Name: a screenable in vivo assay for mitochondrial modulators using transgenic bioluminescent caenorhabditis elegans
Uploaded: 2015-10-16T14:00:00
Duration: 11 min 43 s
Description: Watch this Scientific Journal Video about A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans at JoVE.com

The authors would like to thank David Gray from the Dundee Drug discovery unit for kindly donating firefly luciferase inhibitory compounds DDD00001434, DDD0001477, DDD00000635 and DDD00023047; Tibor Harkani (Medical University of Vienna) for the suggestion of oxaloacetate as a test compound; and Charlie Dear (University of Aberdeen) for illustrations. This work was funded by a BBSRC Pathfinder award (BB/FOF/PF/4/11) and the University of Aberdeen.

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Общая цель этой процедуры является быстрое количественное определение энергетического состояния С. Элеганс в естественных условиях с целью использования этого в качестве конечной точки в соединении скрининга. Обоснованием основан на трансгенной экспрессии гена люциферазы светлячка в нематоды C. Элеганс 1-3 с помощью плазмиды pSLGCV (1 <a href="https://www.addgene.org/49862" target="_blank">https://www.addgene.org/49862~~HEAD=pobj).</a> Люциферазы фермент, слитый с зеленым флуоресцентным белком GFP и выражается конститутивно и повсеместно в цитоплазме (The люциферазы пероксисом ориентации сигнал был удален). Это приводит к эмиссии света, когда подложка люциферин обеспечивается экзогенно. Как червь является прозрачным, свет может быть измерена с помощью люминометра в виде относительных световых единиц (RLU). Сотовые ATP топлива реакция биолюминесценции и ее доступность определяет уровень освещенности, произведенные. Следовательно, luminometry предлагает convenienт средства оценки уровней относительных АТФ и расширение функции митохондрий, так как производство АТФ происходит главным образом в митохондриях. Связь между митохондриальных функциональных уровней и биолюминесценции было продемонстрировано ранее через глушителей митохондриальных генов цепи переноса электронов и сопутствующей снижению светоотдачи. 1 Штаммы биолюминесценции, полученные были обозначены PE254 (feIs4) и PE255 (feIs5) 1 (список материалов) и может быть использован как взаимозаменяемые. 3 Ряд исследований использовали эти штаммы датчика сообщить о сотовых АТФ уровней в естественных условиях после воздействия различных ксенобиотиков химических веществ, таких, как азид натрия 1, кадмия 2, канализации Экстракт осадка 3, 5&#39;-фтор-2-дезоксиуридин 6 и табак-специфичных нитрозаминов 7. Штаммы были также полезны для мониторинга последствий воздействия ультрафиолетового излучения C <sдо&gt; 8 и эффекты нарушая функции митохондрий дыхательной цепи. 1,9 Ранняя версия датчика люминесценции, выражающей ген люциферазы без GFP слияния (PE39) также были использованы в исследовании воздействия тяжелых металлов и респираторных . разобщающий 10 Штаммы PE254 и PE255 нести люциферазы: GFP слияние и GFP флуоресценции показано, что увеличение пропорционально нематод массы, предлагая удобные средства для нормализации значений люминесценции 6,9 Последствия дифференциального количества червей на лунку может быть. учтены в том числе несколько технических повторов в анализе (минимум 5 скважин в состоянии). 3 Протокол предоставляет возможность мониторинга в естественных условиях энергетических уровней (в отличие от более технически трудоемким в пробирке определения АТФ), что позволяет соединение скрининг и изменение в физиологическом контексте целого муlticellular организм. Эта процедура может быть расширена до различных генетических фоны, пересекая хромосомно интегрированный трансген в доступных мутантных штаммов и / или по глушителей генов от РНК-интерференции; Таким образом, в полной мере воспользоваться С. Элеганс в качестве модельного организма. Метод должен помочь уменьшить поздней стадии недостаточности наркотиков кандидатов в связи с митохондриальной токсичности и внести свой вклад в сокращение высшего испытаний на животных.

