资助由美国国家卫生研究院R00-HL116234，U19 AI117941和R56 HL126544提供;国家科学基金会授予DMS-1516675;韩国国家研究基金会（NRF-2011-0030049，NRF-2014M3C9A3064552）;和KRIBB倡议计划。

1.生成上游（左） - 和下游（右） - 连接序列库<ol><li> DNA接头制备: <ol><li>通过加入2μL100μM的LINKER_A寡核苷酸（最终的20μM），2μL的100μM的LINKER_B寡核苷酸（最终的20μM），2μL的5M的NaCl（最终的1M），加入2μL的10μL的接头DNA溶液PCR管中4μL无核酸酶的水。接头序列见表1 。 </li><li>在PCR仪器中将接头DNA溶液在95℃孵育5分钟，停止运行程序，并关闭PCR仪器电源。将接头溶液留在仪器中30分钟以缓慢冷却接头DNA。接头DNA溶液可以在4℃下储存。 </li></ol></li><li> LTR特异性生物素引物延伸: <ol><li>使用UV-Vis分光光度计来确定体内基因组DNA的DNA浓度和260/280 nm值或体外实验。使用无核酸酶的水稀释1-2μg样品基因组DNA至终体积为170μL的基因组DNA溶液。 </li><li>通过混合10μMHIV-1特异性生物素引物（ 表1中的 L-BP和R-BP:四种慢病毒特异性引物共10μL），将20μL10X热稳定剂DNA聚合酶缓冲液，4μL10mM dNTPs（最终每个dNTP200μM），3μL2.5U /μL热稳定DNA聚合酶和163μL基因组DNA。 注意:对于gammaretroviral载体（pMX载体），使用两个10μMpMX特异性生物素引物（L-BP和R-BP:总共为10μL）的5μL，而不是上述四种HIV-1特异性生物素引物。 </li><li>将溶液分成四个PCR管（每个50μL），并在以下条件下进行单个延伸循环:94℃5分钟，56℃3分钟，72℃5分钟min和4℃储存。 </li><li>将所有四个PCR反应物合并到2 mL微量离心管中，并按照PCR纯化试剂盒的PCR纯化程序进行。用50μL洗脱缓冲液（试剂盒中提供的5倍水稀释洗脱缓冲液）洗脱。立即进行步骤1.3.1或将洗脱的DNA储存在-20℃。 </li></ol></li><li> RsaI和CviQI消化 <ol><li>通过加入50μL的DNA（来自步骤1.2.4），10μL的10x缓冲液A和2μL（20U）的RsaI酶，准备100μL的消化反应。在PCR仪中37℃孵育1小时。 </li><li>向反应中加入1μL（10U）CviQI酶，并在PCR仪中于25℃温育30分钟。立即进行第1.4.1节</li></ol></li><li> 钝的结局: <ol><li>制备含有2.5μLDNA聚合酶I大（klenow）片段和2μL10 mM dNTP的4.5μL混合液。转移181;将来自步骤1.3.2的DNA样品的混合物的L。总体积为102μL。通过涡旋混合并在25℃下在PCR仪中孵育1小时。立即进行步骤1.6.1。 </li></ol></li><li> 链霉亲和素珠的制备: <ol><li>简单地涡旋链霉亲和素珠溶液，并将50μL转移到新的2 mL微量离心管中。使用磁性支架取出上清液，用200μL的结合溶液洗涤珠子。 </li><li>将珠子悬浮在100μL的结合溶液中，并将管放置在离磁架上。立即进行步骤1.6.1。 </li></ol></li><li> 链霉抗生物素蛋白珠结合: <ol><li>将100μL样品DNA（步骤1.4.1）转移到100μL再悬浮珠粒溶液（步骤1.5.2），并通过移液小心混合，以避免溶液发生任何发泡。在旋转轮或滚筒上，室温孵育3小时。 </li><li>使用磁性支架捕获珠子并丢弃上清液。在400μL洗涤溶液中洗涤珠子两次，并在400μL1x T4 DNA连接酶缓冲液（从不含核酸酶的水稀释的10X溶液）中洗涤两次。 </li><li>将珠子悬浮于200μL的1×T4 DNA连接酶缓冲液中（使用不含核酸酶的水从10X溶液中稀释），并将其放置在磁性架上。立即继续执行步骤1.7.1。 </li></ol></li><li> 连接器结扎: <ol><li>通过混合0.5μL的DNA接头（步骤1.1.2），10μL的10×T4 DNA连接酶缓冲液，20μL的5X T4 DNA连接酶缓冲液（含有25％的聚乙烯），制备400μL的连接反应溶液于2mL的微量离心管中乙二醇），5μLT4 DNA连接酶，164.5μL无核酸酶的水和200μL再悬浮的珠（步骤1.6.3）。将反应管放在旋转轮上，并在RT（22℃）下孵育3小时（或16°CO / N）。 </li&gt; <li>用洗涤溶液洗涤珠子两次，并用1X耐热DNA聚合酶缓冲液（使用不含核酸酶的水从10x溶液稀释）使用磁性支架两次。 </li><li>将珠子重悬于50μL的1×热稳定DNA聚合酶PCR缓冲液中，并将其放置在离磁架上。立即进行第1.8.1步，或在4℃下储存长达一天。 </li></ol></li><li> 左和右连接DNA的预扩增: <ol><li>通过加入10μL10μM1L引物，10μL10μM1R引物，20μL10μM引物Link1（表1），10μL10X热稳定DNA聚合酶缓冲液，4μL 10mM dNTPs（终体积:每个dNTP为200μM），8μL（20U）热稳定DNA聚合酶和88μL无核酸酶的水加入来自步骤1.7.3的再悬浮珠子（50μL）中。 </li><li>将反应混合物等分成4个PCR管（每个50μL1; L）并进行PCR，具有以下条件:94℃孵育2分钟; 94℃20秒，56℃25秒，72℃2分钟25个循环;最后在72℃延伸5分钟。 </li><li>将所有4个PCR反应混合到2 mL微量离心管中，并按照PCR纯化试剂盒的PCR纯化程序进行。用50μL洗脱缓冲液洗脱。 </li><li>使用UV-Vis分光光度计测定DNA浓度和260/280-nm值; DNA可以保存在-20°C直到准备下一步。继续执行步骤1.9.1 / 1.10.1（可选）或直接执行步骤1.11.1 / 1.12.1。 </li></ol></li><li> 取出左侧内部DNA扩增子（可选）: <ol><li>将高达100ng PCR DNA产物从步骤1.8.4转移到2mL微量离心管中，并使用不含核酸酶的水将体积调节至10μL。 </li><li>准备专门针对左侧内部D的限制酶反应NA扩增子。 注意:反应条件可能因酶的选择而异。例如，当从基于NL4.3的慢病毒载体中除去左侧内部DNA时，加入1μL的pvuII ，2μL的缓冲液B和7μL的无核酸酶的水。在PCR仪中37℃孵育1小时。立即进行步骤1.11.1或存储在-20°C。 </li></ol></li><li> 取出右侧内部DNA扩增子（可选）: <ol><li>继续与步骤1.9.1相同。 </li><li>准备特异性靶向右侧内部DNA扩增子的限制酶反应。 注意:例如，从基于NL4.3的慢病毒载体中取出右侧内部DNA时，加入1μLsfoI ，2μL缓冲液B和7μL无核酸酶的水。在PCR仪中37℃孵育1小时。立即进行步骤1.12.1或储存于-20°C。 </li></ol></li><li> 剩下结特异性扩增: <ol><li>通过混合来自步骤1.8.4（或选自步骤1.9.2）的5μLDNA，5μL10μM2L引物（终体积:1μM），5μL10μM引物Link2，制备50μLPCR反应（最终为1μM），5μL10×耐热DNA聚合酶缓冲液，1μL10mM dNTP（终体积:每个dNTP200μM），2μL（5U）热稳定DNA聚合酶和27μL无核酸酶水。 </li><li>进行以下条件的PCR:94℃孵育3分钟; 94℃20s，56℃25s，72℃2分钟8-15个循环;最后在72℃延伸5分钟。 注:扩增的DNA可以储存在-20°C。 *建议循环次数优化。 </li><li>按照PCR纯化试剂盒的PCR纯化程序。用50μL洗脱缓冲液洗脱。确定DNA浓度和260/280 nm值。 </li></ol></li><li><st荣&gt;右结特异性扩增: <ol><li>所有方法与"左结特异性扩增"相同（步骤1.11.1-1.11.3），不同之处在于使用不同的引物;在该步骤中使用右侧特异性引物（2R-引物， 参见表1 ）。 </li></ol></li><li> PCR扩增子长度变异试验: <ol><li>通过进行2％琼脂糖凝胶电泳或毛细管电泳分析PCR扩增子长度变化（ 图2 ）。 注意:这是确认完成测定程序并基于PCR带图案粗略评估IS模式的重要步骤。来自步骤1.11.3和1.12.3的纯化的PCR扩增子可用于各种DNA测序平台。继续进行经典链终止（Sanger）测序或下一代测序的适当样品制备程序。 DNA可以保存在-20°;C。 </li></ol></li></ol> 2.计算IS序列分析<ol><li> 数据文件的准备: <ol><li>准备三个输入数据文件:fasta格式序列数据文件（补充数据中的Test_data.fa），用于向量和链接器序列的搜索图案的tsv文件（补充数据中的Demultiplexing_Trimming_blunt_GTAC.tsv）和限制酶信息的tsv文件（Enzyme.tsv补充资料）。 注意:这些文件是解复用，修剪向量和链接器序列，以及删除内部向量序列所必需的（ 图3A ）。在补充数据中的README.txt文件中提供了实现计算工作流程的详细分步说明。 </li></ol></li><li> 计算分析: <ol><li>运行解复用和修剪脚本。 注意:原始序列将被处理用于解复用和trimming的载体，接头和引物序列（见补充README.txt文件中的STEP-1）。 </li><li>运行映射命令（参见README.txt文件中的STEP-2），使用类似BLAST的对齐工具（BLAT; www.genome.ucsc.edu）将处理的序列映射到参考基因组上。 </li><li>运行定量IS分析脚本（请参阅README.txt文件中的STEP-3）。 注意:将生成两个输出文件（Initial_count_without_homopolymer_correction.txt和Final_count.txt）。有关映射和序列枚举策略的更多详细信息，请参见"README.txt文件中的补充数据和以前的出版物2,15。 </li></ol></li></ol>

