Fase 1: Scongelare le 293 cellule e piastra per la produzione di lentivirus. <ol><li> Preparare 9 ml di medium DMEM + in un tubo da 15 ml. </li><li> Rimuovere un flacone di 293 titoli congelati dal serbatoio di azoto liquido e mettere la fiala in un bagno d&#39;acqua a 37 ° C fino maggior parte delle cellule (ma non tutti) sono scongelati. </li><li> Rimuovere le cellule dal flacone congelamento e posto nel tubo 15ml dal punto 1. </li><li> Centrifugare a 180g per 5 minuti, e poi scartare il surnatante. </li><li> Risospendere le cellule con 10 ml di terreno DMEM +, e trasferirli in un gelatina rivestita da 100 mm piatto. Incubare le cellule a 37 ° C, 5% CO 2 incubatore fino a quando sono 80-90% confluenti. </li><li> Passaggio delle 293 celle: lavare le cellule con PBS, aspirare il PBS e aggiungere 4 ml per 10 cm piatto dello 0,05% trypsin/0.53 mM EDTA, e incubare per 1 min a 37 ° C. </li><li> Staccare le cellule dai piatti toccando, risospendere con 10 ml di DMEM + medio, e trasferirli in un tubo da 15 ml. Centrifugare a 180g per 5 minuti, e aspirare il surnatante. </li><li> Aggiungere il volume appropriato di DMEM + medio, e rompere le cellule in una sospensione singola cella pipettando su e giù parecchie volte. </li><li> Contare il numero di cellule e regolare la concentrazione di 8x10 5 cellule per ml con media FP. </li><li> Cellule seme a 8x10 6 celle (10 ml) per 100 mm piatto di cultura, e incubare una notte a 37 ° C, 5% di CO 2. </li></ol> Fase 2: Trasfettare 293 cellule con lentivirus plasmidi e virus raccolta <ol><li> Per ogni 10 cm piastra, utilizzare 10 mg di FUW-Teto-cDNA (Oct4, Sox2, Klf4 e c-Myc) dorsale lentivirus con 2.5ug di PMD-G e 7,5 mg di pPax2. </li><li> Mentre aliquoting i tre plasmidi in un tubo, consegnare 30μl di Fugene 6 reagenti di trasfezione in una seconda provetta contenente 500μl di DMEM senza siero e mescolare delicatamente con le dita toccando e incubare per 5 minuti a temperatura ambiente. </li><li> Aggiungere il mix di DNA goccia a goccia nella provetta Fugene 6/DMEM-containing, mescolare delicatamente toccando dito e incubare per 20 min. </li><li> Aggiungere il DNA / Fugene 6 gocce complesso nel piatto di 293, e incubare una notte a 37 ° C, 5% di CO 2. </li><li> La mattina dopo: Aspirare il reagente di trasfezione contenente medio, aggiungere 5 ml di terreno fresco cellule staminali, le cellule e il ritorno alla incubatrice. </li><li> A 48 ore e 72 ore dopo la trasfezione, raccogliere il terreno dalla 293 da parte utilizzando una siringa da 10 ml sterile monouso, filtrandola attraverso un filtro di 0,45 micron dimensioni dei pori di acetato di cellulosa, e il trasferimento in un tubo da 15 ml. </li><li> Concentrare il surnatante virus da ultracentrifugazione. Risospendere il pellet virus nel volume desiderato e fare amixture in parti uguali del supporto contenente Oct-3/4-,-Sox2, Klf4 e c-Myc-lentivirus. </li><li> Pur facendo il virus, preparare Oct4-neo/rtTA o Oct4-GFP/rtTA MEF (&lt;passaggio 3) al 90% di confluenza in 10 cm di piatti (2x10 6 cellule per piatto) </li><li> Aspirare il terreno di coltura e lavare con 10 ml di PBS. </li><li> Eliminare PBS, aggiungere 1 ml per piatto dello 0,05% trypsin/0.53 mM EDTA, e incubare a 37 ° C per 10 min. </li><li> Aggiungere 9 ml di terreno di coltura, di sospendere le cellule a una singola cella, e trasferirli in un tubo da 15 ml. </li><li> Contare il numero di cellule, e regolare la concentrazione di 8x10 4 cellule per ml. Trasferire 10 ml di sospensione cellulare (8x10 5 celle) ad un piatto di 10 cm rivestito con gelatina. Incubare il piatto durante la notte a 37 ° C, 5% di CO 2. </li><li> Aspirare il terreno da un piatto fibroblasti, e aggiungere 10 ml di virus contenenti mezzo. Incubare le cellule da 4 ore a notte a 37 ° C, 5% di CO 2. </li><li> Dopo 24 aspirare il terreno da un piatto fibroblasti, e aggiungere 10 ml di terreno fresco delle cellule ES. </li><li> Sostituire regolarmente medio delle cellule ES con doxiciclina supporto contenente per avviare l&#39;espressione dei quattro geni, in altre