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	<tbody>
		<tr>
			<td>Orion II Microplate Luminometer</td>
			<td>Berthold Detection Systems</td>
			<td></td>
			<td>with injector and the Simplicity 4.2 Software; transfer data to Excel at the end of plate measurement</td>
		</tr>
		<tr>
			<td>FLx800 fluorimeter&#160;</td>
			<td>Biotek</td>
			<td></td>
			<td>with Gen5 Software; transfer data to Excel at the end of plate measurement</td>
		</tr>
		<tr>
			<td>Ks 130 basic shaking platform</td>
			<td>Ika</td>
			<td>#0002980000</td>
			<td></td>
		</tr>
		<tr>
			<td>Innova 4349</td>
			<td>New Brunswick Scientific</td>
			<td></td>
			<td>Refrigerated incubator shaker</td>
		</tr>
		<tr>
			<td>Eppendorf centrifuge 5804R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>with eppendorf rotor A-4-44&#160; (for 50/15ml tubes)</td>
		</tr>
		<tr>
			<td>Stripette, Costar</td>
			<td>Corning</td>
			<td>#4489</td>
			<td>Serological pipette</td>
		</tr>
		<tr>
			<td>Cellstar 50 ml tubes</td>
			<td>Greiner bio-one</td>
			<td>#227661</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellstar 15 ml tubes</td>
			<td>Greiner bio-one</td>
			<td>#188261</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellstar 60 mm tissue culture plates&#160;</td>
			<td>Greiner bio-one</td>
			<td>#628160</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Microscope slides</td>
			<td>VWR</td>
			<td>#631-0909</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile spatula</td>
			<td>Corning</td>
			<td>#3007; #3004</td>
			<td>for weighing out chemicals</td>
		</tr>
		<tr>
			<td>0.22 mM Millex GP filters</td>
			<td>Millipore</td>
			<td>#SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Axygen eppendorf tubes 1.5 ml</td>
			<td>Fisher Scientific</td>
			<td>#MCT-150-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96 well assay plate</td>
			<td>VWR</td>
			<td>#3603</td>
			<td>Black plate clear bottom with lid</td>
		</tr>
		<tr>
			<td>Nunc 96 well assay plate</td>
			<td>Fisher Scientific</td>
			<td>#236105</td>
			<td>White plate with lid</td>
		</tr>
		<tr>
			<td>Reservoir reagent 60 mL</td>
			<td>Thermo Scientific Finnpipette</td>
			<td>#9510027</td>
			<td>used as trough for nematodes&#160; in protocol section 4.4.1)</td>
		</tr>
		<tr>
			<td>Storage box/damp chamber</td>
			<td>Roche Diagnostics</td>
			<td>#10 800 058 001</td>
			<td>174 x101 x 56.6 mm, used as a damp chamber with wet paper towels; 2X 96-well plates with lids can fit into one, the lower sitting on top of a microplate lid</td>
		</tr>
		<tr>
			<td>Bleach: Sodium hypochlorite solution 4-4.99% Chlorine content</td>
			<td>Sigma Aldrich</td>
			<td>#239305</td>
			<td>Store in aliquots at 4&#176;C,&#160; seal with parafilm to prevent loss of chlorine and cover in foil</td>
		</tr>
		<tr>
			<td>D-Luciferin, potassium salt</td>
			<td>Biotium Inc</td>
			<td># 10101-2</td>
			<td>Molecular Probes can also be used as supplier; prepare a 20 mM stock in ddH2O, keep at -20 &#176;C in aliquots</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td># P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>CalBiochem</td>
			<td>#317275</td>
			<td>Purity 99.99%</td>
		</tr>
		<tr>
			<td>Triton X100</td>
			<td>Alfa Aesar</td>
			<td>#A16046</td>
			<td>Diltute to 10% in ddH20</td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>CalBiochem</td>
			<td>#475914</td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans bioluminescent strains</td>
			<td>Author&#39;s own laboratory</td>
			<td>PE254, PE255</td>
			<td>contain integrated arrays feIs4 and feIs5 [Psur-5::luc+::gfp; rol-6(su1006)] respectively on chromossome V and X; select for homogeneous and strong expression of luc::GFP by fluorescence microscopy (e.g. pick 15 worms) every now and then.</td>
		</tr>
		<tr>
			<td>E. coli OP50</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>General chemicals</td>
			<td>Sigma Aldrich/Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a screenable in vivo assay for mitochondrial modulators using transgenic bioluminescent caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

Watch this Scientific Journal Video about A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans at JoVE.com

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans (Video) | JoVE

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

Скринируемый<em&gt; В Vivo</em&gt; Анализ митохондриальных Модуляторы с использованием трансгенных БИОЛЮМИНЕСЦЕНТНЫЕ<em&gt; Caenorhabditis Элеганс</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Research

JoVE Journal

Biology

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

Generation of Stable Transgenic C. elegans Using Microinjection

Generation of Stable Transgenic C. elegans Using Microinjection (Video) | JoVE

Генерация Стабильный Трансгенные Элеганс C. Использование Микроинъекция

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum (Video) | JoVE

В естественных условиях Биолюминесцентный изображения опухоли молочной железы Использование ИВИС Спектра

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Generation of Transgenic C. elegans by Biolistic Transformation

Generation of Transgenic C. elegans by Biolistic Transformation (Video) | JoVE

Поколение трансгенных С. Элеганс По Biolistic преобразования

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans (Video) | JoVE

Покадровый Микроскопия раннего эмбриогенеза в Caenorhabditis Элеганс

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI (Video) | JoVE

Визуализация Caenorhabditis Elegans Кутикулы структур с помощью липофильных Vital краска, DII

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels (Video) | JoVE

Флуоресцентные Анализ Скрининг на выявление каналов модуляторы GIRK

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation (Video) | JoVE

Основной<em&gt; Caenorhabditis Элеганс</em&gt; Методы синхронизации и наблюдения

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Prostaglandin Extraction and Analysis in Caenorhabditis elegans (Video) | JoVE

Добыча простагландинов и анализа в<em&gt; Caenorhabditis Элеганс</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism (Video) | JoVE

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment (Video) | JoVE

Культивирование<em&gt; Caenorhabditis Элеганс</em &gt; В аксенными жидких сред и создания трансгенных червей бомбардировки микрочастицами

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...