This work was supported in part by grants from the National Institute of Environmental Health Sciences (ES014443 and ES017274) and AstraZeneca to KSR.

注意:所有步骤应在室温下进行，除非另有规定。请参考试剂部分如何准备单独的试剂的详细信息。 1.标签L1探头注:探头可以通过化学或PCR标记进行标记。 <ol><li> 化学标记 <ol><li>使在Tris-乙酸-EDTA（TAE）缓冲液，热0.7-1％琼脂糖凝胶在微波直到熔融，使凝胶冷却，然后加入溴化乙啶（0.5微克/毫升）。倒入凝胶托盘，添加梳子和使凝胶在室温下固化。 </li><li>根据制造商的协议标签用Cy3或FITC的SNeo探针（1-2微克），在37℃1小时。 注:SNeo表示在S pliced ​​逆转录转座子的完整周期之后表达新霉素基因。探头的尺寸〜1000 bp之间。 SNeo可以从任何载体或从染色体组进行PCR扩增IC DNA。见材料和试剂表上链霉亲和CY3 / FITC标记试剂的信息。 </li><li>混合用1×DNA上样缓冲液，负载标记SNeo探针溶液导入各孔中，并在75-100伏运行15-20分钟。 </li><li>使用紫外（UV）照明形象化探头频带。注意，SNeo探针的大小可以调整，并且锁核酸（LNA）也可以被添加（ 图2）。 </li><li>切从凝胶标记SNeo探针用干净的刀片，溶解，并且使用按照制造商的方案在PCR清理试剂盒从凝胶中纯化SNeo探针。 注意:查看和切割凝胶时盖在身上与防护盾（即实验室外套和UV明确面罩）的每一个暴露的部分。见材料表和试剂的信息，凝胶纯化PCR产物。 </li><li>重新运行5微升SNeo的标记探针在0.7％琼​​脂糖凝胶一个未标记探针SNeo（参见1.1.1）精读企业规模的增加和信号强度的损失（ 图2）。 </li><li>在无核酸酶的H 2 O，通过测量260nm处的吸光度定量标记SNeo探针的量和稀释SNeo探针10毫微克/微升等份商店等分于-20℃。 </li></ol></li><li> PCR标记（探针标记的另一种方法） <ol><li>结合SNeo特异性引物（5&#39;-ggatagcattgggagatatacct-3&#39;和5&#39;-attgaacaagatg gattgcacgc-3&#39;）与PCR试 ​​剂成一个单一的管:13.6微升不含核酸酶的 H 2 O; 0.1微升的dATP，dCTP，dGTP（10纳米）; 0.05微升的dTTP（10纳米）; 0.05微升的dUTP / dUTP的-FITC / CY3（10纳米）; 4微升去标签5×缓冲; 0.4微升去标签聚合酶; 1微升SNeo模板（10毫微克）。见材料与试剂的表上的PCR试剂的信息。 </li><li>通过样品的总数乘以调整各试剂的量。 </li><li>使用以下PCR循环参数s至放大和标签SNeo探针:95℃，2分钟; 95℃35个循环，30秒，62℃，30秒，72℃60秒; 72℃，2分钟;在4℃保持下去。 注:退火温度可以调节或梯度PCR就可以完成，以确定最佳退火温度为其他探针。 </li><li>让0.7-1％凝胶如第1.1.1节，加载和可视化探测波段，如第1.1.4。 </li><li>剪切和净化标记SNeo探针在第1.1.5。 </li><li>重新运行5微升SNeo标有联合国标记的SNeo探针来确认大小的增加和信号强度的损失探针（ 见图2）。 </li><li>在无核酸酶的H 2 O，通过测量260nm处的吸光度定量标记SNeo探针的量和稀释SNeo探针10毫微克/微升等份</li></ol></li></ol> 2.准备染色体价差<ol><li>产生表达载体骨架稳定克隆（CONTRO升）或反转录主管L1使用标准选择方法。12,16虽然非附加型记者被用于转录的研究的结果相互关联，也可以使用游离型载体。生长细胞稳定表达控制或L1载体（每10cm平板1×10 6个细胞，并在板尺寸所需调整。制造）在完全生长培养基。对HepG2细胞，用RPMI-1600，10％FBS和200微克/毫升潮霉素。生长培养至70％汇合，在37℃和5％的CO 2。 注:生长培养基的选择是依赖于细胞类型。定，这里使用的质粒携带为潮霉素和新霉素选择盒，克隆的稳定的选择也可以与上潮霉素选择后新霉素进行，但需要进行优化，以建立公差水平对于每个小区类型新霉素的量。 </li><li>秋水仙胺增加（0.4微克/毫升），以培养和孵化90分钟以metaph逮捕细胞酶。如果使用不同类型的细胞，确定最佳中期阻滞凭经验秋水仙胺和曝光的时间（60，90，和120分钟）的最佳浓度。 </li><li>洗涤细胞，加入3-5毫升0.25％胰蛋白酶溶液至细胞，并孵育5分钟，以分离细胞用10ml 1×Dulbecco氏PBS（DPBS），trypsinize的2倍。灭活胰蛋白酶与等量的含10％FBS的介质。 </li><li>离心2分钟将细胞以1000 xg离心在4℃下，吸从细胞沉淀中的培养基并用DPBS洗涤细胞。吸出所有的DPBS，留下200微升DPBS来重新悬浮细胞。确保细胞充分混合，所有的团块分散。使用闪烁或轻柔吹打混合和分散团块，避免涡流。 </li><li>添加5毫升低渗溶液的（ 即，75毫米氯化钾），它是当水平旋转15毫升管预加热到37℃，逐滴，并在37℃下孵育20分钟。见材料和Reagen的表TS有关如何获取并低渗溶液信息。 </li><li>在120×g离心在4℃下5分钟，并重复离心细胞步骤2.4 - 2.5 3×留下约200μl的低渗溶液中重新悬浮每次洗涤后的细胞。保证在3次洗涤结束后，将沉淀是明显的白色和肿胀。 </li><li>重悬在200μl的卡诺固定液沉淀和使1:2,1:4,1:8 1:16稀释在卡诺固定液。从大约为1厘米以上下降10微升每个稀释到干干净的玻片，并立即从30秒沸水暴露自由传播，一边热蒸汽。见材料和试剂信息表就如何使卡诺固定液。 </li><li>用0.1微克/干差在室温下，污点毫升的Hoechst-33342通过浸渍在科普林罐15-20分钟，洗3次用DPBS。见材料和试剂信息表上如何使赫斯特-33342的解决方案。 </li> &lt;LI&gt;查看利差荧光/相差显微镜放大40倍。优化传播的准备后，利差不需要用Hoechst-33342 染色 。在放大10倍的相差显微镜查看利差来确定传播的质量和分离，在结果部分概述（ 见图3）。 </li><li>选择并在滑动的相对侧绕有钻石点标记好差（ 即，扩展自由侧）。 </li><li>商店价差在-20°C至一个月。避免接触水分。 </li></ol> 3. 荧光原位杂交（FISH） <ol><li> 稳定和脱水利差 注意:如果储存在-20℃中平衡中期染色体扩散到室温。见材料和信息试剂的表如何获得和使试剂。 <ol><li>孵育中期染色体用200微升RNA酶A的传播在37℃下1小时。 </li><li>洗涤在2×-SSC缓冲液滑动两次，每次5分钟，随后用10mM的HCl溶液漂洗。 </li><li>在37℃下孵育，用1％的胃蛋白酶差为10分钟，用去离子水2 O冲洗，并用2×-SSC缓冲液洗两次，每次5分钟。 </li><li>孵育，用4％多聚甲醛涂抹10分钟，并在2×SSC缓冲液洗两次，每次5分钟。 </li><li> 70％，80％和95％的乙醇:脱水通过2分钟的乙醇系列孵育传播。 </li><li>风干的幻灯片。 </li></ol></li><li> 杂交L1逆转录转座子标记的探针价差（即 SNeo） 注意:对于直接标记探针，避免光线照射远在任何时候。见材料和信息试剂的表如何获得和使试剂。 <ol><li>加入30纳克SNeo的杂交缓冲液中，热在72℃10分钟并冷却，在室温下2分钟。 </li><li>加入30微升SNeo探针溶液到eACH蔓延，盖上盖玻片和密封橡胶水泥的边缘。确保没有泡沫的形成。 </li><li>加热在72℃的滑动用于热块上5分钟，温度逐渐下降至37℃，并在37℃的黑暗潮湿的腔室孵育过夜。 </li></ol></li><li> 洗涤和查看（或二次抗体中添加） 注意:对于直接标记探针，避免光线照射远在任何时候。 注:请参阅材料以及有关如何准备试剂信息表。建议足够的体积，以覆盖整个染色体扩散的用途。请记住，加盖玻片当过量体积都将丢失。 <ol><li>沉浸在幻灯片2X SSC缓冲液去除盖玻片。 </li><li>在45℃下在2X-SSC缓冲液通过浸渍洗涤玻片5分钟。 </li><li>在45℃在洗涤缓冲液中浸泡洗玻片5分钟，2次。 </li><li>在45℃下在0.1×SSC缓冲液通过浸渍洗涤玻片10分钟。 </li><li>在45℃下在2×SSC缓冲液通过浸渍洗涤玻片10分钟。 注意:将包含建议缓冲区水浴科普林罐，调节温度至45℃。 </li><li>凉爽的幻灯片到室温并平衡在检测缓冲幻灯片。 </li><li>对于直接标记探针，跳到步骤3.4.10。 </li><li>对于间接标记探针，在20-30分钟封闭液块DPBS洗3次。 </li><li>用50μl二抗（ 例如 ，5微克/毫升的链霉-FITC或αCY3在封闭缓冲液）1小时孵育。 </li><li>洗载玻片在2×SSC 5分钟两次。 </li><li>染液用Hoechst-33342溶液（0.1微克/毫升）10分钟。 注:此染色完成染色为共定位与探针的染色体。 </li><li>洗在DPBS 3倍，增加安装介质一滴，放置盖玻片并密封用指甲油的边缘。 </li><li>利用荧光显微镜在40倍均线分析L1逆转录转座子gnification。 </li></ol></li></ol>

