Ringraziamo tutti i membri del nostro laboratorio per le discussioni utili. Ringraziamo Joanna Lisztwan e Christina Gewinner per la lettura critica del manoscritto. Questo lavoro è stato finanziato dalla sovvenzione BBSRC BB/I021248/1 e il Wellcome Trust concessione 090090/Z/09/ZAH è un Wellcome Trust Research fellow presso il Career Development UCL Cancer Institute.

1. Clonazione di pcDNA6_TetR_IRES_blast <ol><li> Come illustrato in figura 3, eseguire un digest parziale del pcDNA6/TR plasmide (V1025-20, Invitrogen) con gli enzimi di restrizione Xba I e Nco I per rimuovere il gene tetR e il promotore della resistenza blasticidina (BlastR) gene. Isolare il vettore frammento di 4,6 kb. </li><li> Per rimuovere la sequenza di poliadenilazione del gene tetR, introdurre mediante PCR un sito Xho I subito dopo il codone di stop del cDNA tetR conservando il sito Xba I alla estremità 5 &#39;del cDNA. Subclonare il frammento risultante in pMigR1 17 usando enzimi di restrizione Xba I e Xho I per generare il plasmide pMigR1_TetR. </li><li> Digerire il vettore pMigR1_TetR con gli enzimi di restrizione Xba I e Nco I per liberare il frammento TetR_IRES. Isolare l&#39;inserto 1,25 kb frammento. </li><li> Legare il 4,6 kb pcDNA6/TR e le 1,25 kb frammenti TetR_IRES. Transform il prodotto legatura in competente E.coli e identificare il costrutto pcDNA6_TetR_IRES_blast desiderato utilizzando le normali tecniche di biologia molecolare. </li></ol> 2. Generazione di linee cellulari che esprimono stabilmente tetR <ol><li> Il giorno 1, Seed 1x10 6 celle per 10 cm di piastra di coltura con il supporto di standard di crescita (ad es DMEM supplementato con 10% FCS). Seed due piastre, una per trasfezione con pcDNA6_TetR_IRES_blast, e l&#39;altro come controllo negativo per la selezione blasticidina. Assicurarsi di utilizzare il passaggio più basso possibile e il mezzo di crescita consigliato. </li><li> Il giorno 2, transfect una piastra con 2 mg di pcDNA6_TetR_IRES_blast utilizzando Fugene 6 (E2691, Promega) seguendo le istruzioni del produttore. Non transfettare la seconda piastra. </li><li> Il giorno 3, aggiungere mezzo standard di crescita contenente 5 mg / ml blasticidina ad entrambe le lastre. Si prega di notare che la concentrazione scelta ottimale può variare per una linea cellulare, e di conseguenza potrebbe need da determinare in anticipo. </li><li> Il giorno 4, dividere le celle a 1/5, 1/25, 1/50 e 1/100 con 10 cm di piatti e terreno di coltura standard contenente 5 mg / ml blasticidina. Assicurarsi di etichettare correttamente piastre contenenti cellule non trasfettate o trasfettate. Preparare due piastre per fase di diluizione. </li><li> Controllare le cellule ogni giorno, in modo che tutte le celle sulle piastre non trasfettate sono morti entro la prima settimana. Modificare i media di selezione ogni 2-3 giorni. </li><li> Dopo 1-2 settimane, blasticidina resistenti colonie dovrebbe iniziare a comparire. Selezionare piastre contenenti 5-50 colonie per assicurare che singole colonie possono essere raccolti. </li><li> Quando le colonie hanno raggiunto un diametro di circa 5 mm (o maggiore), isolare almeno 12 cloni indipendenti utilizzando cilindri di clonazione per raccogliere singole colonie. Trasferire ciascun clone in un pozzo separato di una piastra a 24 pozzetti di coltura. Quando confluenti, dividere le cellule di ogni pozzetto in un singolo pozzetto di una piastra da 6 pozzetti di coltura. </li><li> Continuare a cl culturaquelli in mezzi di selezione. Quando confluenti, le cellule di ogni pozzetto divise in due singoli pozzetti di una piastra da 6 pozzetti di coltura. </li><li> Per ogni clone da testare, congelare un pozzetto di cellule confluenti e cellule raccolto nel pozzetto altro per le prove successive mediante immunoblotting. </li><li> Rileva tetR mediante Western blotting utilizzando l&#39;anticorpo monoclonale murino anti-TET02 anticorpo (Mobitec GmbH). Di includere un lisato della linea cellulare parentale come controllo negativo. </li><li> Selezionare 3 cloni con l&#39;espressione più alta tetR ed espandere ogni cultura clonale. Congelare le scorte di ciascun clone promettente appena possibile. Infine, esaminare i parametri molecolari e cellulari biologici di interesse confrontando le linee cellulari parentali con i tetR-cloni che esprimono. Selezionare il &#39;migliore&#39; clone con l&#39;espressione