Name: generation of stable human cell lines with tetracycline-inducible (tet-on) shrna or cdna expression
Uploaded: 2013-03-05T13:00:00
Description: Watch this Scientific Journal Video about Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression at JoVE.com

Мы благодарим всех членов нашей лаборатории за полезные обсуждения. Мы благодарим Джоанна Lisztwan и Кристина Gewinner за критическое прочтение рукописи. Эта работа была поддержана грантом BBSRC BB/I021248/1 и Wellcome Trust грант 090090/Z/09/ZAH является научным Wellcome Trust развития карьеры научный сотрудник Института Рака UCL.

1. Клонирование pcDNA6_TetR_IRES_blast <ol><li> Как показано на рисунке 3, выполнить частичную дайджест pcDNA6/TR плазмиды (V1025-20, Invitrogen) рестриктазами Xba я и сержантского я, чтобы удалить ген TetR и промоутером сопротивление бластицидин (BlastR) гена. Изолировать 4,6 Кб фрагмент вектора. </li><li> Ч...

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Мы считаем, что Тет-систем значительно облегчить анализ функции генов, особенно в клеточных системах, которые трудно манипулировать и / или при манипуляции генов необходима для выживания клеток. Кроме того, способность контролировать экспрессию генов весьма специфический Тет-систем п...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер в каталоге </td><td> Комментарии (по желанию) </td></tr><tr><td> Эмбриональной телячьей сыворотки (FBS) </td><td> Invitrogen </td><td> 16000-044 </td><td> Протестировано Тет-бесплатно </td></tr><tr><td> Бластицидин </td><td> Invivogen </td><td> Муравей-BL-1 </td><td></td></tr><tr><td> Зеоцина </td><td> Invivogen </td><td> Муравей-Zn-5 </td><td></td></tr><tr><td> G418 </td><td> PAA лабораторий </td><td> P31-011 </td><td> 100 мг / мл в СМИ </td></tr><tr><td> анти-TET02 </td><td> Mobitec GmbH </td><td> TET02 </td><td> Используйте на 1/1000 до 1/2000 для WB </td></tr><tr><td> pcDNA6/TR </td><td> Invitrogen </td><td> V1025-20 </td><td></td></tr><tr><td> PT-Rex DEST30 </td><td> Invitrogen </td><td> 12301-016 </td><td></td&gt; </tr><tr><td> Тетрациклин </td><td> Сигма </td><td> 87128 </td><td> 2 мг / мл в этаноле </td></tr><tr><td> Доксициклин </td><td> Сигма </td><td> D9891 </td><td> 2 мг / мл в воде </td></tr><tr><td> Клонирование цилиндров </td><td> Bellco стекла Инк </td><td> 2090-00808 </td><td> повторного использования </td></tr></tbody></table>

generation of stable human cell lines with tetracycline-inducible (tet-on) shrna or cdna expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Например для начальных характеристик ПЭС-1 клеточные линии, стабильно экспрессирующие TetR показано на рисунке 4. Отметим, что все НПП-1 клонов выражать различные уровни TetR (сравните дорожки 2 и 5), в то время как родительская клеточная линия (которая служит в качестве отрицательно?...

Watch this Scientific Journal Video about Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression at JoVE.com

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

Быстрый и простой способ создания клеточных линий человека с индуцибельной и обратимым кДНК гиперэкспрессией или ShRNA-опосредованной нокдаун гена интереса. Этот метод позволяет исследователям надежно и высоко воспроизводимо управлять клеточными линиями, которые трудно изменить переходным методов трансфекции или обычные нокдаун / нокаут стратегии.

Генерация стабильных клеточных линий человека с Тетрациклин-индуцируемых (Тет-на) ShRNA или кДНК выражений

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

Research

JoVE Journal

Biology

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

Пассирования оттенков эмбриональных стволовых клеток человека линий с Трипсин

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Generation of Stable Transgenic C. elegans Using Microinjection

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 ...

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

Подготовка первичных гемопоэтических клеточных культур из костного мозга мышей для Электропорация

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

Окрашивание белков в гелях Кумасси G-250 без органических растворителей и уксусной кислоты

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Generation of Human CD40-activated B cells

Поколение прав CD40 активированных В-клеток

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

РНК Изоляция от эмбриональных данио рерио и кДНК синтеза для анализа экспрессии генов

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

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

Использование генной Pulser MXcell Электропорация системы для трансфекции первичных элементов с высоким КПД

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

Использование автоматизированных счетчиков сотовый для упрощения исследования экспрессии генов: миРНК Нокдаун ИЛ-4 зависимости экспрессии генов в клетках Namalwa

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 ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Трансдукция клеток человека с Полимер-комплекс Ecotropic лентивирус для работы с улучшенными биобезопасности

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...