parole, avviare riprogrammazione. </li><li> Se si utilizza un Oct4 neo-reporter, poi avviare la sezione di droga in qualsiasi momento tra 6 e 9 giorni. </li><li> Cambia il medium ogni giorno fino le colonie sono abbastanza grande per essere prelevati. Colonie dovrebbe prima diventare visibili circa una settimana dopo l&#39;inizio della riprogrammazione. Dovrebbero diventare abbastanza grande da essere ritirato intorno al giorno 20. </li></ol> Fase 3: Raccogliendo le colonie iPS Il giorno prima raccolta: <ol><li> Seed il numero richiesto di gelatina rivestite 24-pozzetti con γ-irradiati DR4 MEF </li></ol> Picking cloni al giorno 1: <ol><li> Avanzamento delle colonie sulle piastre 1-2 ore prima di prendere. </li><li> Aggiungere 15 ml di PBS per i pozzi di un V a fondo piatto da 96 pozzetti. </li><li> Lavare il piatto contenente le colonie di essere raccolti una volta con PBS e aggiungere 10 ml di PBS al piatto. </li><li> Scegli le colonie con le punte piccole pipetta 5μl (impostare il Pipetman P20 il 4 mL). Trasferimento di colonies ad un pozzo nel piatto da 96 pozzetti. </li><li> Aggiungere 20 ml di tripsina in ogni pozzetto utilizzando il multi-canale (12) pipetor. Pipettare su e giù per 3 volte. Incubare a 37 ° C per 4 minuti. </li><li> Aggiungere 100 l di media ES alle colonie trypsinized. Pipettare su e giù per 10 volte. Trasferimento 6 cloni in un momento dal piatto a 96 pozzetti a un 24-ben piatto con una punta in ogni altra posizione sulla pipetor 12 coperti. </li><li> Crescono le celle del 24-pozzetti a 37 ° C, 5% CO 2 incubatore fino a raggiungere le cellule 80-90% confluenza, alimentazione quotidiana con media delle cellule ES. Cellule sarà probabilmente pronto per espandersi in 3-7 giorni. </li></ol> Fase 4 <ol><li> Espansione di cellule iPS <ol><li> Aspirare il mezzo, e lavare le cellule con 1 ml di PBS. </li><li> Rimuovere completamente PBS, aggiungere 0,1 ml di tripsina 0,25% / 1 mM EDTA e incubare a 37 ° C per 10 min. </li><li> Aggiungere 0,4 ml del mezzo ES e sospendere le cellule pipettando su e giù per la sospensione singola cellula. </li><li> Trasferire la sospensione cellulare in un pozzetto della piastra 6-bene, aggiungere 1,5 ml di mezzo di cellule ES, e incubare a 37 ° C, 5% CO 2 incubatore fino a raggiungere le cellule 80-90% confluenza in 6 pozzetti. A questo punto, preparare congelate delle cellule, come segue. </li></ol></li><li> Preparazione del magazzino freeze <ol><li> Aspirare il mezzo, e lavare le cellule con 2 ml di PBS. </li><li> Rimuovere completamente PBS, aggiungere 0,5 ml di tripsina 0,25% / 1 mM EDTA e incubare a 37 ° C per 10 min. </li><li> Aggiungere 2 ml del mezzo ES e sospendere le cellule pipettando su e giù per la sospensione singola cellula. </li><li> Trasferire la sospensione cellulare in un tubo da 15 ml, contare il numero di cellule e spin giù le cellule. </li><li> Eliminare il surnatante, risospendere le cellule ES con il mezzo per la concentrazione a 2x10 6 cellule per ml. </li><li> Preparare 2 celle medie ES congelamento (20% di DMSO in ES media) e aliquota è al 0,5 ml per flaconcino. </li><li> Trasferire 0,5 ml di sospensione cellulare di congelare fiale e mescolare delicatamente. </li><li> Mettere le fiale in una cella-congelamento contenitore e tenerlo a -80 ° C per una notte, trasferimento di azoto liquido il giorno successivo per conservazione a lungo termine. </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 questo video ci dimostra come utilizzare un sistema inducibile lentivirali per generare GFP-positive, le cellule iPS da MEF pluripotenti derivate da topi e Oct4-GFP/R26-M2rtTA Nanog-GFP/R26-M2rtTA. Questa procedura è un metodo utile per generare cellule iPS, perché permette il controllo di espressione del transgene durante il processo di riprogrammazione. Sebbene l&#39;uso di virus nella generazione di cellule iPS impedisce l&#39;uso di queste cellule in un ambiente clinico, questa procedura ci permette di rispondere ad alcune delle grandi questioni biologiche che sottolineano il processo di riprogrammazione ancora definito.

