Name: detection of intracellular gene expression in live cells of murine, human and porcine origin using fluorescence-labeled nanoparticles
Uploaded: 2015-11-13T14:00:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles at JoVE.com

We thank Lynette Henkel for her excellent support in performing the flow cytometric analysis. We thank Dr. Don Weldon and Dr. Victor Koong (Temecula, CA) for critical reading of the manuscript and helpful comments. This work was supported by grants from the German Research Foundation, KR-3770-7/1 and KR-3770-9/1 (M. K.), the Deutsche Stiftung f&#252;r Herzforschung, F/37/11 (M. K.), Dr. Rusche Research Grant 2014 from the Deutsche Stiftung f&#252;r Herzforschung and Deutsche Gesellschaft f&#252;r Thorax-, Herz- und Gef&#228;sschirurgie (M.-A. D.), and the Deutsches Zentrum f&#252;r Herz- und Kreislaufforschung S&#228;ule B Projekt, DZHK B 13-050A and DZHK B 14-013SE (M. K.).

1.准备的媒体，试剂和细胞培养条件<ol><li>人类的HEK 293胚胎肾细胞<ol><li>培养HEK293细胞（CRL-1573，ATCC）在DMEM /含的F-12培养基补充有5％FCS，L-谷氨酰胺（2毫摩尔）和青霉素（100U /毫升）/链霉素（100微克/毫升）。 </li><li>到通道中的细胞，用PBS洗涤一次细胞，加入0.05％胰蛋白酶/ EDTA孵育3分钟，在37℃和5％的CO 2。传递细胞入15ml管中，离心5分钟，在1200转（300× 克）和传送约5×10 5个细胞到新鲜的T25培养瓶中。 </li></ol></li><li>小鼠胚胎成纤维细胞（MEF中） <ol><li>加入1毫升的0.1％明胶溶液，以单独的6孔中，并在室温下孵育（RT）1小时。吸液体和储存在室温备用。 </li><li>培养的MEF的DMEM补充有10％FCS，L-谷氨酰胺（2毫摩尔），L-葡萄糖（4.5克/升），丙酮酸钠（1mM的），和青霉素（100U /毫升）/链霉素（100微克/毫升）在明胶包被的6孔板中。 </li><li> MEF中分配到6孔板，让他们成长，直至汇合。加入丝裂霉素（5微克/毫升）2.5小时逮捕细胞。吸介质，具有完整的MEF培养基洗涤细胞两次，并加3毫升MEF中到每个6孔。这些细胞用作饲养层为鼠和猪iPS细胞。 </li></ol></li><li>鼠ES和iPS细胞的培养<ol><li>为了制备小鼠ES培养基，补充的DMEM培养基中，用15％的FCS，L-谷氨酰胺（2毫摩尔），L-葡萄糖（4.5克/升），MEM非必需氨基酸（1X），β巯基乙醇（0.1毫摩尔）和白血病抑制因子（1.000单位/毫升）。 </li><li>冲洗6孔与生长停滞的MEF一次用无血清的DMEM，加入2 ml小鼠ES培养基和小鼠iPS细胞（汇合培养皿的细胞的20％）。每天洗一次细胞无血清DMEM，并加2ml新鲜小鼠ES网上平台。 </li><li>到通道中的细胞，洗净各孔一次用无血清的DMEM中，添加00.25％胰蛋白酶/ EDTA孵育3分钟，在37℃和5％的CO 2。