Etape 1: Dégeler les cellules 293 et ​​la plaque pour la production de lentivirus. <ol><li> Préparer 9 ml de DMEM + support dans un tube de 15 ml. </li><li> Retirer un flacon de 293 stocks congelés à partir du réservoir d&#39;azote liquide et mettre le flacon dans un bain à 37 ° C jusqu&#39;à ce que la plupart des cellules (mais pas tous) sont décongelés. </li><li> Éliminer les cellules du flacon de gel et le placer dans le tube 15ml de l&#39;étape 1. </li><li> Centrifuger à 180g pendant 5 min, puis jeter le surnageant. </li><li> Resuspendre les cellules avec 10 ml de DMEM + support, et le transfert d&#39;une gélatine à revêtement de 100 mm plat. Incuber les cellules dans un 37 ° C, 5% de CO 2 incubateur jusqu&#39;à ce qu&#39;ils soient confluentes à 80-90%. </li><li> Passage des 293 cellules: les cellules de lavage avec du PBS, aspirer le PBS et ajouter 4 ml par 10 cm de plat de 0,05% trypsin/0.53 mM d&#39;EDTA, et incuber pendant 1 min à 37 ° C. </li><li> Détachez les cellules à partir des plats en tapant, resuspendre avec 10 ml de DMEM + support, et le transfert d&#39;un tube de 15 ml. Centrifuger à 180g pendant 5 min, et aspirer le surnageant. </li><li> Ajouter le volume approprié de DMEM + support, et briser les cellules dans une suspension cellulaire unique par aspiration et refoulement à plusieurs reprises. </li><li> Comptez le nombre de cellules et d&#39;ajuster la concentration à 8x10 5 cellules par ml avec le milieu de PF. </li><li> Cellules des semences à 8x10 6 cellules (10 ml) par boîte de culture de 100 mm et incuber une nuit à 37 ° C, 5% de CO 2. </li></ol> Étape 2: Transfecter des cellules avec des plasmides 293 lentivirus et le virus de la récolte <ol><li> Pour chaque plaque de 10 cm, utiliser 10 ug de FUW-tetO-ADNc (Oct4, Sox2, Klf4 ou c-Myc) épine dorsale des lentivirus avec 2.5ug des PMD-G et 7,5 ug d&#39;pPax2. </li><li> Alors que les trois plasmides aliquotage dans un tube, livrer 30μl de réactif de transfection Fugene 6 dans un second tube contenant 500μl de DMEM sans sérum et mélanger délicatement avec le doigt tapant et incuber pendant 5 min à température ambiante. </li><li> Ajouter le mélange d&#39;ADN au goutte-à-goutte dans le tube Fugene 6/DMEM-containing, mélanger délicatement en tapotant des doigts et incuber pendant 20 min. </li><li> Ajouter l&#39;ADN / 6 Fugene goutte à goutte complexes dans le plat de 293, et incuber une nuit à 37 ° C, 5% de CO 2. </li><li> Le lendemain matin: Aspirer le réactif de transfection contenant du milieu, ajoutez 5 ml de milieu de cellules ES frais, et le retour des cellules dans l&#39;incubateur. </li><li> À 48 heures et 72 heures après la transfection, de recueillir le support de la 293 en utilisant une de 10 ml seringue stérile à usage unique, le filtrant à travers un filtre de 0,45 um de taille de l&#39;acétate de cellulose, des pores, et transférer dans un tube de 15 ml. </li><li> Concentrer le surnageant de virus par ultracentrifugation. Reprendre le culot de virus dans le volume désiré et faire amixture à parts égales du milieu contenant Oct-3/4-, Sox2-, Klf4-et c-Myc-lentivirus. </li><li> Tout en faisant le virus, de préparer ou de Oct4-neo/rtTA Oct4-GFP/rtTA FAE (passage &lt;3) à 90% de confluence dans des plats de 10 cm (2x10 6 cellules par boîte) </li><li> Aspirer le milieu de culture et laver avec 10 ml de PBS. </li><li> Jeter PBS, ajouter 1 ml par boîte de 0,05% trypsin/0.53 mM d&#39;EDTA, et incuber à 37 ° C pendant 10 min. </li><li> Ajouter 9 ml de milieu de culture, de suspendre les cellules à une seule cellule, et le transfert d&#39;un tube de 15 ml. </li><li> Comptez le nombre de cellules, et d&#39;ajuster la concentration à 8x10 4 cellules par ml. Transférer 10 ml de suspension cellulaire (8x10 5 cellules) pour un plat de 10 cm recouvert d&#39;gélatine. Incuber le plat une nuit à 37 ° C, 5% de CO 2. </li><li> Aspirer le support d&#39;un plat de fibroblastes, et ajouter 10 ml de milieu contenant le virus. Incuber les