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

Pluripotenza può essere indotto in murini differenziati per trasduzione virale di Oct4, Sox2, Klf4, e c-Myc (Takahashi e Yamanaka, 2006; Wernig, et al, 2007;. Okita, et al, 2007;.. Maherali, et al, 2007). Abbiamo ideato una strategia di riprogrammazione, in cui sono espressi questi quattro fattori di trascrizione da doxiciclina (dox)-inducibile vettori lentivirali (Brambrink et al., 2008). L&#39;utilizzo di questi costrutti inducibili, possiamo derivare staminali pluripotenti indotte (iPS), cellule di fibroblasti embrionali di topo (MEF). In questo video, dimostriamo la procedura per la generazione di lentivirus inducibile che esprimono i quattro fattori di trascrizione e mostrano come infettare MEF con questi virus al fine di produrre cellule iPS. Utilizzando lentivirus inducibile, l&#39;espressione dei quattro fattori controllati con l&#39;aggiunta di doxiciclina al mezzo di coltura. Il vantaggio di questo sistema oltre l&#39;infezione retrovirale tradizionale è la capacità di trasformare i geni e disattivare in modo che la cinetica di riprogrammazione e di espressione genica possono essere analizzati in dettaglio.

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

Questo video mostra la procedura per la generazione di cellule staminali pluripotenti indotte con lentivirus inducibile che esprimono Oct4, Sox2, c-Myc e Klf4.

Generare cellule iPS dal MEF attraverso l&#39;espressione forzata di Sox-2, Oct-4, c-Myc e 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., ...

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36.1K Views.  Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology. Questo video mostra la procedura per la generazione di cellule staminali pluripotenti indotte con lentivirus inducibile che esprimono Oct4, Sox2, c-Myc e Klf4.

Video: 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.

Dal MEF per Matrigel I: hESC Passaging in presenza di 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.

Dal MEF per Matrigel 2: hESC scissione dal MEF sul 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.

Dal MEF per Matrigel 3: hESC Passaging da Matrigel su 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 preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

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

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

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

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

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

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

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

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

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

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

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

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

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

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

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

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.

Alimentatore-Free adattamento, la cultura e Passaging di cellule IPS utilizzando Completa alimentatore-Free siero sostituzione KnockOut Media

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.

Cryopreserving e recupero di cellule umane iPS usando KnockOut Completa alimentatore-Free siero sostituzione Media

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.

Una piattaforma espressione conveniente e generale per la produzione di proteine ​​secrete da cellule umane

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