Wir danken allen Mitgliedern unseres Labors für hilfreiche Diskussionen. Wir danken Joanna Lisztwan und Christina Gewinner für die kritische Durchsicht des Manuskripts. Diese Arbeit wurde von der BBSRC Gewährung BB/I021248/1 unterstützt und die Wellcome Trust Gewährung 090090/Z/09/ZAH ist ein Wellcome Trust Research Career Development Fellow an der UCL Cancer Institute.

Ein. Klonierung pcDNA6_TetR_IRES_blast <ol><li> Wie in 3 dargestellt, führen einen teilweisen Verdau des Plasmids pcDNA6/TR (V1025-20, Invitrogen) mit den Restriktionsenzymen Xba I und Nco I, die TetR-Gen und den Promotor des Blasticidin Widerstand (BlastR)-Gen zu entfernen. Isolieren Sie die 4,6 kb Vektorfragment. </li><li> Um die Polyadenylierungssequenz aus der TetR-Gen zu entfernen, durch PCR einzuführen eine Xho I-Stelle unmittelbar nach dem Stop-Codon des TetR cDNA gleichzeitiger Schonung der Xba I-Stelle am 5&#39;-Ende der cDNA. Subklonieren das resultierende Fragment in pMigR1 17 unter Verwendung von Restriktionsenzymen XbaI und XhoI zur Erzeugung des pMigR1_TetR Plasmid. </li><li> Verdauen pMigR1_TetR Vektor mit den Restriktionsenzymen Xba I und Nco I-Fragment, um die TetR_IRES freizugeben. Isolieren Sie das 1,25 kb Insert-Fragment. </li><li> Ligieren das 4,6 kb pcDNA6/TR und die 1,25 kb TetR_IRES Fragmente. Transform des Ligationsprodukts in kompetente E.coli und Identifizierung des gewünschten pcDNA6_TetR_IRES_blast Konstrukt unter Verwendung von molekularbiologischen Standardtechniken. </li></ol> 2. Generierung von Zelllinien stabil exprimieren TetR <ol><li> Am Tag 1 Seed 1x10 6 Zellen pro 10-cm Gewebekulturplatte mit Ihrem Standard-Wachstumsmedium (zB DMEM ergänzt mit 10% FCS). Seed zwei Platten, eine für die Transfektion mit pcDNA6_TetR_IRES_blast, und die andere als eine negative Kontrolle für Blasticidin Selektion. Stellen Sie sicher, den günstigsten Durchgang möglich und die empfohlene Wachstumsmedium zu verwenden. </li><li> Am Tag 2 transfizieren eine Platte mit 2 ug pcDNA6_TetR_IRES_blast mit Fugene 6 (e2691, Promega) nach den Anweisungen des Herstellers. Nicht transfizieren die zweite Platte. </li><li> Am Tag 3, fügen Standard Wachstumsmedium mit 5 ug / ml Blasticidin an beide Platten. Bitte beachten Sie, dass die optimale Auswahl Konzentration kann für eine bestimmte Zelllinie variieren und vielleicht need vorher festgelegt werden. </li><li> Am Tag 4 aufgeteilt, die Zellen mit 1/5, 1/25, 1/50 und 1/100 unter Verwendung von 10-cm Platten und Standard-Wachstumsmedium, das 5 mg / ml Blasticidin. Stellen Sie sicher, richtig zu beschriften Platten mit untransfizierte oder transfizierten Zellen. Bereiten Sie zwei Platten pro Verdünnungsstufe. </li><li> Überprüfen Zellen täglich, sicherzustellen, dass alle Zellen auf den transfizierten Platten haben innerhalb der ersten Woche gestorben. Auswahl ändern media alle 2-3 Tage. </li><li> Nach 1-2 Wochen sollte Blasticidin-resistente Kolonien beginnen zu erscheinen. Wählen Platten mit 5 bis 50 Kolonien, sicherzustellen, dass einzelne Kolonien ausgewählt werden können. </li><li> Wenn Kolonien haben einen Durchmesser von ca. 5 mm (oder größer) erreicht, Isolieren mindestens 12 unabhängigen Klonen mittels Klonierung Zylindern, um einzelne Kolonien zu wählen. Übertragen jeder Klon in ein separates Loch einer 24-Well Gewebekulturplatte. Wenn konfluent, spalten die Zellen aus jeder Vertiefung in einem einzigen Well einer 6-Well-Gewebekulturplatte. </li><li> Weiter zu Kultur cldiejenigen in der Auswahl Medien. Wenn konfluent, geteilte Zellen aus jeder Vertiefung in zwei einzelne Vertiefungen einer 6-well Gewebekulturplatten. </li><li> Für jeden zu testenden Klon, eingefroren eine Vertiefung konfluenten Zellen und Ernte Zellen in der anderen gut für nachfolgende Prüfung durch Immunblotting. </li><li> Detect TetR durch Western-Blot mit dem monoklonalen anti-TET02 Antikörper (MoBiTec GmbH). Erinnern, um ein Lysat der parentalen Zelllinie als Negativkontrolle umfassen. </li><li> Wählen Sie 3 Klone mit der höchsten TetR Ausdruck und erweitern Sie jeden klonalen Kultur. Einfrieren Bestände jedes vielversprechende Klon so schnell wie möglich. Schließlich prüft die molekularen und zellbiologischen interessierenden Parameter durch Vergleichen der parentalen Zellinien mit den TetR-exprimierenden Klonen. Wählen Sie den &quot;besten&quot; Klon