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作者宣称没有潜在利益冲突。

方法，如全基因组测序，反向PCR和Southern杂交已用于研究L1逆转录转座子。虽然这些方法是在基因组内发生L1插入定位极有价值，为所有这些混杂的挑战是需要在硅片编程进行重组序列。这里所描述的FISH方法被设计以补充这些方法，特别是在需要在培养细胞中的异位L1反转录的分析研究的情况。该方法可用于反转录事件的定量和定性评价和施加到间期核。这种方法拥有的用于确定异位L1插入位点16随后在硅片序列比对方法的准确性。重复序列的比对可以是不明确的，由于均聚物的形成，而重复sequenCES可造成重复序列的代表性不足模板的引物扩增过程中丢失。 FISH方法可克服这些问题，因为FISH探针可退火到均聚物，而且不可能对完整重复序列16被偏置。此外，相比于基于细胞培养实验逆转录转座子，鱼可以在单核水平确定反转录率。因此，这种方法可以防止两下并用反转录速率为多个插入可以发生成单核16的估计。从NGS获得的最新数据证实，基于细胞培养方法低估了反转录频率25。 其中，L1S年轻人喜欢插入和靶向机制是否存在于基因组中直接插入这些目前还不清楚。使用上述方法，我们已经表明，L1中插入优先入基因差再基因组16 gions。其他人已经表明L1序列的丰度增加与较小基因密度17,18染色体。总之，这些发现表明插入的经调节模式，其中向细胞威胁由L1的插入最小化到基因组中的"较小活性"区域。转座因子运动的低等生物的格局已经支持了这种解释。例如，裂殖酵母座子，Tf1的，集成的95％的时间的基因启动子的上游和大多数这些基因参与胁迫调控19,20。在缺乏通入氮气酵母细胞，TY5集成到ORF的，而不是异区21。在玉米实验已经表明DNA转座子的这种融合导致杂色玉米颜色表型和Hatvine1-RRM的DNA转座子插入VvTFL1A基因的启动子区的结合已被证明influenCE分支模式和葡萄22,23的果实大小。 在这里详述的FISH方法的成功至关重要的步骤是很好的染色体利差和适当的标签，纯化和探针杂交一代。如果探针不正确清洗，将得到的高背景信号将使得难以解决的探针结合到染色体上。优选地，探测应凝胶纯化，以消除的dNTPs剩余荧光。此外，该差在卡诺固定液孵育的时间长度是非常重要的，以准备好染色体差。如果时间过长，细胞膜可能过早爆裂，并导致染色体丢失。如果过短，则可能难以获得，因为缺乏膜破裂的适合的差。甲酰胺的量/浓度决定染色单体的焦点，因此，它以凭经验优化最佳结果是重要的。所有洗涤要彻底，以ENSURE所有未结合的探针和次级抗体洗掉。 总之，这里所描述的FISH方法可用于检测两个全长和截短异位L1反转录事件，并且可以应用到使用绿色荧光蛋白（GFP），为反转录指示器盒其他反转录载体。虽然可以使用这种方法来确定全长度L1插入，它不会识别新的内源性截短的插入由于截短的L1 S中的基因组中的数目。探头的辅助功能也可能会被提高异染色质形成16,24的限制。然而，LNA的FISH探针内包括可以被用来提高探针退火。 SNeo的检测是在反转录的一个完整周期队伍，比插入/缺失可错过的探头更小。 对于whic实验详细说明小时使用这些载体和L1蛋白质和反转录的表达的特征，请参阅出版作品12,16。