传递细胞入15ml管中，离心5分钟，在1200转（300×g）中，悬浮的细胞在500μl小鼠ES培养基，并添加100μl的悬浮液，以2 ml小鼠ES培养基中新鲜6孔用MEF饲养层。 </li></ol></li><li>猪的培养iPS细胞<ol><li>为了制备猪的iPS介质，补充的DMEM /含的F-12培养基，用20％敲除血清，L-谷氨酰胺（2 mM计），MEM非必需氨基酸（1X），β巯基乙醇（0.1毫摩尔），和青霉素（100 U / ml的）/链霉素（100微克/毫升）。通过0.22微米过滤器，并储存在4℃下进行2〜3周进行过滤。在第一次使用时，加入的bFGF（10毫微克/毫升终浓度）。 </li><li>冲洗6孔与生长停滞的MEF一旦与无血清的DMEM /含的F-12，并添加1.5 ml的猪的iPS介质和猪iPS细胞（汇合培养皿的细胞的10％）。每天洗一次细胞无血清的DMEM /联队的F-12，加入1.5毫升新鲜猪的iPS网上平台。 </li><li>为了制备解离缓冲液添加2.5％的胰蛋白酶，胶原酶IV（10毫克/毫升），敲除血清（20％）和氯化钙 （1毫米），以无 Ca 2+的PBS。用PBS洗涤细胞两次，并孵育解离缓冲液进行5分钟，在37℃和5％的CO 2。传递细胞入15ml管中，离心5分钟，在1200转（300× 克）。 </li><li>重悬猪iPS细胞在500μl新鲜的猪的iPS中，加入50微升悬浮液以1.5毫升猪的iPS媒体在一个新的6孔与MEF饲养层。每天洗一次细胞无血清的DMEM /联队的F-12，并添加1.5毫升新鲜猪的iPS网上平台。 </li></ol></li><li>人iPS细胞的无饲养培养<ol><li>解冻含基底膜制备原小瓶在4℃，并用冰冷的无血清DMEM /火腿的F-12〜8毫克/毫升，等分并储存稀释在-20℃直至进一步使用。 </li><li>解冻基的等分试样换货膜制剂在4℃和稀释用冰冷的无血清DMEM /含的F12（90微克/毫升）。吸取1ml该溶液成3厘米培养皿的，摇动轻轻以确保整个表面被覆盖。密封菜封口膜，并保存在4℃下长达一个星期。在使用前的细胞培养孵育菜为在37℃，30分钟或3小时，在室温，以允许基底膜制备的聚合。 </li><li>从碗碟吸介质涂覆有基底膜制剂并用无血清的DMEM /含的F-12洗两次。添加1.5 ml的E8介质到培养皿和人iPS细胞（汇合培养皿的细胞的10％）。每天洗一次细胞无血清的DMEM /联队的F-12，并添加1.5毫升新鲜E8网上平台。 </li><li>要通过细胞，涮菜两次用PBS和加入1毫升的PBS / 0.5毫摩尔EDTA。孵育菜为5分钟，在室温。监视在显微镜下的细胞。注意:当殖民地开始围捕和APPEAR有洞，他们准备转移。 </li><li>吸出液体，通过轻轻地上下吹打添加1.5 ml的E8介质和分离细胞。转移细胞到15ml管中，离心5分钟，在1200转（300×g）中，重悬在500μlE8介质和添加50微升悬浮液至1.5 ml的E8培养基中新鲜培养皿涂覆有基底膜制剂。 </li></ol></li></ol>跨物种边界靶序列的2设计<ol><li>选择参考人，鼠和的靶基因（GAPDH，NANOG，GDF3）猪cDNA序列并执行的CLUSTAL多序列比对分析。 注意:此将指示序列同源物种之间。 <ol><li>用于多重比对以下设置:空位罚（固定）10，缺口罚分（变化）5，过渡加权0，空位分离罚分范围8，对于延迟40％的同一性，最大迭代次数来执行3。 </li></ol></li><li>提交所需的靶序列制造商（ 见表材料 ）探头的设计和制造。 注:那么制造商将确认该技术所需序列的兼容性。如果需要的序列是不兼容，制造商将建议的替代序列。 <ol><li>确保它可以用作潜在捕获链序列具有27碱基的长度，即同源区。 注:这是最好订购多个纳米颗粒不同的捕获股为选定的靶基因，因为只有真正的移动实验会给个人序列的功能有一定的证据。因此，对于GAPDH和GDF3两个不同的序列而三种不同的纳米颗粒进行了测试以评价NANOG的表达进行了评价。它们的位置和精确的序列已经在别处17出版。制造商商业化制造了nanop文章与所需序列，并为他们在冻干格式。 </li></ol></li></ol> 3.重组纳米粒子和除了细胞培养<ol><li>在黑暗中存储在接收的冻干纳米颗粒在4℃下。 </li><li>通过加入50微升双蒸水赋予为100nM的最终浓度重构纳米颗粒。