mit der höchsten TetR Ausdruck, der nicht angezeigt wird keine Veränderungen in den molekularen und zellbiologischen Parameter von Interesse. </li></ol> 3. Pilot Prüfung von pter und pT-Rex DEST30 Vektoren Expressten shRNA oder cDNAs, Jeweils <ol><li> Generieren PTER Plasmid mit shRNA Inserts 12, wie beschrieben. Konstrukt pT-Rex DEST30 Vektor (12.301 bis 016, Invitrogen), enthaltend die cDNA von Interesse mittels Gateway-Technologie (Invitrogen). Im Fall von cDNA-Expression wird die Zugabe eines Tags der cDNA später zu erleichtern Erkennung von exogen exprimierten Proteins. </li><li> Transient zu transfizieren die elterliche Zielzelle Einklang mit PTER oder pT-Rex DEST30 Plasmide (Ausdruck entweder shRNAs oder cDNAs von Interesse) mit dem Transfektionsreagenz der Wahl. Für die Prüfung von shRNA Expressionsplasmide, sorgen für eine hohe Transfektionseffizienz erreicht werden kann. </li><li> Ernte transfizierten Zellen und Verfahren zur Immunoblot mit den entsprechenden Antikörpern in der Erwartung, eine Verarmung oder Überexpression des Gens von Interesse zu erkennen. </li></ol> 4. Erzeugung von stabilen Zelllinien mit Tetrazyklin-induzierbarer (Tet-On) Expression von cDNAs oder shRNAs Verwenden PTER oder pT-Rex DEST30 Vektoren </p&gt; <ol><li> Am Tag 1, Saatgut 1x10 6 Zellen der &#39;besten&#39; TetR-exprimierenden Klon pro 10-cm-Platte mit Ihrem Standard-Wachstumsmedium mit zertifizierten Tetracyclin-Serum (zB FBS von Invitrogen; 16.000-014) ergänzt. Samen einer Platte für die Transfektion mit PTER (oder pT-Rex DEST30) und eine Platte als Negativkontrolle für Doppelselektion. Stellen Sie sicher, den günstigsten Durchgang möglich zu nutzen. </li><li> Am Tag 2 transfizieren eine Platte mit 2 ug PTER (oder pT-Rex DEST30) mit Fugene 6. Nicht transfizieren die zweite Platte. </li><li> Am Tag 3, fügen Standard Wachstumsmedium mit 5 ug / ml Blasticidin und 500 ug / ml Zeocin (oder 1 mg / ml G418). </li><li> Am Tag 4 teilen die Zellen mit 1/5, 1/25, 1/50 und 1/100 mit 10-cm-Platten und Standard Wachstumsmedium mit 5 ug / ml Blasticidin und 500 ug / ml Zeocin (oder 1 mg / ml G418). Bereiten Sie zwei Platten pro Verdünnungsstufe. </li><li> Überprüfen Sie die Zellen täglich, um sicherzustellen, dass innerhalb der ersten Woche alle Zellen auf den transfizierten Platten sind gestorben.Ändern Sie den Standard-Wachstumsmedium mit Blasticidin und Zeocin (oder G418) alle 3-4 Tage. </li><li> Nach 2-3 Wochen, Blasticidin / Zeocin (oder blasticidin/G418) Doppel-resistente Kolonien sollten beginnen zu erscheinen. Wählen Platten mit 5 bis 50 Kolonien, um einzelne Kolonien zu pflücken. </li><li> Wenn Kolonien haben einen Durchmesser von ca. 5 mm (oder größer) erreicht, verwenden Klonen Zylindern, um einzelne Kolonien zu pflücken. Isolieren mindestens 24 einzelnen Klonen. Übertragen jeder Klon in ein separates Loch einer 24-Well Gewebekulturplatte. </li><li> Kultur Klone in Medium, das Wartungs-Konzentrationen von Blasticidin (5 ug / ml) und Zeocin (250 pg / ml) [oder G418 (0,5 mg / ml)]. Wenn konfluent, spalten die Zellen aus jeder Vertiefung in einem einzigen Well einer 6-Well-Gewebekulturplatte. </li><li> Für jeden zu testenden Klon, aufgeteilt ein konfluenter Well einer 6-Well-Platte in drei einzelnen Wells einer 6-Well-Platte. Wenn konfluent, einzufrieren Zellen von einem gut. Verwenden Sie die verbleibenden zwei Kavitäten für die Prüfung der TetracyclinE-induzierbare Expression. Fügen Tetracyclin in einer Konzentration von 1 pg / ml zu einem gut, während die zweite und Tetracyclin kostenlos. </li><li> Nach 24 bis 96 Stunden Inkubation mit Tetracyclin, Ernte-Zellen und Verfahren zur Immunoblotting unter Verwendung geeigneter Antikörper. Kürzere Tetracyclin Inkubationszeiten werden empfohlen, wenn Screenen für die Induktion von cDNA-Expression, während längere Tetracyclin Behandlungen notwendig sein wird, shRNA-vermittelte Abreicherung von endogenen Proteinen zu bestimmen. </li><li> Wählen Sie mindestens zwei Klone mit der gewünschten Abbau / Überexpression und erweitern jede Kultur. Einfrieren Bestände jedes vielversprechende Klon so schnell wie möglich. Beim Anbau von Tet-on-Klone für Experimente immer darauf achten, Medien mit Tetracyclin-freies Serum und den jeweiligen Selektionsantibiotika bei Wartung Konzentrationen ergänzt verwenden. </li></ol>

