Passo 1: Descongelar 293 células e placa para a produção de lentivírus. <ol><li> Prepare 9 ml de DMEM + em um tubo de 15 ml. </li><li> Remover um frasco de congelados 293 ações do tanque de nitrogênio líquido e colocar o frasco em banho-maria a 37 ° C até que a maioria das células (mas não todos) são descongeladas. </li><li> Remove as células de congelamento frasco e colocar no tubo de 15ml a partir do passo 1. </li><li> Centrifugar a 180g por 5 min, em seguida, descartar o sobrenadante. </li><li> Ressuspender as células com 10 ml de DMEM +, e transferir para um prato de gelatina revestidos de 100 mm. Incubar as células a 37 ° C, 5% incubadora de CO 2 até que sejam confluentes 80-90%. </li><li> Passagem dos 293 células: células de lavagem com PBS, aspirar o PBS, e adicionar 4 ml por 10 cm de prato de 0,05% trypsin/0.53 mM EDTA, e incubar por 1 min a 37 ° C. </li><li> Separar células de pratos tocando, ressuspender com 10 ml DMEM + médio, e transferir para um tubo de 15 ml. Centrifugar a 180g por 5 min, e aspirar o sobrenadante. </li><li> Adicionar volume apropriado de DMEM + meio, e acabar com as células em uma suspensão única célula por pipetagem cima e para baixo várias vezes. </li><li> Contar o número de células e ajustar a concentração para 8x10 5 células por ml com meio de FP. </li><li> Células de semente de 8x10 6 células (10 ml) por 100 mm de prato, cultura e incubar durante a 37 ° C, 5% de CO 2. </li></ol> Passo 2: Transfectar 293 células com plasmídeos lentivírus eo vírus da colheita <ol><li> Para cada placa de 10 cm, usar 10 mg de FUW-Teto-cDNA (Oct4, Sox2, KLF4 ou c-Myc) backbone lentivírus com 2.5ug de PMD-G e 7,5 mcg de pPax2. </li><li> Enquanto as três alíquotas plasmídeos em um tubo, entregar 30μl de Fugene reagente transfecção 6 em um segundo tubo contendo 500μl de DMEM sem soro e misture delicadamente com o dedo tocando e incubar por 5 min à temperatura ambiente. </li><li> Adicione a mistura de DNA gota a gota dentro do tubo Fugene 6/DMEM-containing, misture delicadamente tocando dedo e incubar por 20 min. </li><li> Adicione o DNA / Fugene 6 gotas complexo no prato de 293, e incubar durante a 37 ° C, 5% CO 2. </li><li> Na manhã seguinte: Aspirar a transfecção reagentes contendo meio, adicionar 5 ml de meio fresco ES célula, e as células voltem para a incubadora. </li><li> Às 48 horas e 72 horas após a transfecção, recolher o meio do 293, usando um de 10 ml em seringa descartável estéril, filtrando-a através de um poro filtro de tamanho 0,45 mícrons acetato de celulose, e transferir para um tubo de 15 ml. </li><li> Concentrar o sobrenadante vírus por ultracentrifugação. Ressuspender o sedimento vírus no volume desejado e fazer amixture de partes iguais do meio contendo Oct-3/4-, Sox2, KLF4 e c-Myc-lentivírus. </li><li> Ao fazer o vírus, prepare Oct4-neo/rtTA ou Oct4-GFP/rtTA MEFs (passagem &lt;3) a 90% na confluência de 10 cm pratos (2x10 6 células por prato) </li><li> Aspirar o meio de cultura e lavar com 10 ml de PBS. </li><li> Descartar PBS, adicionar 1 ml para cada prato de 0,05% trypsin/0.53 mM EDTA, e incubar a 37 ° C por 10 min. </li><li> Adicionar 9 ml do meio de cultura, suspender as células de uma única célula, e transferir para um tubo de 15 ml. </li><li> Contar o número de células, e ajustar a concentração para 8x10 4 células por ml. Transferência de 10 ml de suspensão de células (8x10 5 células) para um prato de 10 cm revestidas com gelatina. Incubar o prato durante a noite a 37 ° C CO, 5% 2. </li><li> Aspirar o meio de um prato de fibroblastos, e adicione 10 ml do meio-vírus contém. Incubar as células a partir de 4 h durante a noite a 37 ° C, 5% CO 2. </li><li> Após 