一旦重组店纳米粒子在室温在黑暗中。注意:在此条件下它们是稳定至少一年。重构的样本存储在4℃下可在其稳定性造成不利影响。 </li><li>预稀释上施用当天的纳米颗粒储液用PBS到2-（HEK 293细胞）或为8nM（iPS细胞与ES细胞）的浓度。注意:此浓度比在培养皿中的最终浓度的20倍。在ES细胞和i​​PS细胞中，400微米的纳米颗粒诱导的荧光信号，在显微镜下容易检测。为HEK 293细胞中，交流的为100pM oncentration就足够了。 </li><li>吸取12.5微升预稀释纳米粒子以24孔含237.5微升完全细胞培养基。对于不同尺寸的孔中，相应地调整卷。小心摇动板，保证了纳米粒子的均匀分布。过夜（O / N）温育所述细胞在37℃和5％的CO 2中的潮湿空气。 </li><li>第二天分析细胞培养物为基因特异性的Cy3荧光的546nm的（发射575 - 640纳米）的激发波长在荧光显微镜下。注意:为了获得足够强的荧光可以取决于细胞类型和靶基因所需的时间。在这些实验中，这是16和20小时之间的探针的应用后可见。 </li></ol> 4. Cy3的鉴定阳性细胞群流式细胞仪<ol><li>成长HEK 293或第1.1或1.3规定的使用条件下的小鼠胚胎干细胞，Respectively。前一天的FACS分析添加的GAPDH特异性的纳米颗粒以不同的浓度向HEK 293细胞（参见图6A）和在400 1pM至鼠ES细胞。孵育细胞O / N在37℃和5％的CO 2中的潮湿空气。 </li><li>为基因特异性荧光显微镜下的Cy3诱导荧光分析细胞培养物。 </li><li>分离HEK 293细胞下点1.1.2的规定。离心，转移到1.5毫升管，重悬在200μl冰冷的PBS / 0.5％BSA / 2mM EDTA的（FACS缓冲液）。保持细胞的暗冰，直到流式细胞仪分析。 </li><li>分离的小鼠ES细胞下点1.3.3的规定。离心，转移到1.5毫升管，重悬在200μl冰冷的PBS / 0.5％BSA / 2mM EDTA的。保持细胞的暗冰，直到流式细胞仪分析。 </li><li>通过30μM过滤器按细胞，以防止细胞聚集和添加碘化丙啶（2微克/毫升的最终浓度）2分钟前的FACS分析来排除死细胞。 </li><li>为了分析的Cy3阳性细胞首先设置在FSC-A / SSC-A子集层次城门​​，FSC-H / FSC-W-子集和SSC-H / SSC-W子集。使用前向和侧向散射以排除细胞碎片和未散射的细胞。他们的碘化丙啶染色显示PE对PE得克萨斯红排除死细胞。执行上的Cy3-PE阳性细胞的最终排序门（ 图4）。 </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanoma+tumor+cell+heterogeneity:+a+molecular+approach+to+study+subpopulations+expressing+the+embryonic+morphogen+Nodal.">Melanoma tumor cell heterogeneity: a molecular approach to study subpopulations expressing the embryonic morphogen Nodal.</a> Sem. Oncol. 41 (2), 259-266 (2014).</li><li>Khare, 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=The+PYRIN+domain-only+protein+POP3+inhibits+ALR+inflammasomes+and+regulates+responses+to+infection+with+DNA+virus.">The PYRIN domain-only protein POP3 inhibits ALR inflammasomes and regulates responses to infection with DNA virus.</a> Nat. 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HL已经接收自EMD Millipore公司纳米颗粒免费的。所有其他作者什么都没有透露。