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作者没有什么可以披露的。

双向测定能够同时分析上游（左）和下游（右）载体 - 宿主DNA连接序列，并且可用于许多基因治疗，干细胞和癌症研究应用。与以前的基于TCGA基序的酶（ TaqαI ）为基础的检测2,15和其他方法相比，使用GTAC基序酶（RsaI和CviQI）和双向PCR方法显着提高了检测内含子（或克隆群体）的机会单向LM-PCR方法5 。双向测定改善了IS的分析，特别是在临床或小动物模型研究中经常?...

逆转录病毒将其基因组DNA插入各个位点的宿主基因组。这种独特的性质可能有助于癌症和其他形式的病毒发病机制的发展，具有使这些病毒高度适应细胞工程用于基因治疗和基础生物学研究的讽刺意义。病毒整合位点（IS） - 整合了外来DNA（病毒）的宿主基因组上的位置 - 对整合的病毒和宿主细胞的命运具有重要意义。 IS测定已经用于各种生物和临床研究环境，以研究逆转录病毒整合位点选择和发病机理，癌症发展，干细胞生物学和发育生物学1,2,3,4 。检测灵敏度低，数据重现性差，交叉污染频繁将IS测定应用限制在当前和计划研究中的关键因素。 已经开发了许多IS分析技术。包括连接介导的（LM）聚合酶链反应（PCR） 5 ，逆PCR 6和线性扩增介导（LAM）PCR 7的限制性酶基整合位点测定法是最广泛使用的。然而，使用位点特异性限制性酶在IS的检索期间产生偏倚，仅允许在限制性位点附近恢复4个整合到宿主基因组内的整合子（集成到宿主基因组中的外来DNA） 4 。最近几年也引入了更综合评估载体IS的分析技术。这些测定采用各种策略，包括Mu转座子介导的PCR 8 ，非限制性（nr）-LAM PCR 9 ，II型限制性内切酶缓冲消化10 ，机械剪切11和随机六聚体基因PCR（Re-free PCR） 12 ，以克隆基因组DNA并扩增IS。目前的技术具有不同程度的检测灵敏度，基因组覆盖率，靶特异性，高通量能力，测定程序的复杂性以及检测目标位点相对频率的偏差。鉴于现有测定的不同质量和可以使用的各种目的，应仔细选择最佳测定方法。 该工作提供了详细的实验程序和双向测定的计算数据分析工作流程，通过同时分析整合的靶DNA的上游和下游IS，显着提高检测率和序列定量准确度（参见图1的测定程序示意图）。这种方法也提供了是表征逆转录病毒整合过程的手段（例如，靶位点重复的保真度和上游和下游插入的基因组序列的变化）。其他双向方法主要用于克隆和测序目标DNA 11,13,14的两端。使用公认的LM-PCR方法和计算分析映射以及用于量化上游和下游连接点，广泛优化用于载体标记克隆的高通量和可重复定量的测定。已经证明使用TaqαI酶的双向分析对于干细胞基因治疗临床前研究中的高通量克隆定量是有用的。本文介绍了一种使用更频繁的切割器（RsaI / CviQI-主题:GTAC）的修改方法，使两倍与基于TaqαI的测定相比，他检测到整合素的机会。描述了使用GTAC基序酶用于慢病毒（NL4.3及其衍生物）和γ-逆转录病毒（pMX载体）载体IS分析的详细实验和数据分析程序。测定中使用的寡核苷酸列于表1中 。用于IS序列分析的内部编程脚本在补充文档中提供。