24 aspirar o médio de um prato de fibroblastos, e adicione 10 ml de meio fresco ES celular. </li><li> Substituir médio de células ES regulares com meio contendo doxiciclina para iniciar a expressão dos quatro genes, ou seja, iniciar a reprogramação. </li><li> Se estiver usando um repórter Oct4-neo, em seguida, iniciar a seção de drogas a qualquer momento entre 6 e 9 dias. </li><li> Mudar o meio todos os dias até as colônias tornam-se grandes o suficiente para ser pego. Colônias deve primeiro tornar-se visíveis cerca de uma semana após o início da reprogramação. Eles devem tornar-se grande o suficiente para ser pego por volta do dia 20. </li></ol> Passo 3: Pegando as colônias iPS Dia antes de escolher: <ol><li> Semente do número necessário de gelatina revestido placas de 24 poços com γ-irradiados DR4 MEFs </li></ol> Escolher Clones no dia 1: <ol><li> Alimentam as colônias nas placas de 1-2 horas antes de escolher. </li><li> Adicionar 15 mL de PBS aos poços de uma antena V de fundo de 96 poços. </li><li> Lavar a cápsula contendo as colônias para ser escolhido uma vez com PBS e adicione 10 ml de PBS para o prato. </li><li> Escolha as colônias com as pequenas pontas de pipeta 5μl (definir o Pipetman P20 em 4 mL). Transferência de colonies a um poço no prato de 96 poços. </li><li> Adicionar 20 mL de tripsina a cada poço usando o multi-canal (12) pipetor. Pipeta cima e para baixo 3 vezes. Incubar a 37 ° C por 4 minutos. </li><li> Adicionar 100 l de meio de ES para as colônias tripsinizados. Pipeta cima e para baixo 10 vezes. Transferência de seis clones de uma vez do prato de 96 poços para um prato de 24 poços, utilizando uma ponta em todas as outras posições no pipetor lugar 12. </li><li> Cultivar as células na placa de 24 poços a 37 ° C, 5% incubadora de CO 2 até alcançar as células confluência de 80-90%, a alimentação diária com ES meio celular. Células provavelmente irá estar pronto para expandir-se em 3-7 dias. </li></ol> Passo 4 <ol><li> Expansão de células iPS <ol><li> Aspirar o meio, e lavar as células com 1 ml de PBS. </li><li> Remover completamente PBS, adicionar 0,1 ml de 0,25% de tripsina / EDTA 1 mM e incubar a 37 ° C por 10 min. </li><li> Adicionar 0,4 ml do meio de ES e suspender as células mediante pipetando para cima e para baixo para suspensão única célula. </li><li> Transferir a suspensão de célula para um poço de placa 6-bem, adicionar 1,5 ml de meio de células ES, e incubar a 37 ° C, 5% incubadora de CO 2 até atingir 80-90% de células confluência em 6-lamelas. Neste ponto, preparar estoque congelado das células, como se segue. </li></ol></li><li> Preparação de ações congelar <ol><li> Aspirar o meio, e lavar as células com 2 ml de PBS. </li><li> Remover completamente PBS, adicionar 0,5 ml de 0,25% de tripsina / EDTA 1 mM e incubar a 37 ° C por 10 min. </li><li> Adicionar 2 ml do meio de ES e suspender as células mediante pipetando para cima e para baixo para suspensão única célula. </li><li> Transferir a suspensão celular a um tubo de 15 ml, contar o número de células e spin down das células. </li><li> Desprezar o sobrenadante, ressuspender as células com meio ES para a concentração em 2x10 6 células por ml. </li><li> Prepare 2 ES meio de congelamento de células (DMSO 20% em média ES) e alíquota é de 0,5 ml por frasco. </li><li> Transferência de 0,5 ml da suspensão de células para congelar os frascos e misture delicadamente. </li><li> Colocar os frascos em um recipiente de células de congelamento e mantê-lo a -80 ° C durante a noite; transferência para nitrogênio líquido no dia seguinte para armazenamento de longo prazo. </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>

