这些研究工作是由美国国家科学基金会科化工，生物工程，环保，交通（CBET）系统的奖号码下CBET-0853780 CBET-1159344和美国国立卫生研究院，美国国家环境卫生科学研究所（NIEHS的支持）下奖号1R43ES022567-01，美国国家癌症研究所，癌症成像计划在奖号CA127745-01。从美国陆军国防大学研究仪器计划获得IVIS Lumina的仪器，用于这项工作。

1。细胞的制备<ol><li>小瓶从液体氮冷冻库存的生物发光的HEK293细胞的恢复和增长的Dulbecco改进的鹰的培养基（DMEM）中添加10％胎牛血清，0.01 mM的不必要的氨基酸，1X抗生素，抗真菌剂，和0.01 mM的钠丙酮酸用T在75组织培养烧瓶中，在37℃和5％CO 2。 注意:媒体及补充元件会根据细胞系，并因此，应该相应地选择。 </li><li>刷新中期每2-3天，直到达到约80％汇合。 注:所需的细胞的量，将根据实验设计。如果需要，可以通过以下方式获得更多的细胞的传代培养。 </li><li>收获细胞进行测试。 <ol><li>移除介质洗涤细胞，用磷酸盐缓冲盐水（PBS）轻轻摇动烧瓶中，然后弃去使用了PBS。 </li><li>加入胰蛋白酶在37℃孵育2分钟，或直至细胞从烧瓶中脱离。 </li><li>收集在PBS分离的细胞，并将它们传送到一个​​干净的离心管。 注意:如果用于非贴壁细胞系，通过离心分离细胞，并用PBS洗一次。 </li></ol></li><li>确定总的细胞计数，使用血细胞计数器或其他细胞计数系统。 </li><li>离心细胞悬浮液在300×g离心5分钟。弃上清，悬浮细胞在新鲜，预热介质。 </li><li>种子细胞成单独的一个不透明的多孔板的孔中的数目相等。在这个例子中，板5×10 5细胞在1ml体积成一个黑色24孔培养板的各孔中。板等体积的培养基中一式三份井作为阴性对照。 注意:接种于各孔中的细胞数目是灵活的，可优化的个别实验。对于每个生物发光的细胞系，实验确定细胞数和biolumi之间的关系消逝的输出，以及最小可检测细胞计数之前的任何毒性试验。要做到这一点，在很宽的范围的人口规模（例如，1×10 3〜1×10 6个细胞）在标准化的成像条件下测量生物发光。 </li><li>处理细胞与测试化合物（次）（如下文所述）。 </li></ol> 2。化学制剂<ol><li>准备作为浓缩原液被测试的化学品。在这个例子中，使用一个100毫克/毫升贮备液的博莱霉素族抗生素。 注意:为了尽量减少基于车辆的潜在毒性作用，尤其是当使用有机溶剂的化学除了作为车辆，它是建议的库存浓度所需的应用程序后的浓度至少为1,000倍。 </li><li>加入准备好的化学股票直接的细胞。在这个例子中，剂量抗生素的利息，终浓度分别为100，200，300，和400微克/毫升的三折扇井。保留三口井的未经处理的细胞作为对照。 注意:应选择的目标化学品的最终浓度，根据具体的实验目标。广泛的浓度通常是测试，获得全方位的毒副作用。 </li></ol> 3。成像和数据分析<ol><li>紧随化学此外，适当的仪表板在成像室，图像采集和生物发光测量。 </li><li>每隔15分钟测量生物发光在24小时内用10分钟积分时间为每个阅读。 注意:在这个例子中使用的积分时间是在每个板的基础上，可以调整用于测量每个孔的基础上使用等光度计给出。也可以调整积分时间可以根据所选择的生物发光细胞系。由于生物发光自主生产的无细胞的破坏或任何外部的刺激，阅读的INGS可以采取任何所需的时间点，以达到具体的实验目标。 </li><li>定量的生物发光强度从每口井通过吞吐量的区域感兴趣的使用兼容的软件。无论是作为显示输出的光的总光通量（光子/秒）或每个样品的平均辐射（光子/秒/厘米2 / steridian）。 </li></ol>