目前的工作描述了使用荧光标记的纳米颗粒允许细胞内基因表达的可靠的评估直接在来自不同哺乳动物物种用荧光显微镜下多样组织发生起源的活细胞。这种方法有两个优点相比其他公布的方法。首先研究者并不限于与上述任何靶基因或细胞类型。所有基于抗体的检测方法被限制在一个单一的表位。虽然荧光分子信标，也可设计为基因的广谱性，这种方法需要的细胞和随后的核转染15的解离。与此相反，基因特异性纳米颗粒的应用不需要任何操纵哪些吞噬纳米颗粒积极通过内吞作用的靶细胞。在随后的传代金颗粒逐渐离开细胞和培养可以在downstrea使用米实验。 然而，该技术也有一些限制。一些标志物是明显不可检测由于它们的碳水化合物的性质如SSEA-1或TRA-1-81 20。目前，大阵纳米颗粒是市售的。这些颗粒已经预先测试其在细胞实验中的功能。本工作的目的是设计出可以跨越物种边界被用于定制探针。以下这样的方法时，或用于一种新颖的靶基因中的一个设计的探针时必须注意的，在硅片的功能预测探针需要在体外进行全面评估。所述NANOG-SF3探针同源性为100％的序列同源性并没有在所有的工作而NANOG-SF1与一个错配的碱基在鼠和猪的序列产生一个很好的荧光信号。另一个问题是指纳米颗粒的有效吸收。该粒子进入细胞通过内吞作用，这是由纳米颗粒21的尺寸的影响的方法，该细胞类型和分化状态22和蜂窝能力来执行phago-和巨吞23。的最佳浓度，以获得令人满意的荧光信号具有为每个单独的应用或细胞系进行测试。为此目的，第一实验始终应摄取控制的应用上移动到目标特异性检测基因表达之前评估所述探针内的细胞类型和培养条件的相容性。 一个关键点应用该协议时，一个新的细胞系或细胞群是包含适当的控制，以获得可靠的结果。不断荧光摄取控制将提供一个提示该纳米颗粒的浓度将引起在靶细胞群体的足够的荧光信号。荧光必须评估D中的第二天作为信号将随着时间而消逝17。在同一时间，而不在施加以相同浓度的真核基因组中的对应的扰频控制，必须诱导可忽略的背景信号。作为第三控制 ​​最好是使用特异于持家基因的纳米颗粒（如GAPDH，β肌动蛋白 ）。这些纳米颗粒充当阳性对照，该方法可用于检测特定基因的表达在所选择的靶细胞群体。如果这些控制转出令人满意的人们可以期待使用特异于其他靶基因的纳米粒子，以获得可靠的特异性荧光信号。所施加的纳米颗粒的浓度也可以是关键的，因为它们已被证明下调靶序列24。然而，这种效果需要高得多的浓度（5 nm）的比在这里（400分）呈现的实验中使用的浓度。在此浓度下的nanofla水库不影响靶基因的mRNA或蛋白水平和浓度高达为2nM不损害HEK293和鼠胚胎干细胞17的扩散。此外，全基因组表达分析没有发现的基因表达25任何显著改变。因此，在浓度高达1纳米的nanoflares应该不表现出任何重要的不利影响。 这种技术的一个非常有前途的应用是哪个标签带标记的特定基因的任何给定的细胞群体的可能性。最近，基因特异性的纳米颗粒已经成功地被用于选择性地染色活心肌细胞26，黑色素瘤细胞27的亚群，单核细胞28和小脑细胞培养物29。这种荧光标记的细胞群体可以被分类，并通过流式细胞仪作为活细胞中纯化。在肿瘤学领域中它是可以想象的分离活转移性肿瘤细胞在动物模型中基于它们的CEA的表达，针对搜索潜在转移促进基因或靶结构的最有价值的。我们最近表明真正重新编程鼠的iPS集落可以原位标识Nanog的基于所检测的荧光强度17特异性纳米颗粒。因此，真正的重新编程的iPS克隆鉴定可以在高通量平台，它使用机器人荧光检测和分拣设备，以确定最有前途的殖民地进行。此技术似乎是合适的，在许多研究领域以选择特定的细胞亚群，它似乎能够应用纳米颗粒在高通量平台。总之，本协议表示使用荧光标记的纳米颗粒，它允许细胞内基因表达的检测，直接在活细胞中一种新的方法。这种技术可能是一个有价值的工具，以净化，扩大和进一步字符IZE在许多研究领域罕见的细胞亚群。

