Name: high efficiency, site-specific transfection of adherent cells with sirna using microelectrode arrays (mea)
Uploaded: 2012-09-13T13:00:00
Description: Watch this Scientific Journal Video about High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA at JoVE.com

1. МЭС подготовка <ol><li> МЭС для использования в экспериментах электропорации может либо быть изготовлены с использованием стандартной техники фотолитографии, как описано ранее 18 или приобрести непосредственно у поставщиков производителей MEA, такие как многопользовательская си...

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В этом видео статье мы демонстрации использования МПС для конкретных участков трансфекции клеток HeLa с яичницей последовательности миРНК. Одним из преимуществ данной технологии является ее применимость к различным клеточных линий, включая первичные клеточные линии. Наша лаборатория ...

<table border="1"><tr><td> Название реагента </td><td> Компания </td><td> Номер в каталоге </td><td> Комментарии (по желанию) </td></tr><tr><td> Сотовые СМИ: Расширенный MEM L-Glutamine 200 мМ Пенициллина / стрептомицина Эмбриональной бычьей сывороткой </td><td> Gibco / Invitrogen HIMEDIA Лаборатории / VWR Lonza Group ООО Gibco / Invitrogen </td><td> 12492-013 95057-448 09-757F 16000-044 </td><td> Клеточного состава сред: 2% FBS, 2% L-глутамина и 2% Pennstrep заранее MEM </td></tr><tr><td> Трипсин EDTA </td><td> Mediatech, Inc </td><td> 25-053-CL </td><td></td></tr><tr><td> PBS </td><td> Mediatech, Inc </td><td> 21-040-CV </td><td></td></tr><tr><td> Alexa 488 и родамина отмеченных яичницу SEQUENCE миРНК </td><td> Qiagen, Inc </td><td> 1027292 </td><td></td></tr><tr><td> Electroporation буфер </td><td> Biorad лабораторий </td><td> 165-2677 </td><td></td></tr><tr><td> Генератор сигналов </td><td> Прагматический </td><td> 2414A </td><td> Любой сигнал / импульсный генератор, который может обеспечить желаемые импульсы могут быть использованы. </td></tr></table>

high efficiency, site-specific transfection of adherent cells with sirna using microelectrode arrays (mea)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

Watch this Scientific Journal Video about High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA at JoVE.com

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

В статье подробно протокол для сайт-специфической трансфекции яичницу последовательности миРНК в приверженцем культуры млекопитающих ячейки с помощью микроэлектродов массива (МПС).

Высокая эффективность, определенных сайтов Трансфекция прилипшие клетки с помощью миРНК Микроэлектродные массивы (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Research

JoVE Journal

Bioengineering

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

Доставка генов и Трансфекция в человеческий рак поджелудочной железы клетки, используя эпидермального фактора роста рецепторов ориентированных Желатин основе Engineered Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

Сайт-специфическая бактериальная хромосома Инженерно: ΦC31 интегразы опосредованная кассеты бирже (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

Инженерные Приверженец бактерии путем создания единого синтетического Curli Operon

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Заказ отдельных клеток и Single зародышами в 3D конфайнмента: Новое устройство для высокого содержания скрининга

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Пористого кремния Микрочастицы для доставки миРНК терапии

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Легкая и точная механотерапии профилирование на Micropost массивов

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Доставки терапевтических миРНК в ЦНС с использованием катионных и анионных липосом

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

Всеобъемлющая оценка эффективности и безопасности препарата плаценты целевой доставки с помощью трех взаимодополняющих методов

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Микрофлюидика сопряжения с микроэлектродные массивы для изучения нейронные связи и распространения аксональное сигнала

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...