cellules à partir de 4 h à une nuit à 37 ° C, 5% de CO 2. </li><li> Après 24 aspirer le support d&#39;un plat de fibroblastes, et ajouter 10 ml de milieu de cellules ES frais. </li><li> Remplacer régulièrement moyennes de cellules ES avec la doxycycline milieu contenant de lancer l&#39;expression des quatre gènes; en d&#39;autres termes, initier une reprogrammation. </li><li> Si vous utilisez un journaliste néo-Oct4, puis de lancer la section des médicaments à tout moment entre 6 et 9 jours. </li><li> Changer le support tous les jours jusqu&#39;à les colonies deviennent assez gros pour être ramassé. Colonies doit d&#39;abord devenir visibles environ une semaine après le début de la reprogrammation. Ils devraient devenir suffisamment importante pour être ramassé autour du jour 20. </li></ol> Étape 3: Reprenant les colonies iPS La veille des vendanges: <ol><li> Seed le nombre requis de gélatine revêtement des plaques 24 puits avec des γ-irradiés DR4 FAE </li></ol> Cueillette des clones sur le Jour 1: <ol><li> Flux des colonies sur les plaques de 1-2 heures avant la cueillette. </li><li> Ajouter 15 ul de PBS dans les puits d&#39;un plat à fond en V de 96 puits. </li><li> Laver la capsule contenant les colonies à être cueillies une fois avec du PBS et ajouter 10 ml de PBS dans le plat. </li><li> Choisissez les colonies avec les petits conseils pipette 5uL (réglez le Pipetman P20 sur 4 pi). Transfert colonies d&#39;un puits dans le plat de 96 puits. </li><li> Ajouter 20 ul de trypsine dans chaque puits en utilisant le multi-canal (12) pipetor. Pipet haut et en bas trois fois. Incuber à 37 ° C pendant 4 minutes. </li><li> Ajouter 100 pi de milieu ES pour les colonies trypsinisées. Pipet haut et en bas 10 fois. Transfert 6 clones à une époque de l&#39;antenne de 96 puits dans un plat de 24 puits en utilisant une pointe à chaque position d&#39;autres sur le pipetor 12 place. </li><li> Cultiver les cellules sur la plaque de 24 puits dans un 37 ° C, 5% de CO 2 de l&#39;incubateur jusqu&#39;à atteindre 80-90% des cellules de confluence, l&#39;alimentation quotidienne avec un milieu de cellules ES. Cellules sera probablement prêt à se développer dans 3-7 jours. </li></ol> Etape 4 <ol><li> Expansion des cellules iPS <ol><li> Aspirer le milieu, et laver les cellules avec 1 ml de PBS. </li><li> Retirer du PBS complètement, ajouter 0,1 ml de 0,25% de trypsine / EDTA 1 mM et incuber à 37 ° C pendant 10 min. </li><li> Ajouter 0,4 ml de milieu ES et les cellules en suspension de pipetage haut et bas pour suspension cellulaire unique. </li><li> Transférer la suspension cellulaire à un puits de la plaque de 6 puits, ajouter 1,5 ml de cellules ES moyennes, et incuber dans un 37 ° C, 5% de CO 2 incubateur jusqu&#39;à atteindre 80-90% des cellules de confluence dans des plaques 6 puits. A ce point, préparer stock congelé des cellules, comme suit. </li></ol></li><li> Préparation de stock gel <ol><li> Aspirer le milieu, et laver les cellules avec 2 ml de PBS. </li><li> Retirer du PBS complètement, ajoutez 0,5 ml de 0,25% de trypsine / EDTA 1 mM et incuber à 37 ° C pendant 10 min. </li><li> Ajouter 2 ml de milieu ES et les cellules en suspension de pipetage haut et bas pour suspension cellulaire unique. </li><li> Transférer la suspension cellulaire dans un tube de 15 ml, comptez le nombre de cellules et de spin-down des cellules. </li><li> Jeter le surnageant, remettre en suspension les cellules avec le milieu ES à la concentration à 2x10 6 cellules par ml. </li><li> Préparer 2 milieu de congélation de cellules ES (DMSO 20% en ES moyenne) et il aliquote de 0,5 ml par flacon. </li><li> Transfert de 0,5 ml de la suspension cellulaire de geler les flacons et mélanger doucement. </li><li> Mettez les flacons dans un contenant de congélation des cellules et le conserver à -80 ° C pendant la nuit; transfert à l&#39;azote liquide le lendemain pour stockage à long terme. </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>

