组分析。 在过去，LAM-PCR方法已被用于鉴定致病逆转录病毒IS白血病引起在少数患者中的临床基因治疗试验13-15。从那时起，LAM-PCR已被改编以确定从其它整合载体（慢病毒载体，转座子）和还识别的被动结合像腺相关载体（AAV）或整合酶缺陷型慢病毒载体（IDLV）载体的集成模式IS 16 -21。 LAM-PCR方法的应用是广泛的传播：传统LY，该技术被广泛地用于研究基因修饰的细胞的克隆性组合物中已经历基因疗法的患者或解开他们的整合行为15,16,22-24评估新型载体系统的生物安全。近日，LAM-PCR启用确定特异性和脱靶设计师核酸酶的活性由IDLV诱捕法25。 此外，LAM-PCR方法可以非常方便地跟随转导的细胞的命运随着时间的推移在一个有机体。这允许识别原癌基因以及肿瘤抑制基因和还研究造血或癌症干细胞生物26-28。最后但并非最不重要的，LAM-PCR法适于研究T细胞受体多样性的人类29（和未公布的数据）。 该技术的固有功率是由所述方法连接到深度测序技术，使表征百万未知侧翼DNA的单核苷酸Ř增强esolution在整个基因组。在下面的协议中，我们描述了一步一步的扩增和鉴定未知侧翼DNA的模范，以确定慢病毒载体IS的。在协议中使用的寡核苷酸列于表1。提取的DNA的任何来源的或cDNA可以用作用于LAM-PCR和nrLAM-PCR的DNA模板。

<table border="0" cellpadding="0" cellspacing="0" width="1209">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:120px;">Catalog Number</td>
			<td style="width:527px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Taq DNA Polymerase</td>
			<td style="width:203px;">Genaxxon Bioscience GmbH</td>
			<td style="width:120px;">M3001.5000</td>
			<td>Alternative Taq Polymerases may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PCR Buffer</td>
			<td style="width:203px;">Qiagen</td>
			<td style="width:120px;">201203</td>
			<td>Use of this buffer is recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">dNTP-Mixture</td>
			<td style="width:203px;">Genaxxon Bioscience GmbH</td>
			<td style="width:120px;">M3015.4020</td>
			<td>or any other dNTPs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Oligonucleotides (Primers)</td>
			<td style="width:203px;">MWG Biotech</td>
			<td style="width:120px;"></td>
			<td>HPLC purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Dynabeads M-280 Streptavidin&#160;</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:120px;">11206D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PBS</td>
			<td style="width:203px;">Gibco</td>
			<td style="width:120px;">14190-086</td>
			<td>0.1 % wt/vol BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">6M LiCl</td>
			<td style="width:203px;">Roth</td>
			<td style="width:120px;">3739.1</td>
			<td>10 mM Tris-HCl&#160; (pH 7.5)/1 mM EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tris-HCl, pH 7.5</td>
			<td style="width:203px;">USB Corporation&#160;</td>
			<td style="width:120px;">22637</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">EDTA</td>
			<td style="width:203px;">Applichem</td>
			<td style="width:120px;">A1103,0250</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Klenow Polymerase</td>
			<td style="width:203px;">Roche Diagnostics</td>
			<td style="width:120px;">10104523001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Hexanucleotide mixture</td>
			<td style="width:203px;">Roche Diagnostics</td>
			<td style="width:120px;">11277081001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Restriction endonuclease</td>
			<td style="width:203px;">NEB</td>
			<td style="width:120px;"></td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Fast-Link DNA ligation kit</td>
			<td style="width:203px;">Epicentre Biotechnologies</td>
			<td style="width:120px;">LK11025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">CircLigase ssDNA Ligase Kit</td>
			<td style="width:203px;">Epicentre Biotechnologies</td>
			<td style="width:120px;">CL4111K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NaOH</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">72068</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Agarose LE</td>
			<td style="width:203px;">Roche Diagnostics</td>
			<td style="width:120px;">11685660001</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">TBE buffer</td>
			<td style="width:203px;">Amresco</td>
			<td style="width:120px;">0658</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ethidium bromide</td>
			<td style="width:203px;">Applichem</td>
			<td style="width:120px;">A2273,0005</td>
			<td>Ethidium bromide is mutagenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">100 bp DNA Ladder</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:120px;">15628-050</td>
			<td>or any other DNA ladder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">20 mM NaCl</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">71393-1L</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Magna-Sep Magnetic Particle Separator&#160;</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:120px;">K158501</td>
			<td>for use with 1.5 ml Tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Magna-Sep Magnetic Particle Separator&#160;</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:120px;">K158696</td>
			<td>for use with 96 well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-0.5, Ultracel-30 membrane</td>
			<td style="width:203px;">Millipore</td>
			<td>UFC503096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PerfectBlue Gelsystem Midi S</td>
			<td style="width:203px;">PeqLab</td>
			<td style="width:120px;">40-1515</td>
			<td>or other electrophoresis system&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">TProfessional 96</td>
			<td style="width:203px;">Biometra</td>
			<td style="width:120px;">050-551</td>
			<td>or other Thermocycler for 96-well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Orbital shaker KS 260 basic</td>
			<td style="width:203px;">IKA</td>
			<td style="width:120px;">2980200</td>
			<td>or other horizontal shaker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PCR softtubes 0.2 ml</td>
			<td style="width:203px;">Biozym Scientific GmbH</td>
			<td style="width:120px;">711082</td>
			<td>or other 0.2 ml PCR tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">1.5 ml tubes</td>
			<td style="width:203px;">Eppendorf</td>
			<td style="width:120px;">12682</td>
			<td>or other 1.5 ml tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Gel documentation system</td>
			<td style="width:203px;">PeqLab</td>
			<td style="width:120px;"></td>
			<td>or any other gel documentation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Nanodrop ND-1000 spectrophotometer</td>
			<td style="width:203px;">Thermo Scientific</td>
			<td style="width:120px;">ND-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Spreadex EL1200 precast gel</td>
			<td style="width:203px;">Elchrom Scientific</td>
			<td style="width:120px;">3497</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:360px;">Submerged gel electrophoresis apparatus SEA 2000&#160;</td>
			<td style="width:203px;">Elchrom Scientific</td>
			<td style="width:120px;">2001E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">2100 Electrophoresis Bioanalyzer</td>
			<td style="width:203px;">Agilent Technologies</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
	</tbody>
</table>