più alta tetR che non visualizza alcuna modifica nei parametri molecolari e biologiche delle cellule di interesse. </li></ol> 3. Test pilota di pter e pT-Rex DEST30 vettori EsprimiING shRNA o cDNA, rispettivamente, <ol><li> Generare pter plasmide con inserti shRNA come descritto 12. Costruire pT-Rex DEST30 vettore (12.301-016, Invitrogen) contenente il cDNA di interesse utilizzando la tecnologia Gateway (Invitrogen). In caso di espressione di cDNA, l&#39;aggiunta di un tag al cDNA sarà poi facilitare l&#39;individuazione di esogenamente proteina espressa. </li><li> Transitoriamente transfettare la linea cellulare parentale bersaglio con pter o pT-Rex DEST30 plasmidi (che ha espresso shRNAs o cDNA di interesse) con il reagente di trasfezione di scelta. Per il test di shRNA plasmidi di espressione, garantire elevate efficienze di trasfezione possono essere raggiunti. </li><li> Harvest cellule trasfettate e procedimenti per immunoblotting usando gli anticorpi appropriati, in attesa di rilevare un esaurimento o sovraespressione del gene di interesse. </li></ol> 4. Generazione di linee cellulari stabili con tetraciclina-inducibile (Tet-on) Espressione di shRNAs o cDNA utilizzando pter o pT-Rex DEST30 vettori </p&gt; <ol><li> Il giorno 1, seme 1x10 6 celle del &#39;migliore&#39; tetR esprimono clone per 10 cm di lastra, con il supporto di standard di crescita integrato con certificato Tetracycline senza siero (ad esempio da FBS Invitrogen, 16000-014). Seme una piastra per la trasfezione con pter (o pT-Rex DEST30), e una piastra come controllo negativo per doppia selezione. Assicurarsi di utilizzare il più basso possibile passaggio. </li><li> Il giorno 2, transfect una piastra con 2 pg di pter (o pT-Rex DEST30) utilizzando Fugene 6. Non transfettare la seconda piastra. </li><li> Il giorno 3, aggiungere mezzo standard di crescita contenente 5 mg / ml blasticidina e 500 mcg / ml zeocin (o 1 mg / ml G418). </li><li> Il giorno 4, dividere le celle a 1/5, 1/25, 1/50 e 1/100 utilizzando piastre da 10 cm e terreno di coltura standard contenente 5 mg / ml di blasticidina e 500 pg / ml zeocin (o 1 mg / ml G418). Preparare due piastre per fase di diluizione. </li><li> Controllare le celle di tutti i giorni, facendo in modo che entro la prima settimana tutte le cellule sulle piastre non trasfettate sono morti.Cambia il mezzo standard di crescita contenente blasticidina e zeocin (o G418) ogni 3-4 giorni. </li><li> Dopo 2-3 settimane, blasticidina / zeocin (o blasticidin/G418) double-resistenti colonie dovrebbe iniziare a comparire. Selezionare piastre contenenti 5-50 colonie di scegliere singole colonie. </li><li> Quando le colonie hanno raggiunto un diametro di circa 5 mm (o maggiore), utilizzare cilindri di clonazione per raccogliere singole colonie. Isolare almeno 24 cloni individuali. Trasferire ciascun clone in un pozzo separato di una piastra a 24 pozzetti di coltura. </li><li> Cloni cultura in concentrazioni medie di manutenzione contenenti blasticidina (5 mg / ml) e zeocin (250 mcg / ml) [o G418 (0,5 mg / ml)]. Quando confluenti, dividere le cellule di ogni pozzetto in un singolo pozzetto di una piastra da 6 pozzetti di coltura. </li><li> Per ogni clone da testare, dividere un confluente pozzetto di una piastra da 6 pozzetti in tre singoli pozzetti di una piastra da 6 pozzetti. Quando confluenti, congelare le cellule da un pozzetto. Usare gli altri due pozzi per la verifica della tetraciclinae-inducibile espressione. Aggiungere tetraciclina alla concentrazione di 1 ug / ml ad un pozzetto, lasciando la tetraciclina secondo pozzo libero. </li><li> Dopo 24-96 ore di incubazione con le tetracicline, le cellule raccolto e di processo per immunoblotting utilizzando anticorpi appropriati. Brevi tempi di incubazione tetracicline sono raccomandati quando screening per l&#39;induzione di espressione di cDNA, mentre più trattamenti tetraciclina sarà necessario determinare shRNA-mediata deplezione di proteine ​​endogene. </li><li> Selezionare almeno due cloni con l&#39;esaurimento desiderato / sovraespressione ed espandere ogni cultura. Congelare le scorte di ciascun clone promettente appena possibile. Quando cresce Tet-on cloni per gli esperimenti sempre assicurarsi di utilizzare supporti integrati con Tetracycline senza siero e gli antibiotici di selezione relativi a concentrazioni di manutenzione. </li></ol>