<ol>
	<li>U.S. Food and Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+it+really+FDA+approved?.">Is it really FDA approved?.</a> FDA Consumer Health Information. 2, (2009).</li><li>Hartung, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicology+for+the+twenty-first+century.">Toxicology for the twenty-first century.</a> Nature. 460, 208-212 (2009).</li><li>Olson, H., 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=Concordance+of+the+toxicity+of+pharmaceuticals+in+humans+and+in+animals.">Concordance of the toxicity of pharmaceuticals in humans and in animals.</a> Regul. Toxicol. Pharmacol. 32, 56-67 (2000).</li><li>Stacey, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+developments+in+cell+culture+technology.">Current developments in cell culture technology.</a> Adv. Exp. Med. Biol. 745, 1-13 (2012).</li><li>Li, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+human+ADME/Tox+drug+properties+in+drug+discovery.">Screening for human ADME/Tox drug properties in drug discovery.</a> Drug Discov. Today. 6, 357-366 (2001).</li><li>Li, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+approaches+to+evaluate+ADMET+drug+properties.">In vitro approaches to evaluate ADMET drug properties.</a> Curr. Top. Med. Chem. 4, 701-706 (2004).</li><li>Tannous, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaussia+luciferase+reporter+assay+for+monitoring+biological+processes+in+culture+and+in+vivo.">Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo.</a> Nat. Protoc. 4, 582-591 (2009).</li><li>Thompson, E. M., Nagata, S., Tsuji, F. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+cDNA+for+the+luciferase+from+the+marine+ostracod+Vargula+hilgendorfii.">Cloning and expression of cDNA for the luciferase from the marine ostracod Vargula hilgendorfii.</a> Proc. Natl. Acad. Sci. U.S.A. 86, 6567-6571 (1989).</li><li>Lupold, S. E., Johnson, T., Chowdhury, W. H., Rodriguez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real+time+Metridia+luciferase+based+non-invasive+reporter+assay+of+mammalian+cell+viability+and+cytotoxicity+via+the+beta-actin+promoter+and+enhancer.">A real time Metridia luciferase based non-invasive reporter assay of mammalian cell viability and cytotoxicity via the beta-actin promoter and enhancer.</a> PLoS ONE. 7, (2012).</li><li>Meighen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+bacterial+bioluminescence.">Molecular biology of bacterial bioluminescence.</a> Microbiol. Rev. 55, 123-142 (1991).</li><li>Close, D. M., 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=Autonomous+bioluminescent+expression+of+the+bacterial+luciferase+gene+cassette+(lux)+in+a+mammalian+cell+line.">Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line.</a> PLoS ONE. 5, e12441 (2010).</li><li>Close, D. M., Hahn, R. E., Ripp, S. A., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+toxicant+bioavailability+using+a+constitutively+bioluminescent+human+cell+line.">Determining toxicant bioavailability using a constitutively bioluminescent human cell line.</a> , 1-4 (2011).</li><li>Oliva-Trastoy, M., Defais, M., Larminat, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+the+antibiotic+Zeocin+by+stable+expression+of+the+Sh+ble+gene+does+not+fully+suppress+Zeocin-induced+DNA+cleavage+in+human+cells.">Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells.</a> Mutagenesis. 20, 111-114 (2005).</li><li>Close, D. M., Webb, J. D., Ripp, S. A., Patterson, S. S., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+detection+of+human+toxicants+in+real+time+using+a+human+optimized+bioluminescent+bacterial+luciferase+gene+cassette+bioreporter.">Remote detection of human toxicants in real time using a human optimized bioluminescent bacterial luciferase gene cassette bioreporter.</a> Proc. SPIE 8371, Sensing Technologies for Global Health, Military Medicine, Disaster Response, and Environmental Monitoring II; and Biometric Technology for Human Identification IX. 837117, (2012).</li><li>Close, D. M., Hahn, R. E., Patterson, S. S., Ripp, S. A., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+optimized+bacterial+luciferase,+firefly+luciferase,+and+green+fluorescent+protein+for+continuous+imaging+of+cell+culture+and+animal+models.">Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models.</a> J. Biomed. Opt. 16, e12441 (2011).</li></ol>