2006年，一种新的方法被描述为体细胞重编程到多能状态。预筛选潜在重编程的阵列的后因子鼠成纤维细胞，可以成功地转换到所谓的诱导多能干细胞（iPS）通过逆转录病毒转导和四种转录因子的过度表达（Oct4的，SOX2，KLF4，c-Myc的 ）1。随后该技术被有效地使用不同的哺乳动物物种包括人类在内的2，3猴子，大鼠4，狗5，6羊和猪7的体细胞再生。此外，改性的协议漏报或替换潜在致癌转录因子C- MYC已经出版8,9。 不管初始重新编程接近生长的菌落的真正的多能性已被确认，费力和耗时的任务。主要缺点大多数这些分析的是，它们不能被活细胞上进行的。建立多能性因素，如OCT4，SOX2，NANOG或REX1代表位于细胞核转录因子和它们的检测要求到免疫组织化学或细胞裂解用于RT-PCR分析10之前的细胞的固定- 12之后，这些细胞被丢失供将来实验要求各殖民地的复制培养。 因此，一个技术来分析多能性活细胞上是非常需要的。几种方法确实可以在使用抗体的活细胞针对基于膜的抗原直接进行（例如。TRA-1-60，SSEA4）12，荧光报道染料来检测碱性磷酸酶13或小分子，其特异性地标记胚胎干（ES）和iPS细胞14。然而，这些抗体可能仍然不利地细胞和每甲基影响外径被限制为单个多能性标志物。最后，荧光分子信标，双反义寡核苷酸具有发夹结构，其允许细胞活染色，已经描述了15。虽然这种方法可以被应用到许多基因，该技术需要一个单一的细胞悬浮液，这是不适用的iPS生长菌落核转染。 几年前基因特异的纳米粒子已被描述分析Survivin表达人类SKBR3乳腺癌细胞16。这份手稿描述了一种新的创新的方法通过利用金纳米粒子结合物检测现场ES和iPS细胞多能性标志物。这些纳米颗粒由带耦合捕获链携带互补的RNA序列和Cy3标记记者链中的核心金颗粒。记者链的荧光信号是由中央金颗粒骤冷。另外，适当纳米颗粒用作阴性和阳性对照，分别为（ 图1）。一个应用程序的纳米粒子被简单地加入到培养基中（图2），并通过内吞作用进入细胞（图3）。如果靶RNA表达它的排水量记者链，然后发出荧光信号。此方法不需要操纵靶细胞和靶基因的表达可以被光学地在荧光显微镜下在活细胞中监测直接。此外，该技术可以适用于各种物种中，iPS细胞的情况下，任何期望的靶基因，因此可以在许多不同的研究领域被采用。最后，流式细胞仪分析允许的Cy3阳性细胞的识别和充实。

