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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-flares+for+mRNA+regulation+and+detection.">Nano-flares for mRNA regulation and detection.</a> ACS Nano. 3 (8), (2009).</li><li>Mehta, 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=Re-trafficking+of+hERG+reverses+long+QT+syndrome+2+phenotype+in+human+iPS-derived+cardiomyocytes.">Re-trafficking of hERG reverses long QT syndrome 2 phenotype in human iPS-derived cardiomyocytes.</a> Cardiovasc. Res. 102 (3), 497-506 (2014).</li><li>Seftor, E. 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. Immunol. 15 (4), 343-353 (2014).</li><li>Kratz, 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=Digital+expression+profiling+of+the+compartmentalized+translatome+of+Purkinje+neurons.">Digital expression profiling of the compartmentalized translatome of Purkinje neurons.</a> Genome Res. 24 (8), 1396-1410 (2014).</li></ol>

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 border="0">
	<tbody>
		<tr>
			<td>DMEM/Ham&#39;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 (107 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 CO2 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

Research

JoVE Journal

Biology

10.9K 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

Bacterial Gene Expression Analysis Using Microarrays

使用微阵列的细菌基因表达分析

科研

生物学

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

等位基因特异性基因表达检测，测试的遗传协会的功能基础

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

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 ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...