490生物技术公司的创始人和所有者，SA瑞普DM关闭和GS Sayler

这种方法演示了如何使用自主生物发光的哺乳动物细胞， 在体外细胞毒性筛选试验，使活细胞进行连续监测，对他们的一生。此协议是灵活的，并且可以被修改，以满足特定的实验条件下，根据需要。例如，这里提出的实验适合用于跟踪急性毒性作用，但可适应评估通过反复成像在增加的时间间隔（ 例如每24个小时），并维持在标准培养条件下的细胞之间的缓慢作用的或长期的影响...

在美国，药品和其他产品用于人类消费需要大量的评估，才供消费者使用的由美国食品和药物管理局批准。为执行此测试的财政负担被放置在显影剂1，大大提高了新化合物的开发成本，因此，转化为对消费者的成本增加。虽然传统的多筛查已利用受试动物作为代表人类宿主，这已被证明是大的财务负担，一个估计$ 2.8十亿每年用于ADME / Tox的（吸收，分布，代谢，排泄，和毒性）筛查，越来越多的证据表明，动物模型不能可靠地预测人类的毒性反应。因此， 在体外人体细胞培养为基础的测试已经获得了普及，在过去二十年来，由于其相对较低的成本，更高的吞吐量，更好地代表人类的生物利用度和毒理学4。电流细胞培养为基础的毒性筛选方法采用各种端点，如ATP水平的测量，提供细胞质内源性酶的活性筛选，探测细胞膜的完整性，或跟踪线粒体活性的水平，来评估细胞的活力5 ，6。但是，无论所选择的端点，这些方法都需要在试样破坏之前，都可以测量，因此只在一个单一的时间点产生数据。其结果是，大量的样品需要是平行的基本毒理动力学研究制备和处理，再次加入到所需的新化合物的开发成本和劳动力。另外，测定使用荧光素酶的分泌，如Gaussia荧光素酶7，8 Vargula荧光素酶和荧光素酶的Metridia 9已经开发了，不再需要进行细胞裂解，并需要一小部分的端点测量的介质，但是，这些仍然有限，以在预定的时间点取样，还需要加入外源的光活化基板。 为了避免损害所需样品的破坏，以及消除基板的成本，已设计的人细胞系表达的完整细菌的生物发光（ 勒克司 ）基因盒（luxCDABEfrp）使活细胞的连续监测是类似于基于荧光染料的活细胞成像，但没有额外的光子激活和微观调查程序的。该细胞系能够组成型生产为连续的，直接检测的光信号，而不需要外部的刺激，从而避免破坏样品。机械，这些细胞所产生的生物发光信号，导致s时的发光酶形成的荧光素酶催化氧化的长链脂肪酸的醛（的的基因产物luxCDE使用内源性物质的合成和再生）的存在下还原核黄素磷酸（FMNH 2，这是由玻璃钢基因从FMN再循环产品）和分子氧10。 勒克斯磁带盒在宿主细胞中表达，因此，使光被制造，以及细胞破坏或外源性底物除了没有检测到。同样地，将所得的生物发光信号之间的相互作用的勒克司的基因和内源性提供FMN，和O 2 cosubstrates的，维护的环境中，可以支持的转换的FMN FMNH 2的要求，确保只能检测从活，代谢活跃的细胞。 这些要求先前已被利用来证明LUX-BASED生物发光输出密切相关蜂窝人口规模11和有毒的化合物暴露损害autobioluminescent的生产在时尚的剂量-反应12。这里，我们使用一个以前的特点autobioluminescent的人类胚胎肾（HEK293）细胞系11演示自动化毒理学的筛选与已知的DNA损伤活性作为一个代表性的例子来验证的应用的autobioluminescent哺乳动物细胞毒性试验的博莱霉素族抗生素。