<table><tbody><tr><td>DMEM/Ham&#039;s F12 medium with stable glutamine</td><td>Biochrome GmbH</td><td>FG4815</td><td></td></tr><tr><td>Fetal calf serum</td><td>Life Technologies</td><td>SV30160.03</td><td></td></tr><tr><td>Penicillin/Streptomycin (100x)</td><td>Millipore</td><td>A2213</td><td></td></tr><tr><td>Sodium pyruvate (100x)</td><td>Life Technologies</td><td>11360-039</td><td></td></tr><tr><td>DMEM high glucose with stable glutamine</td><td>Biochrome GmbH</td><td>FG0435</td><td></td></tr><tr><td>Mitomycin C</td><td>Roche</td><td>10 107 409 001</td><td>prevents cell division of MEFs</td></tr><tr><td>MEM non-essential amino acids (100x)</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>b-Mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>Leukemia inhibitory factor (10&lt;sup&gt;7&lt;/sup&gt; U/ml)</td><td>Millipore</td><td>ESG1107</td><td>growth factor for murine ES and iPS cells, prevents differentiation</td></tr><tr><td>0.25% Trypsin/EDTA (1x)</td><td>Life Technologies</td><td>25200-056</td><td>detaches adherent cell cultures</td></tr><tr><td>Knockout serum</td><td>Invitrogen</td><td>10828-028</td><td>supplement for human and porcine iPS cell culture</td></tr><tr><td>bFGF</td><td>Peprotech</td><td>100-18B</td><td>growth factor for human and porcine iPS cells</td></tr><tr><td>Collagenase IV</td><td>Worthington</td><td>LS004188</td><td>dissociation of pig iPS cells</td></tr><tr><td>Matrigel</td><td>Corning</td><td>354277</td><td>effective surface for feeder-free culture of human iPS cells, referred to as basement preparation membrane in the manuscript</td></tr><tr><td>E8 medium</td><td>Stem Cell Technologies</td><td>05940</td><td>defined medium for feeder-free culture of human iPS cells on Matrigel</td></tr><tr><td>EDTA 0.5 M</td><td>Invitrogen</td><td>15575-038</td><td></td></tr><tr><td>SmartFlare Probes</td><td>EMD Millipore Corporation</td><td>customized item</td><td>detection of intracellular gene expression in live cells, referred to as nanoparticles in the manuscript</td></tr><tr><td>Fluorescence microscope</td><td>Zeiss</td><td>Axiovert 200M</td><td></td></tr><tr><td>HeraCell 150i CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Scientific</td><td>51026282</td><td></td></tr><tr><td>Gelatine (from porcine skin)</td><td>Sigma</td><td>G6144</td><td>enhances attachment of MEFs to surface</td></tr><tr><td>Propidium iodide (1 mg/ml)</td><td>Sigma</td><td>P4864</td><td>labels dead cells in flow cytometric analyses</td></tr><tr><td>BD FACS Aria Illu</td><td>Becton Dickinson</td><td>A4544</td><td>referred to as device 1 in Figure 4</td></tr><tr><td>Navios</td><td>Beckman Coulter</td><td>775213</td><td>referred to as device 2 in Figure 4</td></tr><tr><td>FloJo vX 10.0.7r2</td><td>Treestar</td><td></td><td>software for analysis of flow cytometry data</td></tr></tbody></table>