人长散布核元件-1（ 行1或L1）是通过基因组内传播"复制和粘贴"反转录机制的自主移动元素。一个典型的人类L1是〜6 kb的，由充当启动子的5&#39;UTR（非翻译区）的，两个开放阅读框（ORF）:L1 -ORF1和L1 -ORf2，并与聚腺苷酸一个3&#39;UTR 。尾巴L1 -ORF1蛋白质具有三个不同的区域:一个线圈螺旋结构域，RNA识别基序和C末端结构域，而L1 -ORf2蛋白质具有核酸内切酶，逆转录酶和富含半胱氨酸的结构域1,2,3,4,5。 L1 -ORF1表现出的核酸分子伴侣的活动，而L1 -ORf2提供酶活性，与逆转录转座子1,2,6所需的两种蛋白。 L1逆转录转座子的周期从L1的5&#39;UTR转录启动</EM&gt;由RNA聚合酶II，易位进入细胞质和翻译表达双顺反子。在细胞质中，L1 -ORF1展品无论是顺式结合位它打包了自己的RNA或反式结合位它打包其他的RNA（ 即 SINE / SVAS /假）与L1 -ORF2p 7形成核糖核蛋白颗粒（RNP） 8。的RNP易位进入细胞核其中L1 -ORF2p刻痕基因组DNA（gDNA的）的核酸内切酶活以暴露的OH -基团9，即轮流使用逆转录酶素和反向合成的RNA，从3&#39;末端的DNA。期间反转的合成，DNA的第二链被缺口从原始切口部位7-20碱基创建交错符它们填充以形成签名L1插入序列（ 例如 ，TTTTAA）称为目标部位重复（TSD）9,10。这个过程被称为目标素逆转录（TPRT）并导致inserti上在插入序列的两端的L1 /与TSDS其他DNA的完整或截短的副本。L1反转录也已经显示，通过介导TPRT状非同源末端接合在一些细胞类型11。 在我们的研究中所用的L 1反转录载体是非附加型和由标签L1 -ORF1＆2P通过组合CMV-L1-5&#39;UTR启动子驱动（ 图1）12的。这种结构的早期版本已在使用酵母和人类细胞培养13,14,15研究进行了描述。位于5&#39;和3&#39;两个不同的CMV启动子的载体的结束时，用3&#39;末端放置在反向方向，剪接和融合后带动新霉素的表达。在3&#39;末端的反转录指示符盒由一个新霉素基因插入的反义到L1 -ORFs的和的glob呈现由分离非活性成两半在与拼接供体（SD）和剪接受体（SA）位点（ 图1）的内含子。一旦整合到染色体中，L1是从共同的启动子转录产生，它由一个双顺反子mRNA的和不活动的新霉素基因的mRNA。期间RNA加工，球蛋白内含子剪接出来的新霉素基因的恢复一个全功能的新霉素基因。该混合的mRNA包装成在顺一个RNP和易位进入它被整合到基因组作为使用TPRT任全长或截短的插入的核。 在这里，我们描述了使用荧光原位杂交（FISH）用专门针对拼接新霉素基因（SNeo）探针在单核水平来跟踪L1 -retrotransposiition型态和插入率的方法。效率和检测的特异性是使用反转录称职和不称职的结构和探针DET证实等SNeo或新霉素和珠蛋白内含子交界处16。这种方法占一些细胞培养基于反转录测定法，如多次插入，菌落电阻和有利的克隆扩充的缺点。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Labeling Probes&lt;/strong&gt;</td></tr><tr><td>Go Tag DNA polymerase</td><td>Promega</td><td>M3178</td><td></td></tr><tr><td>GO Tag 10x colorless buffer</td><td>Promega</td><td>M3178</td><td></td></tr><tr><td>Individual dNTP</td><td>Sigma</td><td>DNTP10</td><td>Adjust the concentration of dATP, dGTP, dCTP to 2&amp;nbsp;mM and dTTP to 1.5&amp;nbsp;mM in nuclease free water.</td></tr><tr><td>dUTP-16-Biotin (0.5&amp;nbsp;mM)</td><td>Roche</td><td>11093070910</td><td></td></tr><tr><td>dUTP-FITC (0.5&amp;nbsp;mM)</td><td>Thermo Scientific</td><td>R0101</td><td></td></tr><tr><td>dUTP-CY3 (0.5&amp;nbsp;mM)</td><td>Sigma</td><td>GEPA53022</td><td></td></tr><tr><td>MIRUS FISH Labeling Kit</td><td>MIRUS</td><td>MIR3625</td><td></td></tr><tr><td>MIRUS FISH Labeling Kit</td><td>MIRUS</td><td>MIR3225</td><td></td></tr><tr><td>NucleoSpin PCR Clean-Up kit&amp;nbsp;</td><td>Qiagen</td><td>1410/0030</td><td>Any PCR clean reagent can be used in place of NucleoSpin PCR Clean Up Kit</td></tr><tr><td>PCR Machine</td><td></td><td></td><td>Any Thermocycler machine can be used to amplify the label product.</td></tr><tr><td>Agarose</td><td>Sigma</td><td>A9539</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Preparing chromosome Spreads&lt;/strong&gt;</td></tr><tr><td>Colcemid</td><td>Life Technologies&amp;nbsp;</td><td>15212-012</td><td>Add Colcemid directly to growth media to a final concentration of 0.4&amp;nbsp;&amp;micro;g/ml</td></tr><tr><td>1&amp;nbsp;M KCl</td><td>Sigma</td><td>P9541</td><td>Dilute 1&amp;nbsp;M KCl solution to 75&amp;nbsp;mM solution to make Hypotonic Solution</td></tr><tr><td>Carnoy Fixative Solution</td><td></td><td></td><td>Mix 3 volumes of methanol and 1 volume of acetic acid to make Carnoy Fixative Solution. Make Fresh everytime.</td></tr><tr><td>Methanol</td><td>Sigma</td><td>322415</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma</td><td>320099</td><td></td></tr><tr><td>Hoechst-33342</td><td>Thermo Scientific</td><td>62249</td><td>Dissolve Hoechst-33342 in PBS to final concentration of 1&amp;nbsp;mg/ml.</td></tr><tr><td>Diamond Point Marker</td><td>Thermo Scientific</td><td>750</td><td></td></tr><tr><td>Frosted Slides</td><td>Thermo Scientific</td><td>2951-001</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Life Technologies&amp;nbsp;</td><td>R001100</td><td>To detech cells, incubate cell in Trypsin for 5 min and inactive with equal volume of complete media.</td></tr><tr><td>Cover slides</td><td>VWR</td><td>48366067</td><td></td></tr><tr><td>Coplin Jars</td><td>Thermo Scientific</td><td>107</td><td></td></tr><tr><td>Heat Block</td><td></td><td></td><td>Any heat block can be used for this purpose, though blocks fitted for heating slides are recommended.