Dans cette vidéo, nous montrent comment utiliser une inductible, système lentiviral pour générer GFP-positives, pluripotentes à partir de cellules iPS FAE provenant de souris et de Oct4-GFP/R26-M2rtTA Nanog-GFP/R26-M2rtTA. Cette procédure est une méthode utile pour générer des cellules iPS, car il permet le contrôle de l&#39;expression du transgène pendant le processus de reprogrammation. Bien que l&#39;utilisation de virus dans la génération de cellules iPS empêche l&#39;utilisation de ces cellules dans un milieu clinique, cette procédure ne nous permettent de répondre à certaines des grandes questions biologiques qui sous-tendent le processus de reprogrammation encore défini.

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

Pluripotence peut être induite dans murines différenciées par transduction virale de Oct4, Sox2, Klf4, et c-Myc (Takahashi et Yamanaka, 2006; Wernig, et al, 2007;. Okita, et al, 2007;.. Maherali, et al, 2007). Nous avons élaboré une stratégie de reprogrammation dans laquelle ces facteurs de transcription quatre sont exprimés à partir de doxycycline (Dox) inductible par des vecteurs lentiviraux (Brambrink et al., 2008). En utilisant ces constructions inductible, on peut dériver souches pluripotentes induites (iPS) à partir de fibroblastes embryonnaires de souris (MEF). Dans cette vidéo, nous démontrons la procédure pour la génération des lentivirus inductibles qui expriment les quatre facteurs de transcription et de montrer comment à infecter MEFs par ces virus, afin de produire des cellules iPS. En utilisant des lentivirus inductible, l&#39;expression des quatre facteurs dans contrôlées par l&#39;ajout de doxycycline au milieu de culture. L&#39;avantage de ce système au cours de l&#39;infection rétrovirale est traditionnelle de la capacité de transformer les gènes sur et en dehors de sorte que la cinétique de la reprogrammation et les exigences expression des gènes peut être analysé en détail.

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

Cette vidéo montre la procédure pour générer des cellules souches pluripotentes induites à l&#39;aide de lentivirus inductibles qui expriment Oct4, Sox2, c-Myc et Klf4.

Générer des cellules iPS de MEFS travers l&#39;expression forcée de Sox-2, Oct-4, c-Myc et 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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36.1K vues.  Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology. Cette vidéo montre la procédure pour générer des cellules souches pluripotentes induites à l'aide de lentivirus inductibles qui expriment Oct4, Sox2, c-Myc et Klf4.

Vidéo: 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.

De MEFs à I Matrigel: CSEh repiquage en présence du MEF

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

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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.

De FAE au Matrigel 2: Fractionnement des CSEh MEFs sur 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.

De FAE au Matrigel 3: Le repiquage du Matrigel CSEh sur 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.

La préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

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.

Coloration des protéines dans les gels de Coomassie G-250, sans solvant et l&#39;acide acétique organique

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.

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

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.

Aide d&#39;un compteur de cellules automatisé pour simplifier les études d&#39;expression génique: Knockdown siRNA de l&#39;IL-4 dans l&#39;expression des gènes dépendant cellules 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 ...

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.

Chargeur sans adaptation, de la Culture et de repiquage des cellules iPS humaines à l&#39;aide complète d&#39;alimentation sans remplacement KnockOut Sérum 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.

Cryoconservation et la récupération des cellules iPS humaines utilisant KnockOut complète Chargeur sans sérum remplacement moyen

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.

Une plate-forme d&#39;expression pratique et général pour la production de protéines sécrétées à partir de cellules humaines

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