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Nessun conflitto di interessi dichiarati.

Crediamo che Tet-sui sistemi facilitare notevolmente l&#39;analisi della funzione genica, in particolare in sistemi cellulari che sono difficili da manipolare e / o quando il gene manipolato è essenziale per la sopravvivenza cellulare. Inoltre, la capacità di controllare l&#39;espressione genica altamente specifici Tet-sui sistemi offre l&#39;opportunità di studiare funzioni geniche in diverse fasi (per esempio durante la progressione del ciclo cellulare o ben definiti processi di differenziazione). Il metodo qui pre...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Siero bovino fetale (FBS) </td><td> Invitrogen </td><td> 16000-044 </td><td> Testato Tet-free </td></tr><tr><td> Blasticidina </td><td> Invivogen </td><td> ant-bl-1 </td><td></td></tr><tr><td> Zeocin </td><td> Invivogen </td><td> ant-Zn-5 </td><td></td></tr><tr><td> G418 </td><td> PAA laboratori </td><td> P31-011 </td><td> 100 mg / ml in media </td></tr><tr><td> anti-TET02 </td><td> Mobitec GmbH </td><td> TET02 </td><td> Utilizzare a 1/1000 a 1/2000 per 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> Tetracycline </td><td> Sigma </td><td> 87128 </td><td> 2 mg / ml in etanolo </td></tr><tr><td> Doxycycline </td><td> Sigma </td><td> D9891 </td><td> 2 mg / ml in acqua </td></tr><tr><td> Clonazione di cilindri </td><td> Bellco Glass Inc. </td><td> 2090-00808 </td><td> riutilizzabile </td></tr></tbody></table>