</td></tr><tr><td>Beaker (600&amp;nbsp;ml)</td><td>Sigma</td><td>CLS1003600</td><td>Fill the beaker with water, heat to boil, use the hot steam to burst chromosomes.</td></tr><tr><td>Nikon Microscope</td><td>Nikon</td><td>125690</td><td>Any microscope can be used to look at spread quality.</td></tr><tr><td colspan="4">&lt;strong&gt;Reagents and Materials for FISH&lt;/strong&gt;</td></tr><tr><td>Trisodium citrate</td><td>Sigma</td><td>S1804</td><td></td></tr><tr><td>Sodium Chroride</td><td>Sigma</td><td>S3014</td><td></td></tr><tr><td>Formamide</td><td>Sigma</td><td>F9037</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>F1379</td><td></td></tr><tr><td>Detran Sulfate</td><td>Sigma</td><td>D8906</td><td></td></tr><tr><td>SDS</td><td>Sigma</td><td>L3771</td><td></td></tr><tr><td>Salmon Sperm DNA</td><td>Life Technologies&amp;nbsp;</td><td>15632-011</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A8531</td><td></td></tr><tr><td>HCl (N)</td><td>Sigma</td><td>38283</td><td></td></tr><tr><td>Ethyl Alcohol</td><td>Sigma</td><td>459844</td><td></td></tr><tr><td>Methanol</td><td>Sigma</td><td>322415</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies&amp;nbsp;</td><td>14190-250</td><td></td></tr><tr><td>NaOH</td><td>Sigma</td><td>S8045</td><td></td></tr><tr><td>Rnase A</td><td>Sigma</td><td>R4642</td><td></td></tr><tr><td>Pepsin</td><td>Sigma</td><td>P6887</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Hoechst-33342</td><td>Thermo Scientific</td><td>62249</td><td>Dissolve Hoechst-33342 in PBS to final concentration of 1&amp;nbsp;mg/ml.</td></tr><tr><td>Rubber Cement/Cytobond Sealant&amp;nbsp;</td><td></td><td>2020-00-1</td><td></td></tr><tr><td>Seven Coplin Jars</td><td>Thermo Scientific</td><td>107</td><td></td></tr><tr><td>Dark Humidified chamber</td><td>Sigma</td><td>CLS2551</td><td></td></tr><tr><td>Cover slides</td><td>VWR</td><td>48366067</td><td></td></tr><tr><td>Dry Digital Heat Block</td><td>VWR</td><td>13259</td><td></td></tr><tr><td>Fluorescence Microscope</td><td></td><td></td><td></td></tr><tr><td>20x Saline-Sodium Citrate (20x SSC)</td><td></td><td></td><td>Combine 175 g of NaCl, and 88.2&amp;nbsp;g of trisodium citrate with 800 ml of molecular grade H&lt;sub&gt;2&lt;/sub&gt;O. Stir while adjusting to pH 7. Once the all salts dissolve, adjust the volume to 1 L and filter through 0.22 &amp;micro;m Filter paper. Store at 4 &amp;deg;C.</td></tr><tr><td>1&amp;nbsp;mg/ml of Rnase A</td><td></td><td></td><td>Dilute 20x SSC buffer to 2x SSC buffer. Dissolve RNase A in 2x SSC buffer to a final concentration of 1&amp;nbsp;mg/ml. Make fresh solution every time.</td></tr><tr><td>1% Pepsin</td><td></td><td></td><td>Dissolve pepsin (W/V) in 10 mM HCl to a final concentration of 1% solution. Make fresh every time.</td></tr><tr><td>4% Paraformaldehyde (4% PFA)</td><td></td><td></td><td>Weigh 4.0 g of PFA in fume hood, add 50 ml of 1xPBS, heat to 60&amp;nbsp;&amp;deg;C while stirring and adjust the pH with drops of NaOH until all PFA dissolves and the solution becomes clear. Adjust the volume to 100&amp;nbsp;ml, filter through 0.22 &amp;micro;m Filter paper and store 5 ml aliquots at -20&amp;nbsp;&amp;deg;C. Thaw aliquot of PFA at 37 &amp;deg;C for 10-15 min for subsequent uses. Adjust filter size depending on amount of paraformaldehyde.</td></tr><tr><td>Wash Buffer</td><td></td><td></td><td>Combine 100 ml of Formamide (20%) with 2.5 ml of 20x SSC, adjust to pH 7 with 1.0 M HCl and bring the volume to 500 ml with molecular grade H&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Hybridization Buffer</td><td></td><td></td><td>Combine 50% formamide, 10% dextran sulfate, 0.1% SDS, and 300 ng/ml Salmon Sperm DNA in 2x SSC buffer. Amount of Formamide determine how focused chromatids are; titrate the amount.</td></tr><tr><td>Detection Buffer</td><td></td><td></td><td>Dilute 20x SSC buffer to 4x and add 0.2% Tween-20 to make detection buffer.</td></tr><tr><td>Blocking Buffer</td><td></td><td></td><td>Combine 5% bovine serum albumin (BSA) with 0.2% Tween-20 in 4x SSC buffer.</td></tr><tr><td>Dehydrating Solution</td><td></td><td></td><td>Make 70%, 80%, and 95% ethanol solutions.</td></tr></tbody></table>

