Schritt 1: Auftauen 293-Zellen und die Platte für Lentivirus Produktion. <ol><li> Bereiten 9 ml DMEM + Medium in einer 15-ml-Tube. </li><li> Nehmen Sie ein Fläschchen eingefroren 293 Aktien aus dem flüssigen Stickstoff-Tank und steckte die Flasche in einem 37 ° C Wasserbad, bis die meisten (aber nicht alle) Zellen aufgetaut werden. </li><li> Entfernen Zellen vor dem Einfrieren Fläschchen und Ort in die 15ml Tube aus Schritt 1. </li><li> Zentrifugation bei 180g für 5 min, und dann den Überstand verwerfen. </li><li> Resuspendieren der Zellen mit 10 ml DMEM + Medium, und in ein mit Gelatine beschichtete 100-mm-Schale. Inkubieren Sie die Zellen in einem 37 ° C, 5% CO 2-Inkubator bis sie 80-90% konfluent sind. </li><li> Passage des 293-Zellen: Zellen mit PBS waschen, saugen Sie den PBS und 4 ml pro 10-cm-Spiegel von 0,05% trypsin/0.53 mM EDTA und Inkubation für 1 min bei 37 ° C. </li><li> Lösen Sie Zellen aus Gerichten, indem Sie mit 10 ml DMEM + Medium resuspendieren und Transfer zu einem 15-ml-Tube. Zentrifugation bei 180g für 5 min, und saugen Sie den Überstand. </li><li> Add entsprechenden Volumen DMEM + Medium und die Zellen aufzubrechen zu einer einzigen Zelle Suspension durch Auf-und Abpipettieren mehrmals. </li><li> Zählen Sie die Anzahl der Zellen und stellen Sie die Konzentration auf 8x10 5 Zellen pro ml mit FP-Medium. </li><li> Seed-Zellen bei 8x10 6 Zellen (10 ml) pro 100-mm Kulturschale, und bei 37 ° C über Nacht, 5% CO 2. </li></ol> Schritt 2: Transfizieren 293 Zellen mit Lentiviren Plasmide und Ernte-Virus <ol><li> Für jeden 10-cm-Platte, verwenden 10 ug FUW-tetO-cDNA (Oct4, Sox2, Klf4 oder c-Myc) Lentivirus Backbone mit 2.5ug der PMD-G und 7,5 ug pPax2. </li><li> Während Aliquotierung der drei Plasmide in ein Rohr, liefern 30μl der Fugene 6 Transfektionsreagenz in ein zweites Röhrchen mit 500μl DMEM ohne Serum und vorsichtig mischen mit dem Finger tippen und Inkubation für 5 min bei Raumtemperatur. </li><li> Fügen Sie die DNA-Mix Drop-by-drop in die Fugene 6/DMEM-containing Rohr, mischen Sie durch Fingertapping und Inkubation für 20 min. </li><li> Fügen Sie die DNA / Fugene 6-Komplex tropfenweise in die Schüssel von 293 ist, beimpft und über Nacht bei 37 ° C, 5% CO 2. </li><li> Am nächsten Morgen: Saugen Sie das Transfektionsreagenz-haltigem Medium, 5 ml frischem ES-Zell-Medium, und kehren die Zellen in den Inkubator. </li><li> Nach 48 Stunden und 72 Stunden nach der Transfektion, sammeln das Medium von den 293 ist mit einem 10-ml sterilen Einwegspritze, Filtrieren durch ein 0,45-um Porengröße Celluloseacetat-Filter, und die Übertragung in einen 15-ml-Tube. </li><li> Konzentrieren Sie sich die Virus-Überstand durch Ultrazentrifugation. Resuspendieren Viruspellet in gewünschte Lautstärke und stellen ein Gemisch von gleichen Teilen aus dem Medium, das Oct-3/4-, Sox2-, Klf4 und c-Myc-Lentiviren. </li><li> Während der Arbeit an dem Virus, bereiten Oct4-neo/rtTA oder Oct4-GFP/rtTA MEFs (Durchgang &lt;3) bis 90% Konfluenz in 10-cm-Schalen (2x10 6 Zellen pro Schale) </li><li> Absaugen des Kulturmediums und Waschen mit 10 ml PBS. </li><li> Discard PBS, 1 ml pro Schale von 0,05% trypsin/0.53 mM EDTA, und bei 37 ° C für 10 min. </li><li> Add 9 ml des Kulturmediums, Aussetzung der Zellen zu einer einzigen Zelle, und in ein 15-ml-Tube. </li><li> Zählen Sie die Anzahl der Zellen, und passen Sie die Konzentration auf 8x10 4 Zellen pro ml. Transfer 10 ml Zellsuspension (8x10 5 Zellen) zu einer 10-cm-Spiegel mit Gelatine beschichtet. Inkubieren Sie die Schüssel über Nacht bei 37 ° C, 5% CO 2. </li><li> Saugen Sie das Medium aus Fibroblasten Schüssel und fügen Sie 10 ml