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Keine Interessenskonflikte erklärt.

Wir glauben, dass Tet-On-Systeme erheblich erleichtern die Analyse der Genfunktion, insbesondere in Zellsysteme, die schwierig zu manipulieren und / oder wenn die manipulierte Gen ist essentiell für das Überleben der Zelle sind. Darüber hinaus bietet die Fähigkeit, die Genexpression durch hochspezifische Tet-On-Systeme kontrollieren die Möglichkeit Genfunktionen in verschiedenen Phasen (zum Beispiel während der Zellzyklusprogression oder wohldefinierten Differenzierungsprozesse) studieren. Die hier vorgestellten V...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Fötales Rinderserum (FBS) </td><td> Invitrogen </td><td> 16000-044 </td><td> Überprüft Tet-free </td></tr><tr><td> Blasticidin </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 Laboratories </td><td> P31-011 </td><td> 100 mg / ml im Medium </td></tr><tr><td> Anti-TET02 </td><td> MoBiTec GmbH </td><td> TET02 </td><td> Verwenden Sie bei 1/1000 bis 1/2000 für 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 Ethanol </td></tr><tr><td> Doxycycline </td><td> Sigma </td><td> D9891 </td><td> 2 mg / ml in Wasser </td></tr><tr><td> Cloning Zylinder </td><td> Bellco Glass Inc. </td><td> 2090-00808 </td><td> wieder verwendbare </td></tr></tbody></table>