analysis of line-1 retrotransposition at the single nucleus level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

在L1反转录载体的示意图示于图1中 。该载体包括在反义方向的L1的ORF新霉素基因，该基因是通过在有义方向一个球蛋白内含子中断，由SD和SA位点夹持的。当稳定地整合入染色体中，L1基因是从组合CMV和L1-5&#39;UTR启动子（ 图1）转录。在RNA加工，珠蛋白内含子拼接出新霉素基因。的新- L1 RNA被打包，易位和整合进基因组，可以是全长或截短的插入。正如所指出的，L 1反转录可以通过FISH使用特异于拼接新霉素（ 图1）的探针跟踪。 图2示出了新霉素探针的示意图，与labeleð探测，由于在激发波长的差异是在大小和荧光缺陷较大。注意，CY3和FITC激发在不同波长比溴化乙锭染色的DNA。 2，1:4,1:8和1:16的稀释液，并在10倍的放大倍率对从细胞核差的密度，分布和距离来评价每个扩频（中期染色体差的密度通过稀释原始原液成1中确定图3A）。结果显示高密度，块状和放声差（ 图3A-I-II），以及均匀地分布和良好的间距差（ 图3A-ⅲ-ⅳ）。低密度蔓延与均匀分布被选为随后的分析。 染色体差被用Hoechst染料和传播质量进行评价的基础上的染色体的长度，圆度在S的染色preads和染色体间距离（ 图3B）。这些指数是由时间的细胞用秋水仙胺，低渗溶液，卡诺固定液和/或其中的细胞被捣毁释放染色体的方式处理的长度的影响。如果细胞培养在低渗溶液中的周期短，染色体差变得紧打结和个体染色体难以显现（ 图3B-1）。另一方面，在低渗溶液再孵育导致晶核破裂，染色体的散射，和/或染色体丢失（ 图3B-ⅱ）。在秋水仙胺再孵育在中期增加细胞的数量，但会导致染色体缩合（ 图3B-ⅲ）。因此，良好的品质染色体传播需要在这两个秋水仙碱和低渗KCl溶液最佳的潜伏期。在我们的手中，一个90分钟孵育秋水仙碱，20在37低渗溶液℃，使用热蒸汽爆裂的细胞被认为是用于产生高品质的差（ 图3B-ⅳ）的最佳条件。 染色体利差进行染色使用的目标SNeo探头L1逆转录转座子。所述探针标记有两个FITC和CY3表明在这两种情况下的L1反转录在表达野生型L1细胞只看到 （ 图4）。没有染色见于单独表达载体骨架细胞（ 图4;比较顶板和底板的每个荧光团）。在我们的手中，核80-90％的检测探头的信号，以压倒多数较单一反转录事件的信号。我们只打进了逆转录转座子在细胞核与多个插入和鱼类面前，总是检查蔓延质量。 图1. L1 逆转录转座子载体 的示意图 。图描绘的目标主要逆转录导致完全或截短的L1插入的过程。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53753/53753fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53753/53753fig2.jpg" /> 图2. 标记SNeo和未标记的新霉素探头。标记探针具有荧光少，是相对于非标记探针大小。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53753/53753fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3.相衬（明场）和赫斯特染色染色体的价差。 A）显示利差的稀释，以获得良好的间距准备。比例尺为50微米。B）显示秋水仙碱，低渗溶液，卡诺固定液和传播质量爆棚的影响赫斯特染色染色体。比例尺为10微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53753/53753fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53753/53753fig4.jpg" /> L1 逆转录转座子 的 图4. FISH分析 。SNeo染色是控制细胞不存在，但目前在L1表达野生型载体细胞。比例尺为10微米。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53753/53753fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Analysis of LINE-1 Retrotransposition at the Single Nucleus Level at JoVE.com