<table border="0" cellpadding="0" cellspacing="0" width="1004">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Thermostable DNA polymerase</td>
			<td style="width:208px;">Agilent</td>
			<td style="width:119px;">600424</td>
			<td style="width:347px;">PicoMaxx Polymerase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermostable DNA polymerase buffer</td>
			<td style="width:208px;">Agilent</td>
			<td>600424</td>
			<td style="width:347px;">PicoMaxx Polymerase buffer</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Deoxynucleotide (dNTP) solution mix</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>N0447L</td>
			<td>dNTP solution mix (10mM each)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR tubes</td>
			<td style="width:208px;">VWR International</td>
			<td style="width:119px;">53509-304</td>
			<td style="width:347px;">PCR Strip Tubes With Individual Attached Caps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">2ml microcentrifuge tube</td>
			<td style="width:208px;">Molecular Bioproducts</td>
			<td style="width:119px;">3453</td>
			<td>microcentrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">PCR purification kit</td>
			<td style="width:208px;">Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">RsaI&#160;</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>R0167L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">CviQI</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>R0639L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Buffer A</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>B7204S</td>
			<td>NEB CutSmart buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA Polymerase I large (klenow) fragment&#160;</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>M0210L</td>
			<td>Blunting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">streptavidin beads solution</td>
			<td>Invitrogen&#160;</td>
			<td>60101</td>
			<td>Dynabeads kilobaseBINDER kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Binding Solution</td>
			<td>Invitrogen&#160;</td>
			<td>60101</td>
			<td>Dynabeads kilobaseBINDER kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Washing Solution</td>
			<td>Invitrogen&#160;</td>
			<td>60101</td>
			<td>Dynabeads kilobaseBINDER kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">magnetic stand</td>
			<td style="width:208px;">ThermoFisher</td>
			<td>12321D</td>
			<td>DynaMag&#8482;-2 Magnet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">T4 DNA ligase</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>M0202L</td>
			<td>T4 DNA ligase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">10X T4 DNA ligase buffer</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>B0202S</td>
			<td>T4 DNA ligase reaction buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">5X T4 DNA ligase buffer</td>
			<td style="width:208px;">Invitrogen&#160;</td>
			<td>46300-018</td>
			<td>T4 DNA ligase buffer with polyethylene glycol-8000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV-Vis spectrophotometer</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td>S06497</td>
			<td style="width:347px;">Nanodrop 2000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">pvuII</td>
			<td style="width:208px;">new England Biolabs</td>
			<td>R0151L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">sfoI</td>
			<td style="width:208px;">new England Biolabs</td>
			<td>R0606L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buffer B</td>
			<td style="width:208px;">new England Biolabs</td>
			<td>B7203S</td>
			<td>NEB buffer 3.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Nuclease free water</td>
			<td style="width:208px;">Integrated DNA Technologies</td>
			<td>11-05-01-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Capillary electrophoresis</td>
			<td style="width:208px;">Qiagen</td>
			<td>9001941</td>
			<td style="width:347px;">QIAxcel capillary electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Veriti 96-well Fast Thermal Cycler</td>
			<td style="width:208px;">Thermo Fisher Scientific</td>
			<td>4375305</td>
			<td>PCR Instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Rotating wheel (or Roller)</td>
			<td style="width:208px;">Eppendorf</td>
			<td>M10534004</td>
			<td>Cell Culture Roller Drums</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">DNA size marker</td>
			<td style="width:208px;">Qiagen</td>
			<td>929559</td>
			<td>QX size marker (100-2,500 bp)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">DNA size marker</td>
			<td style="width:208px;">Qiagen</td>
			<td>929554</td>
			<td>QX size marker (50-1,500 bp)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">DNA alignment markers</td>
			<td style="width:208px;">Qiagen</td>
			<td>929524</td>
			<td>QX DNA Alignment Marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">genomc DNA</td>
			<td style="width:208px;">Not Available</td>
			<td style="width:119px;">Not Available</td>
			<td>Sample genomc DNA from in vivo or in vitro experiments</td>
		</tr>
	</tbody>
</table>

bidirectional retroviral integration site pcr methodology and quantitative data analysis workflow

