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>

<ol>
	<li>Mathias, S. L., Scott, A. F., Kazazian, H. H., Boeke, J. D., Gabriel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+transcriptase+encoded+by+a+human+transposable+element.">Reverse transcriptase encoded by a human transposable element.</a> Science. 254, 1808-1810 (1991).</li><li>Feng, Q., Moran, J. V., Kazazian, H. H., Boeke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+L1+retrotransposon+encodes+a+conserved+endonuclease+required+for+retrotransposition.">Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition.</a> Cell. 87, 905-916 (1996).</li><li>Clements, A. P., Singer, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+LINE-1+reverse+transcriptase:effect+of+deletions+outside+the+common+reverse+transcriptase+domain.">The human LINE-1 reverse transcriptase:effect of deletions outside the common reverse transcriptase domain.</a> Nucl Acids Res. 26, 3528-3535 (1998).</li><li>Martin, S. L., Branciforte, D., Keller, D., Bain, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimeric+structure+for+an+essential+protein+in+L1+retrotransposition.">Trimeric structure for an essential protein in L1 retrotransposition.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 13815-13820 (2003).</li><li>Khazina, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimeric+structure+and+flexibility+of+the+L1ORF1+protein+in+human+L1+retrotransposition.">Trimeric structure and flexibility of the L1ORF1 protein in human L1 retrotransposition.</a> Nat Struct Mol Biol. 18, 1006-1014 (2011).</li><li>Martin, S. L., Li, J., Weisz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+analysis+defines+distinct+functional+domains+for+protein-protein+and+nucleic+acid+interactions+in+the+ORF1+protein+of+mouse+LINE-1.">Deletion analysis defines distinct functional domains for protein-protein and nucleic acid interactions in the ORF1 protein of mouse LINE-1.</a> J Mol Biol. 304, 11-20 (2000).</li><li>Martin, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonucleoprotein+particles+with+LINE-1+RNA+in+mouse+embryonal+carcinoma+cells.">Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells.</a> Mol Cell Biol. 11 (9), 4804-4807 (1991).</li><li>Hohjoh, H., Singer, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+ribonucleoprotein+complexes+containing+human+LINE-1+protein+and+RNA.">Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA.</a> EMBO J. 15, 630-639 (1996).</li><li>Cost, G. J., Feng, Q., Jacquier, A., Boeke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+L1+element+target-primed+reverse+transcription+in+vitro.">Human L1 element target-primed reverse transcription in vitro.</a> EMBO J. 21, 5899-5910 (2002).</li><li>Szak, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+archeology+of+L1+insertions+in+the+human+genome.">Molecular archeology of L1 insertions in the human genome.</a> Genome Biol. 3, (2002).</li><li>Sen, S. K., Huang, C. T., Han, K., Batzer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endonuclease-independent+insertion+provides+an+alternative+pathway+for+L1+retrotransposition+in+the+human+genome.">Endonuclease-independent insertion provides an alternative pathway for L1 retrotransposition in the human genome.</a> Nucl Acids Res. 35, 3741-3751 (2007).</li><li>Bojang, P., Roberts, R. A., Anderton, M. J., Ramos, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+the+HepG2+genome+by+long+interspersed+nuclear+element-1.">Reprogramming of the HepG2 genome by long interspersed nuclear element-1.</a> Mol Oncol. 7, 812-825 (2013).</li><li>Boeke, J. D., Garfinkel, D. J., Styles, C. A., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ty+elements+transpose+through+an+RNA+intermediate.">Ty elements transpose through an RNA intermediate.</a> Cell. 40, 491-500 (1985).</li><li>Heidmann, T., Heidmann, O., Nicolas, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+indicator+gene+to+demonstrate+intracellular+transposition+of+defective+retroviruses.">An indicator gene to demonstrate intracellular transposition of defective retroviruses.</a> PNAS. 85, 2219-2223 (1988).</li><li>Moran, J. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+retrotransposition+in+cultured+mammalian+cells.">High frequency retrotransposition in cultured mammalian cells.</a> Cell. 87, 917-927 (1996).</li><li>Bojang, P., Anderton, M. J., Roberts, R. A., Ramos, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+LINE-1+retrotransposition+in+HepG2+cells+preferentially+targets+gene+poor+regions+of+chromosome+13.">De novo LINE-1 retrotransposition in HepG2 cells preferentially targets gene poor regions of chromosome 13.</a> Genomics. 104, 96-104 (2014).</li><li>Rozen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundant+gene+conversion+between+arms+of+palindromes+in+human+and+ape+Y+chromosomes.">Abundant gene conversion between arms of palindromes in human and ape Y chromosomes.</a> Nature. 423, 873-876 (2003).</li><li>Graves, J. A., Koina, E., Sankovic, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+gene+content+of+human+sex+chromosomes+evolved.">How the gene content of human sex chromosomes evolved.</a> Curr Op Gen Develop. 16, 219-224 (2006).</li><li>Leem, Y. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrotransposon+Tf1+is+targeted+to+Pol+II+promoters+by+transcription+activators.">Retrotransposon Tf1 is targeted to Pol II promoters by transcription activators.</a> Mol Cell. 30, 98-107 (2008).</li><li>Guo, Y., Levin, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+sequencing+of+retrotransposon+integration+provides+a+saturated+profile+of+target+activity+in+Schizosaccharomyces+pombe.">High-throughput sequencing of retrotransposon integration provides a saturated profile of target activity in Schizosaccharomyces pombe.</a> Genome Res. 20, 239-248 (2010).</li><li>Dai, J., Xie, W., Brady, T. L., Gao, J., Voytas, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+regulates+integration+of+the+yeast+Ty5+retrotransposon+into+heterochromatin.">Phosphorylation regulates integration of the yeast Ty5 retrotransposon into heterochromatin.</a> Mol Cell. 27, 289-299 (2007).</li><li>McClintock, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+behavior+of+mutable+loci+in+maize.">The origin and behavior of mutable loci in maize.</a> Proc. Natl. Acad. Sci. U.S.A. 36, 344-355 (1950).</li><li>Fernandez, L., Torregrosa, L., Segura, V., Bouquet, A., Martinez-Zapater, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-induced+gene+activation+as+a+mechanism+generating+cluster+shape+somatic+variation+in+grapevine.">Transposon-induced gene activation as a mechanism generating cluster shape somatic variation in grapevine.</a> Plant J Cell Mol Biol. 61, 545-557 (2010).</li><li>Garcia-Perez, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+silencing+of+engineered+L1+retrotransposition+events+in+human+embryonic+carcinoma+cells.">Epigenetic silencing of engineered L1 retrotransposition events in human embryonic carcinoma cells.</a> Nature. 466 (7307), 769-773 (2010).</li><li>Rodic, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrotransposon+insertions+in+the+clonal+evolution+of+pancreatic+ductual+adenocarcinoma.">Retrotransposon insertions in the clonal evolution of pancreatic ductual adenocarcinoma.</a> Nat Med. 21 (9), (2015).</li></ol>