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

在单核级LINE-1逆转录转座子分析

在这里，我们采用FISH方法在单核级跟踪LINE-1逆转录转座子在肝癌细胞系稳定表达合成LINE-1的染色体价差 ​​。

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

analysis-of-line-1-retrotransposition-at-the-single-nucleus-level

Nanyang Technological University

Research

JoVE Journal

Biology

8.3K 次观看.  University of Arizona College of Medicine. 在这里，我们采用FISH方法在单核级跟踪LINE-1逆转录转座子在肝癌细胞系稳定表达合成LINE-1的染色体价差 ​​。 

视频: Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

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 ...

科研

遗传学

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 ...

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

细胞谱系分析和基因功能研究使用双点MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

发育生物学

Detection of Retrotransposition Activity of Hot LINE-1s by Long-Distance Inverse PCR

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so by a process called retrotransposition wherein they transcribe to form an mRNA intermediate which is then consequently inserted into the genome by reverse transcription. Despite being silent in normal cells, LINE-1s are highly active in different epithelial tumors. De novo LINE-1 insertions can potentially drive tumorigenesis, and hence it is important to systematically study LINE-1 retrotransposition in cancer. Out of ~150 retrotransposition-competent LINE-1s present in the human genome, only a handful of LINE-1 loci, also referred to as &#34;hot&#34; LINE-1s, account for the majority of de novo LINE-1 insertion in different cancer types. We have developed a simple polymerase chain reaction (PCR)-based method to monitor retrotransposition activity of these hot LINE-1s. This method, based on long-distance inverse (LDI)-PCR, takes advantage of 3&#180; transduction, a mechanism by which a LINE-1 mobilizes its flanking non-repetitive region, which can subsequently be used to identify de novo LINE-1 3&#180; transduction events stemming from a particular hot LINE-1.