linear amplification mediated pcr &#8211; localization of genetic elements and characterization of unknown flanking dna

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify&#160;unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3&#8217;- and 5&#8217;- sequences adjacent to the integrated lentiviral vector.

LAM-PCR结果在载体基因组路口放大与定义的片段大小为每个结。个体的PCR片段的大小取决于在基因组中的已知的DNA的位置与最近的限制性内切酶识别位点之间的距离。这允许通过凝胶电泳， 例如可视化扩增结的多样性分析的样品中。如果只有单一的（单克隆），几个（寡克隆）或多个（多克隆）频带上存在的凝胶。 LAM-PCR的结果，最好是由高分辨率凝胶电泳（ 图2A）被观看，但...

Watch this Scientific Journal Video about Linear Amplification Mediated PCR â€“ Localization of Genetic Elements and Characterization of Unknown Flanking DNA at JoVE.com

Linear Amplification Mediated PCR &amp;#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

线性扩增介导的（LAM）-PCR方法是开发，以确定基因组中的整合型病毒载体的精确位置的方法。该技术已经发展成为学习中的基因治疗的患者中，新颖的矢量技术，T-细胞的多样性，癌症干细胞模型等生物安全克隆动力学的优越方法

线性扩增介导的PCR  - 未知侧翼DNA的遗传物质和表征本地化

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with ...

linear-amplification-mediated-pcr-localization-genetic-elements

Research

JoVE Journal

Biology

Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

29.9K 次观看.  National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ). 线性扩增介导的（LAM）-PCR方法是开发，以确定基因组中的整合型病毒载体的精确位置的方法。该技术已经发展成为学习中的基因治疗的患者中，新颖的矢量技术，T-细胞的多样性，癌症干细胞模型等生物安全克隆动力学的优越方法

视频: Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

腺病毒介导的基因去除信号分子在原代培养的小鼠胚胎成纤维

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

确定基因表达概况 C。线虫使用微阵列和实时PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA电穿孔，肌纤维的分离和成像

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

体内碱性彗星试验和酶修饰碱性彗星试验测量DNA链断裂和DNA氧化损伤大鼠肝

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.