Neste vídeo, que demonstram como usar um sistema induzível, lentiviral para gerar GFP-positivas, células pluripotentes a partir de iPS MEFs derivadas de camundongos Oct4-GFP/R26-M2rtTA e Nanog-GFP/R26-M2rtTA. Este procedimento é um método útil para gerar células iPS, pois permite o controle de expressão do transgene durante o processo de reprogramação. Embora o uso de vírus na geração de células iPS impede o uso dessas células em um ambiente clínico, este procedimento nos permite responder a algumas das grandes questões biológicas que sublinhar o processo ainda definido reprogramação.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Doxycycline hyclate</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>D9891-100G</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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

Pluripotência pode ser induzida em murinos diferenciados por transdução viral de Oct4, Sox2, KLF4, e c-Myc (Takahashi e Yamanaka, 2006; Wernig, et al, 2007;. Okita, et al, 2007;.. Maherali, et al, 2007). Nós planejamos uma estratégia de reprogramação em que estes quatro fatores de transcrição são expressos de doxiciclina (Dox)-induzível lentivirais (Brambrink et al., 2008). Usando essas construções induzível, podemos derivar-tronco pluripotentes induzidas (iPS) células de fibroblastos de rato embrionárias (MEFs). Neste vídeo, demonstramos o procedimento para a geração de lentivírus induzível que expressam os quatro fatores de transcrição e mostrar como a infectar MEFs com estes vírus, a fim de produzir células iPS. Usando lentivírus induzível, a expressão dos quatro fatores na controlada pela adição de doxicilina ao meio de cultura. A vantagem deste sistema sobre a infecção retroviral tradicionais é a capacidade de transformar os genes dentro e fora de forma que a cinética de reprogramação e os requisitos de expressão gênica podem ser analisados ​​em detalhe.

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

Este vídeo mostra o procedimento para gerar células-tronco pluripotentes induzidas usando lentivírus induzível que expressam Oct4, Sox2, c-Myc e KLF4.

Geração de iPS a partir de células MEFS através da expressão forçada de 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., ...

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

Research

JoVE Journal

Biology

36.1K visualizações.  Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology. Este vídeo mostra o procedimento para gerar células-tronco pluripotentes induzidas usando lentivírus induzível que expressam Oct4, Sox2, c-Myc e KLF4.

Vídeo: 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 para Matrigel I: hESCs Passaging na Presença de MEFs

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

Biologia

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 MEFs para Matrigel 2: hESCs Divisão de MEFs para 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 MEFs para Matrigel 3: hESCs Passaging de Matrigel em 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.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

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.

Usando o Sistema de Eletroporação Gene Pulser MXcell para transfectar pilhas com Alta Eficiência

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.

Usando um contador de células automatizadas para simplificar Estudos da Expressão Gênica: Knockdown siRNA de Expressão IL-4 Gene Dependente em células 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.

Alimentador livre adaptação, Cultura e Passaging de células iPS humanas usando Completa KnockOut Alimentador-Free substituição Serum Médio

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.

Criopreservação e recuperação das células humanas iPS usando KnockOut Completa Feeder livre de substituição de soro Médio

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.

A Plataforma de expressão adequada e geral para a produção de proteínas secretadas a partir de células humanas

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