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>IVIS Lumina</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Other IVIS models and PMT-based plate readers can also be used</td>
		</tr>
		<tr>
			<td>Living Imaging 2.0</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Newer updates of this software is available</td>
		</tr>
		<tr>
			<td>75 cm2 cell culture treated flasks</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture-treated plates</td>
			<td>Costar</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 24-well plate</td>
			<td>Greiner Bio-One</td>
			<td>662174</td>
			<td>96- or 384-well plates can be used for higher throughput applications</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Hyclone</td>
			<td>SH30910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), phenol red-free</td>
			<td>Hyclone</td>
			<td>SH30284</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH3091003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids, 100x</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic, 100x</td>
			<td>Life Technologies</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate, 100mM</td>
			<td>Life Technologies</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin, 100 mg/ml</td>
			<td>Life Technologies</td>
			<td>R25001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tripsin, 0.05%</td>
			<td>Life Technologies</td>
			<td>25300062</td>
			<td></td>
		</tr>
	</tbody>
</table>

autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity

已广泛采用哺乳动物细胞为基础的体外实验毒理学研究动物试验的替代品，但已经有限的，由于高金钱和时间成本的平行样品制备，必要由于破坏性的萤火虫荧光素酶的筛选方法， 。本视频介绍的利用自主生物发光的哺乳动物细胞中，不需要的萤光素基板破坏性此外，作为廉价且简便的方法监测感兴趣的化合物的细胞毒作用。哺乳动物细胞中稳定表达基因盒的完整细菌的生物发光（luxCDABEfrp）自主产生的光信号，而不增加一个昂贵的，并可能干扰的虫荧光素底物，由外部能量源，或破坏的示例是传统的激发峰在490nm处期间进行光学成像过程秒。这种独立于外界刺激保持仅在细胞的生物发光反应的负担，这意味着，所得到的信号在代谢活跃期间只检测到。这一特性使得该勒克斯 -表达细胞系生物发光生产变化的一个很好的候选人使用作为biosentinel对细胞毒素的作用，因为是表示对细胞生长和代谢的不利影响。同样地，自主性和缺乏所需的样本的破坏，允许重复毒物接触的整个期间内对同一样品进行实时成像，可以使用现有的成像设备，在多个样品以自动化的方式进行。

在这项研究中，的动态autobioluminescent HEK293细胞抗生素暴露（ 图1），在24小时内连续监测。这种抗生素的毒性作用，这是一种已知的杀死活细胞通过结合和裂解DNA 13的博莱霉素家族的成员，与未经处理的细胞相比，通过减少在生物发光生产，它可以直接通过伪彩色图像可视化进行了论证（ 图2）。这些细胞中，在不同的处理条件下，允许样品的反复平行筛选的的性...

Watch this Scientific Journal Video about Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity at JoVE.com

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

哺乳动物细胞表达生物发光细菌基因盒（<em&gt;勒克斯</em&gt;），自主产生光。化学品接触后产生的生物发光动力学已被证明，以反映细胞生长和代谢​​的治疗效果，使这些细胞的一种廉价的，连续的，实时的毒性筛选的工具，可以很容易地适应高通量自动化。

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

autonomously-bioluminescent-mammalian-cells-for-continuous-real-time

Research

JoVE Journal

Biology

10.0K 次观看.  Oak Ridge National Laboratory. 哺乳动物细胞表达生物发光细菌基因盒（勒克斯），自主产生光。化学品接触后产生的生物发光动力学已被证明，以反映细胞生长和代谢​​的治疗效果，使这些细胞的一种廉价的，连续的，实时的毒性筛选的工具，可以很容易地适应高通量自动化。

视频: Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing和传代的哺乳动物细胞

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

科研

生物学

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

实时监控的配体 - 受体相互作用的荧光共振能量转移

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

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) ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

基于探针实时荧光定量PCR途径的小分子RNA定量检测

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

细菌、酵母和人类细胞中瞬态形成的分子茶龙组件的原位监测

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...