des Virus-haltigem Medium. Inkubieren Sie die Zellen von 4 h bis über Nacht bei 37 ° C, 5% CO 2. </li><li> Nach 24 Saugen Sie das Medium aus Fibroblasten Schüssel, und mit 10 ml frischem ES-Zell-Medium. </li><li> Ersetzt einen regulären ES-Zell-Medium mit Medium mit Doxycyclin die Expression der vier Gene initiieren, mit anderen Worten, zu initiieren Neuprogrammierung. </li><li> Bei Verwendung eines Oct4-neo-Reporter, dann starten Medikament Abschnitt jederzeit zwischen 6 und 9 Tagen. </li><li> Ändern Sie das Medium jeden Tag, bis die Kolonien groß genug, um abgeholt zu werden. Kolonien sollten zuerst sichtbar werden etwa eine Woche nach Beginn der Umprogrammierung. Sie sollten groß genug, um bis etwa Tag 20 abgeholt werden. </li></ol> Schritt 3: Picking up the iPS Kolonien Am Tag vor Picking: <ol><li> Seed die erforderliche Anzahl von Gelatine beschichtete 24-Well-Platten mit γ-bestrahlten DR4 MEFs </li></ol> Picking Clones an Tag 1: <ol><li> Feed the Kolonien auf den Platten 1-2 Stunden vor Kommissionierung. </li><li> Add 15 ul PBS in die Vertiefungen eines V-Boden 96-well Schale. </li><li> Waschen Sie die Schale mit den Kolonien, einmal mit PBS aufgenommen und fügen Sie 10 ml PBS in die Schale. </li><li> Wählen Sie die Kolonien mit dem kleinen 5μl Pipettenspitzen (stellen Sie den P20 Pipetman auf 4 ul). Transfer-colonies zu einem Brunnen in der 96-well Schale. </li><li> Add 20 ul Trypsin in jede Vertiefung mit dem Multi-Channel-(12) pipetor. Pipet nach oben und unten 3 mal. Bei 37 ° C für 4 Minuten. </li><li> 100 l von ES Medium, um die trypsiniert Kolonien. Pipet nach oben und unten 10-mal. Transfer-6 Klone in einer Zeit, aus dem 96-Loch-Spiegel auf eine 24-Well-Schale mit einer Spitze an jedem anderen Position auf den 12 Platz pipetor. </li><li> Wachsen die Zellen auf der 24-Well-Platte in einem 37 ° C, 5% CO 2-Inkubator bis die Zellen 80-90% Konfluenz erreichen, Fütterung täglich mit ES-Zell-Medium. Cells dürfte bereit sein, innerhalb von 3-7 Tagen zu erweitern. </li></ol> Schritt 4 <ol><li> Ausbau der iPS-Zellen <ol><li> Saugen Sie das Medium, und waschen Sie die Zellen mit 1 ml PBS. </li><li> Entfernen PBS vollständig, fügen Sie 0,1 ml 0,25% Trypsin / 1 mM EDTA und Inkubation bei 37 ° C für 10 min. </li><li> Fügen Sie 0,4 ml der ES-Medium und setzt die Zellen durch Auf-und Abpipettieren, einzelne Zell-Suspension. </li><li> Übertragen Sie die Zellsuspension in eine Vertiefung 6-Well-Platte, 1,5 ml ES-Zell-Medium und Inkubation bei 37 ° C, 5% CO 2-Inkubator bis die Zellen zu erreichen 80-90% Konfluenz in 6-well Platten. An dieser Stelle vorzubereiten eingefroren Lager der Zellen, wie folgt. </li></ol></li><li> Vorbereitung von freeze Lager <ol><li> Saugen Sie das Medium, und waschen Sie die Zellen mit 2 ml PBS. </li><li> Entfernen PBS vollständig, 0,5 ml 0,25% Trypsin / 1 mM EDTA und Inkubation bei 37 ° C für 10 min. </li><li> 2 ml des ES-Medium und setzt die Zellen durch Auf-und Abpipettieren, einzelne Zell-Suspension. </li><li> Übertragen Sie die Zellsuspension auf eine 15-ml-Tube, die Anzahl von Zellen und Spin-down der Zellen. </li><li> Überstand verwerfen, resuspendieren Sie die Zellen mit ES-Medium, um die Konzentration auf 2x10 6 Zellen pro ml. </li><li> Bereiten Sie 2 ES-Zelle Einfriermedium (20% DMSO in ES-Medium) und aliquote es bei 0,5 ml pro Fläschchen. </li><li> Übertragen 0,5 ml der Zellsuspension Fläschchen einfrieren und vorsichtig mischen. </li><li> Legen Sie die Flaschen in einer Zelle-Gefrierbehälter und halten Sie sie bei -80 ° C über Nacht; in flüssigen Stickstoff Transfer am nächsten Tag für eine langfristige Lagerung. </li></ol></li></ol>