This article outlines a simple PCR-based assay to monitor the activity of an active LINE-1 retrotransposon and to map de novo retrotranspositions in a given genome. Using the MCF7 cell line, we demonstrate herein how this method can be applied to detect activity of a LINE-1 located at 22q12.1.

长距离反向PCR对热线1的逆变活性的检测

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so ...

癌症研究

RNA Next-Generation Sequencing and a Bioinformatics Pipeline to Identify Expressed LINE-1s at the Locus-Specific Level

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and mutagenesis. Understanding the expression patterns of L1 loci at the individual level will lend to the understanding of the biology of this mutagenic element. This autonomous element makes up a significant portion of the human genome with over 500,000 copies, though 99% are truncated and defective. However, their abundance and dominant number of defective copies make it challenging to identify authentically expressed L1s from L1-related sequences expressed as part of other genes. It is also challenging to identify which specific L1 locus is expressed due to the repetitive nature of the elements. Overcoming these challenges, we present an RNA-Seq bioinformatic approach to identify L1 expression at the locus specific level. In summary, we collect cytoplasmic RNA, select for polyadenylated transcripts, and utilize strand-specific RNA-Seq analyses to uniquely map reads to L1 loci in the human reference genome. We visually curate each L1 locus with uniquely mapped reads to confirm transcription from its own promoter and adjust mapped transcript reads to account for mappability of each individual L1 locus. This approach was applied to a prostate tumor cell line, DU145, to demonstrate the ability of this protocol to detect expression from a small number of the full-length L1 elements.

Here, we present a bioinformatic approach and analyses to identify LINE-1 expression at the locus specific level.

RNA 下一代测序和生物信息学管道, 以识别点对位的表达 Line-1

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

<meta charset="utf8"></head><body>用于分析 DNA 复制和叉停滞事件的 2D 凝胶电泳

Electrophoretic Analysis of Replication Through Structure-Prone DNA Repeats Within the SV40-Based Human Episome

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This protocol describes how to analyze replication fork progression through structure-prone, expandable DNA repeats within the simian virus 40 (SV40)-based episome in human cells. In brief, upon plasmid transfection into human cells, replication intermediates are isolated by the modified Hirt protocol and treated with the DpnI restriction enzyme to remove non-replicated DNA. Intermediates are then digested by appropriate restriction enzymes to place the repeat of interest within the origin-distal half of a 3-5 kb-long DNA fragment. The replication intermediates are separated into two perpendicular dimensions, first by size and then by shape. Following Southern blot hybridization, this approach allows researchers to observe fork stalling at various structure-forming repeats on the descending half of the replication Y-arc. Furthermore, this positioning of the stall site allows the visualization of various outcomes of repeat-mediated fork stalling, such as fork reversal, the advent of a converging fork, and recombinational fork restart.

<meta charset="utf8"></head><body>作者聚焦:表征致病性重复序列的 DNA 复制以揭示复制叉停滞和扩增的机制

Here, we outline the procedure for analyzing replication progression through pathogenic, structure-prone repeats using 2-dimensional gel electrophoresis.

通过基于 SV40 的人游离体内易结构 DNA 重复序列的电泳分析进行复制

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This ...

以单细胞分辨率从小鼠心脏祖细胞中分离细胞核

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

作者聚焦:从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率下的表观基因组和基因表达分析  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率的表观基因组和基因表达谱分析

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

在单核级LINE-1逆转录转座子分析

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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

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

细胞谱系分析和基因功能研究使用双点MARCM

长距离反向PCR对热线1的逆变活性的检测

RNA 下一代测序和生物信息学管道, 以识别点对位的表达 Line-1

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

通过基于 SV40 的人游离体内易结构 DNA 重复序列的电泳分析进行复制

通过基于 SV40 的人游离体内易结构 DNA 重复序列的电泳分析进行复制

通过基于 SV40 的人游离体内易结构 DNA 重复序列的电泳分析进行复制

从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率的表观基因组和基因表达谱分析