Name: autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity
Uploaded: 2013-10-28T20:00:00
Description: Watch this Scientific Journal Video about Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity at JoVE.com

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...

<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>

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 ...

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Biology

Trypsinizing and Subculturing Mammalian 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

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MISSION esiRNA for RNAi Screening in Mammalian Cells

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

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

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

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

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

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

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

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

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

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 ...