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Wernig, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al+.+In+vitro+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state.">et al . In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448, 318-324 (2007).</li><li>Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+germline-competent+induced+pluripotent+stem+cells.">Generation of germline-competent induced pluripotent stem cells.</a> Nature. 448, 313-317 (2007).</li><li>Maherali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epigenetic+remodeling+in+directly+reprogrammed+fibroblasts.">Global epigenetic remodeling in directly reprogrammed fibroblasts.</a> Cell Stem Cell. 1, 55-70 (2007).</li><li>Brambrink, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+expression+of+pluripotency+markers+during+direct+reprogramming+of+somatic+cells.">Sequential expression of pluripotency markers during direct reprogramming of somatic cells.</a> Cell Stem Cell. 2, 151-159 (2008).</li></ol>

In diesem Video zeigen wir, wie man einen induzierbaren, lentiviralen System nutzen, um GFP-positive, pluripotente iPS-Zellen aus MEFs aus Oct4-GFP/R26-M2rtTA und Nanog-GFP/R26-M2rtTA Mäusen abgeleitet generieren. Dieses Verfahren ist eine nützliche Methode zur Erzeugung von iPS-Zellen, weil es für die Steuerung von Transgenexpression während der Umprogrammierung Prozess ermöglicht. Obwohl der Einsatz von Viren bei der Erzeugung von iPS-Zellen verhindert die Verwendung dieser Zellen in einem klinischen Umfeld, hat dieses Verfahren ermöglichen es uns, einige der wichtigsten biologischen Fragen, die noch nicht definiert Reprogrammierung unterstreichen beantworten.

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generating ips cells from mefs through forced expression of sox-2, oct-4, c-myc, and klf4

Pluripotenz in differenzierten murinen durch virale Transduktion von Oct4 induziert werden, Sox2, Klf4 und c-Myc (Takahashi und Yamanaka, 2006; Wernig, et al, 2007;. Okita, et al, 2007;.. Maherali, et al, 2007). Wir haben eine Umprogrammierung Strategie, in der diese vier Transkriptionsfaktoren von Doxycyclin ausgedrückt werden erarbeitet (dox)-induzierbaren lentiviralen Vektoren (Brambrink et al., 2008). Mit Hilfe dieser induzierbaren Konstrukten, können wir ableiten induzierte pluripotente Stammzellen (iPS-Zellen) aus embryonalen Fibroblasten (MEFs). In diesem Video zeigen wir, das Verfahren für die Erzeugung von induzierbaren Lentiviren, dass die vier Transkriptionsfaktoren auszudrücken und zu zeigen, wie man MEFs mit diesen Viren zu infizieren, um iPS-Zellen zu produzieren. Durch die Verwendung von induzierbaren Lentiviren, der Ausdruck der vier Faktoren in durch die Zugabe von Doxycyclin zum Kulturmedium kontrolliert. Der Vorteil dieses Systems gegenüber den herkömmlichen retrovirale Infektion ist die Möglichkeit, die Gene ein-und ausschalten, so dass die Kinetik der Neuprogrammierung und Genexpression Anforderungen im Detail analysiert werden können.

Watch this Scientific Journal Video about Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4 at JoVE.com

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Dieses Video zeigt das Verfahren zur Erzeugung von induzierten pluripotenten Stammzellen mit induzierbaren Lentivirus, dass ausdrückliche Oct4, Sox2, c-Myc und Klf4.

Generieren von iPS-Zellen aus MEFS durch Forced Expression von Sox-2, Oct-4, c-Myc und Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

generating-ips-cells-from-mefs-through-forced-expression-sox-2-oct-4

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JoVE Journal

Biology

36.1K Aufrufe.  Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology. Dieses Video zeigt das Verfahren zur Erzeugung von induzierten pluripotenten Stammzellen mit induzierbaren Lentivirus, dass ausdrückliche Oct4, Sox2, c-Myc und Klf4.

Serie: Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.


This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

Von MEFs auf Matrigel I: Die Passage hESCs in Gegenwart von MEFs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

Forschung

Biologie

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

Von MEFs auf Matrigel 2: Splitting hESCs aus MEFs auf Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

Von MEFs auf Matrigel 3: Die Passage hESCs von Matrigel auf Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

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 ...

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 ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

Feeder-freie Adaption, Kultur und Passagierung der Menschenrechte iPS-Zellen mit Komplett KnockOut Serum Ersatz Feeder-freiem Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

Kryokonservierung und Wiederherstellen von Human iPS-Zellen unter Verwendung Komplette KnockOut Serum Ersatz Feeder-freiem Medium

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Eine bequeme und allgemein Expression Plattform für die Produktion von sekretierten Proteinen aus menschlichen Zellen

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...