整合位点（IS）测定是逆转录病毒整合位点研究的关键组成部分及其生物学意义。在最近的逆转录病毒基因治疗研究中，IS测定结合下一代测序已被用作细胞跟踪工具来表征共享相同IS的克隆干细胞群体。为了准确比较不同样品内和跨越不同样品的干细胞克隆，检测灵敏度，数据重现性和高通量能力均为最重要的检测标准之一。这项工作为双向IS分析提供了详细的协议和数据分析工作流程。双向测定可以同时排列上游和下游载体 - 宿主结。与常规单向IS测序方法相比，双向方法显着提高了IS检测率和t两端积分事件的表征他瞄准DNA。这里描述的数据分析流程通过多个比较步骤准确地识别和列举相同的IS序列，将IS序列映射到参考基因组上并确定测序误差。使用优化的测定程序，我们最近在恒河猴猕猴移植后发表了数千个造血干细胞（HSC）克隆的详细重新填充模式，首次证实了HSC重新增殖的精确时间点和HSC在功能异质性中的功能异质性灵长类动物系统。以下协议描述了准确识别和量化相同的IS序列的逐步实验过程和数据分析工作流程。

双向IS测定为上游（左）和下游（右）载体宿主结（ 图2 ）产生不同大小的PCR扩增子。 PCR扩增子的大小取决于最后的GTAC基序上游和下游的位置。该测定还产生内部DNA PCR扩增子:分别在左侧和右侧连接PCR期间在多巴胺附近的逆转录病毒序列和引物结合位点同时扩增。通过毛细管或琼脂糖（2％）电泳可以显示PCR扩增子条带。 <p class="jove_content" fo:keep-to...

Watch this Scientific Journal Video about Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow at JoVE.com

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

该手稿描述了可以同时分析上游和下游载体 - 宿主连接DNA的双向整合位点测定的实验程序和软件分析。双向PCR产品可用于任何下游测序平台。所得数据对于综合DNA靶标的高通量，定量比较是有用的。

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

bidirectional-retroviral-integration-site-pcr-methodology

Research

JoVE Journal

Genetics

10.7K 次观看.  University of California at Los Angeles (UCLA). 该手稿描述了可以同时分析上游和下游载体 - 宿主连接DNA的双向整合位点测定的实验程序和软件分析。双向PCR产品可用于任何下游测序平台。所得数据对于综合DNA靶标的高通量，定量比较是有用的。 

视频: Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

合并绝对和相对定量PCR数据来量化STAT3剪接变体成绩单

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

科研

遗传学

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

逆转录病毒扫描:映射MLV集成站点以定义细胞特异性监管区域

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

斑马鱼 RNASeq-based 基因表达数据的样品制备与分析

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

间歇酵母 2-混合筛序列数据的信息化分析

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

使用纳米流体技术的单体和散装 C. 电子样品 中的高通量定量 RT-PCR

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

Quantitative Real-Time PCR Evaluation of microRNA Expressions in Mouse Kidney with Unilateral Ureteral Obstruction

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to partially complementary target sites in the 3&#39; untranslated region (UTR) of messenger RNA (mRNA), which reduces the mRNA&#39;s translation and stability. The miRNA expression profiles in various organs and tissues of mice have been investigated, but standard methods for the purification and quantification of miRNA in mouse kidney have not been available. We have established an effective and reliable method for extracting and evaluating miRNA expression in mouse kidney with renal interstitial fibrosis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The protocol required five steps: (1) creation of sham and unilateral ureteral obstruction (UUO) mice; (2) extraction of kidney samples from the UUO mice; (3) extraction of total RNA, which includes miRNA, from the kidney samples; (4) complementary DNA (cDNA) synthesis with reverse transcription from miRNA; and (5) qRT-PCR using the cDNA. Using this protocol, we successfully confirmed that compared to the controls, the expression of miRNA-3070-3p was significantly increased and those of miRNA-7218-5p and miRNA-7219-5p were significantly decreased in the kidneys of a mouse model of renal interstitial fibrosis. This protocol can be used to determine the miRNA expression in the kidneys of mice with UUO.

We describe a method for evaluating the microRNA expression in the kidneys of mice with unilateral ureteral obstruction (UUO) by quantitative reverse-transcription polymerase chain reaction. This protocol is suitable for studying kidney microRNA expression profiles in mice with UUO and in the context of other pathological conditions.

小鼠肾中微RNA表达与单体尿阻的定量实时PCR评价

MicroRNAs (miRNAs) are single stranded, non-coding RNA molecules that typically regulate gene expression at the post-transcriptional level by binding to ...

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the &#966;C31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces &#966;C31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the &#966;C31 integrase. We report two representative results of &#966;C31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. &#966;C31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the &#966;C31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the &#966;C31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

位点导向 的φC31介导的疟疾 按蚊 载体中的整合和盒式交换

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the ...