detection of intracellular gene expression in live cells of murine, human and porcine origin using fluorescence-labeled nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

在第一次尝试，以验证并建立使用纳米粒子活细胞的细胞内检测特异性基因表达，我们决定在人类HEK 293细胞使用组成性表达的管家基因（GAPDH）进行试点实验。根据制造商的建议，纳米颗粒在这些实验中使用的浓度被选择。在诱导了鲜明的Cy3荧光在这些细胞中O / N培养（图5A）后在荧光显微镜下是可见的一个为100pM最终浓度增加了一个GAPDH特异性探针与培养基。两个对照包括在这些实验中证实的结果。一种控制包括一个所谓的"加扰"纳米颗粒，其中所述记者链不具有在哺乳动物或真核细胞的匹配序列。在"争夺"纳米决定了非特异性背景fluorescenCE由于可能探针降解或荧光淬灭不全。加扰频探针产生的只有边际的荧光信号，这是可以忽略的（图5B）。甲永久荧光"摄取"控制有助于确定细胞通过内吞作用可能不同的细胞类型之间变化，其中包含纳米颗粒的能力。这些积极的控制颗粒在HEK 293细胞（图5C）产生的明亮的荧光信号。因此，该技术的可行性，记录细胞内基因与可忽略的背景信号的一个特定的荧光信号进行了确认。必须提到的是，背景信号和所述特定荧光之间的差异随时间降低。由于探针降解或不完全淬火而特定荧光变淡逐渐（图5D）的背景提高。同样GAPDH特异性纳米粒子在鼠，猪和羊的产地17的初级成纤维细胞培养进行了测试。这些细胞产生高得多的背景信号比HEK293细胞，而目标特异性荧光相当弱，这可能是由于这些培养物的相对低的增殖能力。 这些导频实验促使我们把这一技术用于多能性基因表达的来自不同物种来源的iPS的检测。为此目的，NANOG和GDF3进行了分析，这两者都是已知待表达的多能细胞18。在从一个心脏的Nkx2.5增强剂的转基因小鼠系19的尾尖成纤维细胞衍生的第一iPS细胞进行了研究。对于这两个基因得到强的荧光信号（图6A）和可忽略的Cy3荧光加扰控制（数据未显示）。类似的结果对人类和猪iPS细胞（图6中获得B和C）。重要的是，荧光信号被限制到的iPS集落，并没有标注用作饲养层小鼠和猪的iPS（参见图 6A 和 C的箭头）的成纤维细胞。 NANOG基因表达的分析也表明，并不是每一个序列，在硅片预测是"好"终于能正常工作。之一的诱导几乎没有荧光信号相比的加扰控制的NANOG的特异性的纳米颗粒的三种设计尽管对人类和猪来源的iPS细胞得到了类似的结果的100％的序列匹配的小鼠iPS细胞（图6D）。与此探针（数据未显示）。因此，纳米粒子可用于检测跨物种边界多​​能性基因的表达而需要的每一个设计探针的功能将被事先评估。 的原位可喜的成果活细胞在细胞培养皿的标记促使我们定量分析通过流式细胞术的基因表达。为此GAPDH特异性的纳米颗粒在分级浓度添加到HEK 293细胞单层，以比较所述目标细胞群中所述标记效率。所评定的荧光显微镜相比，没有接受纳米颗粒的细胞的增强的Cy3诱导荧光被认为与最低浓度。纳米颗粒的浓度增加引起的HEK 293细胞的原位 （图7A）一个明确浓度依赖性荧光。使这些细胞以流式细胞分析证实了浓度依赖性标记效率。的Cy3阳性细胞的频率显著增加（比七倍以上）时纳米颗粒的最高浓度施加（图7B）。在进一步的实验的ES从的Nkx2.5心脏细胞增强转基因小鼠线 19被用于通过流式细胞术来分析多能性基因的表达。试点实验400分的积极摄取控制取得了明显的Cy3积极的生活ES菌落在显微镜下和流式细胞仪检测到的Cy3阳性细胞（ 图7C）的近40％。与此相反，可比未经处理的细胞与加扰染色阳性处理的细胞的约0.1％（数据未显示）。类似地，除了特定的GAPDH，和Nanog的GDF3纳米颗粒诱导在细胞培养皿的单个菌落明显的荧光信号。的Cy3阳性细胞为GAPDH作为通过流式细胞术确定的频率是可比较所看到的两个多能性基因（图7C）。这一结果是可预期的Nanog的和GDF3也被认为可以在多能胚胎干细胞组成型表达。因此，这些数据清楚地印第美食的细胞表达特定的基因可以通过流式细胞鉴定分析，定性和通过他们的相对量。 <img alt="图1" src="/files/ftp_upload/53268/53268fig1.jpg" /> 图1:纳米粒子的结构的示意图。基因特异的纳米粒子（A）的结构。（B）争夺纳米粒子（阴性对照）。（C）吸收纳米粒子（阳性对照）。从拉姆等人 17改编，转载来自威利许可。 <img alt="图2" src="/files/ftp_upload/53268/53268fig2.jpg" /> 图2:纳米颗粒的应用以细胞培养物的纳米颗粒的适当稀释液用移液管简单地进入培养基中。 Ø后/ N培养基因特异荧光细胞是在AF可见<html> luorescent显微镜。 <img alt="图3" src="/files/ftp_upload/53268/53268fig3.jpg" /> 图3:纳米颗粒的通过经由内吞细胞主动摄取（A）的细胞积极地通过内吞作用吞噬纳米颗粒（B）的目标mRNA的结合到捕获链和置换荧光报道。 <img alt="图4" src="/files/ftp_upload/53268/53268fig4.jpg" /> 图4:门控策略流式细胞仪。 （一）战略HEK 293细胞。（B）战略小鼠胚胎干细胞。使用装置1为HEK 293细胞或装置2的鼠胚胎干细胞获得的数据和由一个软件工具（见表材料 ）随后分析。 /53268fig5.jpg"/&gt; 图5:在HEK 293细胞的GAPDH -expression分析阿GAPDH特异性的纳米颗粒加入到该细胞为100pM（终浓度）和荧光O / N培养后记录（A）中 的GAPDH特异性的纳米颗粒（B。 ）争夺（阴性对照）。（C）吸收（阳性对照）。（D）争夺和控制的吸收动力学。荧光在指定的时间点加成GAPDH特异性的纳米颗粒后进行测定。刻度条表示50微米。 <img alt="图6" src="/files/ftp_upload/53268/53268fig6.jpg" /> 图6:的多能性基因表达的不同种纳米粒子特异于NANOG或GDF3加入到细胞中，在400 微米的iPS细胞分析（最终concentration）和荧光O / N培养后记录。（A）鼠iPS细胞。（二）人类 iPS细胞。（C）猪iPS细胞。在（A）和（C）的箭头添加NANOG-设计SF3纳米颗粒后指定未标记的成纤维细胞饲养层细胞。（D）的荧光在小鼠iPS细胞。刻度条表示50微米。 <img alt="图7" src="/files/ftp_upload/53268/53268fig7.jpg" /> 图7:纳米标记的活细胞可通过流式细胞仪来识别。流式细胞仪检测Cy3的阳性细胞（A）的Cy3的积极HEK 293细胞中加入不同浓度的纳米粒子后微观角度。（B）频率。（C）的小鼠ES细胞集落直播染色和决心的Cy3积极的频率细胞。加入纳米粒子在400微米的最终浓度。刻度条表示50微米。

Watch this Scientific Journal Video about Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles at JoVE.com

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

detection-intracellular-gene-expression-live-cells-murine-human

Nanyang Technological University

Research

JoVE Journal

Biology

11.0K 次观看.  Technische Universität München. This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species. 

视频: Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

在生物系统中的离子浓度测量荧光纳米粒子

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

科研

生物工程

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

癌症研究

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

锁核酸流式细胞仪，荧光原位杂交（LNA流FISH）：细菌小RNA检测方法

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

免疫与感染

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

通过胞内细菌，研究内溶酶体系统的改造荧光纳米粒子中的应用

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

对活细胞成像的SNAP - Tag融合蛋白的荧光标记的COS - 7

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

生物学

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

使用体外活体细胞成像技术研究脂纳米粒子化疗

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

医学

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

在Live哺乳动物细胞中质粒复制多代荧光显微镜监测

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

通过电穿孔内在和生物分子荧光观察生活中的微生物

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...