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

Ein wichtiger Ansatz im Bereich der Säugerzelle Biologie ist die Manipulation der die Expression von Genen von Interesse in ausgewählten Zellinien, mit dem Ziel, zu enthüllen einem oder mehreren der des Gens Funktion (en) unter Verwendung von transienten / stabile Überexpression oder Knockdown des Gens von Interesse. Leider für verschiedene zellbiologische Untersuchungen dieser Ansatz ist ungeeignet, wenn Manipulationen der Genexpression führen Zellwachstum / Zellproliferation Mängel oder unerwünschte Zelldifferenzierung. Daher haben Forscher die Tetracyclin-Repressorprotein (TetR), genommen aus dem E. angepaßt coli Tetracyclin-Resistenz-Operons 1, zu einer sehr effizienten und engen regulatorischen Systeme zu erzeugen, um cDNAs in Säugerzellen 2,3 auszudrücken. Kurz gesagt, hat TetR um entweder (1) Block Initiation der Transkription wurde durch Bindung an das Tet-Operator (TO) in der Promotorregion nach Zugabe von Tetracyclin (Tet-off bezeichnet System) oder (2) binden an die AN in modifizierter tEr Abwesenheit von Tetracyclin (sog. Tet-On-System) (Abbildung 1). Angesichts der Unannehmlichkeiten, das Tet-Off-System die kontinuierliche Anwesenheit von Tetracyclin (welches eine Halbwertszeit von etwa 24 Stunden in Gewebe Zellkulturmedium) erfordert das Tet-On-System wurden ausführlicher optimiert, was in der Entwicklung von sehr engen und effiziente Vektor-Systeme für die cDNA-Expression, wie hier verwendet. Kurz nach der Gründung der RNA-Interferenz (RNAi) für die Gen-Knockdown in Säugetierzellen 4, Vektoren, kurz-hairpin RNAs (shRNAs) beschrieben wurden diese Funktion sehr ähnlich siRNAs 5-11. Jedoch haben diese shRNA-vermittelte Knockdown Ansätze die gleiche Einschränkung wie herkömmliche Knockout Strategien, da stabile Abreicherung nicht durchführbar, wenn Genziele für zelluläre Überleben essentiell. Um diese Einschränkung zu umgehen, van de Wetering et al. 12 veränderte die shRNA Expressionsvektor pSUPER 5 </sup&gt; durch Einfügen eines TO in der Promotorregion, die sie zu stabilen Zelllinien mit Tetracyclin-induzierbare Depletion ihrer Zielgene von Interesse zu erzeugen aktiviert. Hier beschreiben wir ein Verfahren effizient zu erzeugen stabilen menschlichen Tet-On Zell-Linien, die zuverlässig fahren entweder induzierbare Überexpression oder Abreicherung des Gens von Interesse. Mit dieser Methode haben wir erfolgreich generiert Tet-on-Zelllinien, die wesentlich erleichtert die Analyse der MST / hMOB / NDR Kaskade in Zentrosom 13,14 und Apoptose Signalisierung 15,16. In diesem Bericht beschreiben wir unsere Vektoren der Wahl, neben der Beschreibung der zwei aufeinander folgenden Manipulationen Schritte, die erforderlich sind, um effizient zu erzeugen sind menschliche Tet-on-Zelllinien (Abbildung 2). Außerdem neben umreißt ein Protokoll für die Herstellung von menschlichen Tet-On Zell-Linien werden wir kritischen Aspekte hinsichtlich der technischen Verfahren und die Charakterisierung von Tet-On-Zellen zu diskutieren.

Ein Beispiel für die erstmalige Beschreibung RPE-1-Zelllinien stabil exprimieren TetR ist in Abbildung 4 dargestellt. Beachten Sie, dass alle RPE-1 Klone unterschiedlicher TetR ausdrücken (vgl. Bahnen 2 und 5), während die elterliche Zelllinie (die als negative Kontrolle) nicht exprimiert die exogene TetR-Protein (Abbildung 4, Spur 1). Diese Variation in TetR Expression unter RPE-1 Tet-On Zell-Klone wird erwartet, da die Expression des TetR exprimierenden Plasmiden ist stark abhängi...

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

Eine schnelle und einfache Art und Weise die menschliche Zelllinien mit induzierbarer und reversible cDNA Überexpression oder shRNA-vermittelte Knock-down des Gens von Interesse zu erzeugen. Diese Methode ermöglicht es Forschern, zuverlässig und hoch reproduzierbar zu manipulieren Zelllinien, die nur schwer durch transiente Transfektion Methoden oder konventionellen knockdown / Knockout-Strategien zu verändern sind.

Generierung von stabilen menschlichen Zelllinien mit Tetracyclin-induzierbarer (Tet-on) shRNA oder 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 ...

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

Research

JoVE Journal

Biology

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

35.0K Aufrufe.  UCL Cancer Institute. Eine schnelle und einfache Art und Weise die menschliche Zelllinien mit induzierbarer und reversible cDNA Überexpression oder shRNA-vermittelte Knock-down des Gens von Interesse zu erzeugen. Diese Methode ermöglicht es Forschern, zuverlässig und hoch reproduzierbar zu manipulieren Zelllinien, die nur schwer durch transiente Transfektion Methoden oder konventionellen knockdown / Knockout-Strategien zu verändern sind.

Serie: 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.

Passagierung FARBEN humanen embryonalen Stammzellen-Linien mit 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 ...

Forschung

Biologie

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.

Generierung von stabilen transgenen C. elegans mit Mikroinjektion

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.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

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.

Färbung von Proteinen in Gelen mit Coomassie G-250 ohne organische Lösungsmittel und Essigsäure

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.

Generation of Human CD40-aktivierten B-Zellen

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.

RNA Isolation aus embryonalen Zebrafisch und cDNA-Synthese für die Genexpressionsanalyse

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.

Mit dem Gene Pulser MXcell Elektroporation System zur Primary Cells mit hohem Wirkungsgrad zu transfizieren

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.

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

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.

Transduktion humaner Zellen mit Polymer-Komplex ecotropen Lentivirus für 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 ...

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.