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

Un approccio importante nel campo della biologia cellulare di mammifero è la manipolazione dell&#39;espressione dei geni di interesse in linee cellulari selezionate, con lo scopo di rivelare una o più funzione del gene (s) con sovraespressione transiente / stabile o knockdown del gene di interesse. Purtroppo, per le indagini cellulari diversi biologici questo approccio non è adatto quando le manipolazioni del risultato dell&#39;espressione genica in cellule di crescita / proliferazione difetti o differenziazione cellulare indesiderata. Pertanto, i ricercatori hanno adattato la proteina repressore tetraciclina (tetR), tratto dal E. coli resistenza alla tetraciclina operone 1, per generare sistemi di regolazione molto efficienti e stretto per esprimere cDNA in cellule di mammifero 2,3. In breve, tetR è stato modificato a uno (1) blocco di inizio trascrizione legandosi al Tet-operatore (TO) nel promotore per aggiunta di tetraciclina (Tet-off definito sistema) o (2) si legano al TO in tegli assenza di tetraciclina (Tet-definito sul sistema) (Figura 1). Data l&#39;inconveniente che il Tet-off sistema richiede la presenza continua di tetraciclina (che ha una emivita di circa 24 ore in terreno di coltura cellulare tissutale) il Tet-il sistema è stato più ampiamente ottimizzata, con conseguente sviluppo di molto stretto ed efficienti sistemi vettore di espressione di cDNA come usato qui. Poco dopo la creazione di RNA interference (RNAi) per atterramento gene in cellule di mammifero 4, vettori che esprimono breve hairpin RNA (shRNA) sono stati descritti che funzione molto simile a siRNA 5-11. Tuttavia, questi approcci shRNA-mediati atterramento presenta la stessa limitazione come strategie knockout convenzionali, in quanto l&#39;esaurimento stabile non è fattibile se gli obiettivi di geni sono essenziali per la sopravvivenza cellulare. Per superare questa limitazione, van de Wetering et al 12. Modificato l&#39;espressione shRNA vettore pSUPER 5 </su&gt; p inserendo un TO nella regione del promotore, che ha permesso loro di generare linee cellulari stabili con tetraciclina inducibile deplezione dei loro geni bersaglio di interesse. Qui, si descrive un metodo per generare efficientemente umana stabile Tet-on linee cellulari che guidano affidabile iperespressione inducibile o deplezione del gene di interesse. Utilizzando questo metodo, siamo riusciti generato Tet-on linee cellulari che hanno notevolmente facilitato l&#39;analisi del MST / hMOB / NDR cascata in centrosoma 13,14 e apoptosi segnalazione 15,16. In questo rapporto, descriviamo i nostri vettori di scelta, oltre a descrivere i due passaggi consecutivi di manipolazione che sono necessari per generare efficientemente umana Tet-on linee cellulari (Figura 2). Inoltre, oltre a delineare un protocollo per la generazione di umana Tet-on linee cellulari, discuteremo aspetti critici riguardanti le procedure tecniche e la caratterizzazione di cellule Tet-on.

Un esempio per la caratterizzazione di RPE-1 linee cellulari che esprimono stabilmente tetR è mostrato in Figura 4. Si noti che tutti i cloni RPE-1 esprimono diversi livelli di tetR (confronta corsie 2 e 5), mentre la linea cellulare parentale (che serve come controllo negativo) non esprime la proteina esogena tetR (Figura 4, corsia 1). Questa variazione nell&#39;espressione tetR tra RPE-1 Tet-on cloni cellulari è previsto, poiché l&#39;espressione del tetR esprimere plasmidi è alta...

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

Un modo rapido e semplice per generare linee cellulari umane di inducibile e reversibile sovraespressione cDNA o shRNA-mediata knock-down del gene di interesse. Questo metodo consente ai ricercatori di linee cellulari altamente affidabile e riproducibile manipolare che sono difficili da modificare con metodi di trasfezione transiente o convenzionali atterramento / knockout strategie.

Generazione di linee cellulari umane stabili con tetraciclina-inducibile (Tet-on) shRNA o espressione di cDNA

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 ...

generation-stable-human-cell-lines-with-tetracycline-inducible-tet-on

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Biology

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

35.0K Views.  UCL Cancer Institute. Un modo rapido e semplice per generare linee cellulari umane di inducibile e reversibile sovraespressione cDNA o shRNA-mediata knock-down del gene di interesse. Questo metodo consente ai ricercatori di linee cellulari altamente affidabile e riproducibile manipolare che sono difficili da modificare con metodi di trasfezione transiente o convenzionali atterramento / knockout strategie.

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

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

RIFLESSI Passaging sulle cellule staminali embrionali umane-linee con tripsina

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

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Biologia

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Generazione di Stabile transgenici C. elegans Utilizzando Microiniezione

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

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

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

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

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

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Generazione di Human CD40 delle cellule B attivate

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

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Isolamento RNA da embrionali Sintesi Zebrafish e cDNA per l&#39;analisi di espressione genica

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

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

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

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 to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle 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

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Trasduzione di cellule umane con Polymer-complessato Lentivirus Ecotropic per Enhanced biosicurezza

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

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

Preparazione di linee cellulari per Knockdown condizionale dell&#39;espressione genica e valutazione degli effetti sulla Knockdown E4orf4 morte cellulare indotta