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?...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Labeling Probes</td>
			<td></td>
			<td></td>
			<td></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 2mM and dTTP to 1.5mM&#160; in nuclease free water.</td>
		</tr>
		<tr>
			<td>&#160;dUTP-16-Biotin (0.5mM)</td>
			<td>Roche</td>
			<td>11093070910</td>
			<td></td>
		</tr>
		<tr>
			<td>dUTP-FITC (0.5mM)</td>
			<td>Thermo Scientific</td>
			<td>R0101</td>
			<td></td>
		</tr>
		<tr>
			<td>dUTP-CY3 (0.5mM)</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&#160;</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Preparing chromosome Spreads.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Colcemid&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>15212-012</td>
			<td>Add Colcemid directly to growth media&#160; to a final concentration of 0.4 &#181;g/mL</td>
		</tr>
		<tr>
			<td>1M KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>Dilute 1M KCl solution to 75 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 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&#160;</td>
			<td>R001100</td>
			<td>To detech cells, incubate cell in Trypsin for 5 mins and inactive with equal volume of complete media</td>
		</tr>
		<tr>
			<td>Cover slides&#160;</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>&#160;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>&#160;Beaker (600ml)</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents and Materials for FISH</td>
			<td></td>
			<td></td>
			<td></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&#160;</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&#160;</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 mg/mL.</td>
		</tr>
		<tr>
			<td>Rubber Cement/Cytobond Sealant&#160;</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>&#160; Dark Humidified chamber</td>
			<td>Sigma</td>
			<td>CLS2551</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slides&#160;</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>&#160;Fluorescence Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></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 g of trisodium citrate with 800 mL of molecular grade H2O. Stir while adjusting to pH 7. Once the all salts dissolve, adjust the volume to 1 L and filter through 0.22 &#181;m Filter paper. Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>&#160;1mg/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 mg/mL. Make fresh solution every time.</td>
		</tr>
		<tr>
			<td>1% Pepsin</td>
			<td></td>
			<td></td>
			<td>Dissolve&#160; 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 &#176;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 mL, filter through 0.22 &#181;m Filter paper and store 5 mL aliquots at -20 &#176;C. Thaw aliquot of PFA at 37 &#176;C for 10-15 min for subsequent uses.&#160; 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 H2O.</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.&#160; Amount of Formamide determine how focused chromatids are, titrate the amount&#160;</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，?...

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

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

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

科研

生物学

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

在大气压力质谱直接分析单细胞

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

斑马鱼胰岛单细胞分辨率钙成像对β细胞功能的分析

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &#955; DNA at the Single Molecule Level

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. In single molecule assays for studying DNA-protein interactions, there are two important factors for consideration: the DNA substrate with enough length for easy observation and labeling a protein with a suitable fluorescent probe. 48.5 kb &#955; DNA is a good candidate for the DNA substrate. Quantum dots (Qdots), as a class of fluorescent probes, allow long-time observation (minutes to hours) and high-quality image acquisition. In this paper, we present a protocol to study DNA-protein interactions at the single-molecule level, which includes preparing a site-specifically modified &#955; DNA and labeling a target protein with streptavidin-coated Qdots. For a proof of concept, we choose ORC (origin recognition complex) in budding yeast as a protein of interest and visualize its interaction with an ARS (autonomously replicating sequence) using TIRFM. Compared with other fluorescent probes, Qdots have obvious advantages in single molecule studies due to its high stability against photobleaching, but it should be noted that this property limits its application in quantitative assays.

Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified &#955; DNA substrate and a Quantum-dot labeled protein.

Qdot 标记蛋白与现场特异修饰的λ DNA 在单分子水平上的相互作用可视化

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

利用两色单分子荧光在原位杂交中Escherichia coli探索大肠杆菌中空间和时间中的 mRNA 动力学

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.