Paso 1: Descongelar 293 células y la placa para la producción de lentivirus. <ol><li> Prepare 9 ml de medio DMEM + en un tubo de 15 ml. </li><li> Eliminar un vial de 293 acciones congeladas desde el tanque de nitrógeno líquido y poner el frasco en un baño de agua a 37 ° C hasta su mayoría de las células (pero no todos) se descongelan. </li><li> Eliminar las células de la ampolla de congelación y el lugar en el tubo de 15 ml en el paso 1. </li><li> Centrifugar a 180 g por 5 min, y luego descartar el sobrenadante. </li><li> Resuspender las células con 10 ml de medio DMEM +, y la transferencia de una gelatina con cubierta de 100 mm plato. Se incuban las células en un 37 ° C, 5% de CO 2 incubadora hasta que el 80-90% confluente. </li><li> La aprobación de la 293 células: las células de lavado con PBS, aspirar el PBS y añadir 4 ml por cada 10 cm plato de 0,05% trypsin/0.53 mM EDTA y se incuba durante 1 min a 37 ° C. </li><li> Separar las células de los platos a la grabación, resuspender con 10 ml de DMEM + medio, y transferir a un tubo de 15 ml. Centrifugar a 180 g por 5 min, y aspirar el sobrenadante. </li><li> Añadir un volumen adecuado de DMEM + medio, y se rompen las células en una suspensión de células individuales pipeteando arriba y abajo varias veces. </li><li> Cuente el número de células y ajustar la concentración de 8x10 5 células por ml con medio FP. </li><li> Las células de las semillas a 8x10 6 células (10 ml) por placa de cultivo de 100 mm, y se incuba durante la noche a 37 ° C, 5% de CO 2. </li></ol> Paso 2: Transfectar 293 células con plásmidos lentivirus y virus de la cosecha <ol><li> Por cada placa de 10 cm, el uso de 10 mg FUW-tetO-cDNA (Oct4, Sox2, Klf4 y c-Myc), columna vertebral de lentivirus con 2.5ug del PMD-G y 7,5 g de pPax2. </li><li> Mientras que los tres plásmidos alícuotas en un tubo, entregar 30μl de reactivo de transfección Fugene 6 en un segundo tubo que contiene 500μl del DMEM sin suero y mezclar suavemente con el dedo tocando e incubar durante 5 minutos a temperatura ambiente. </li><li> Añadir la mezcla de ADN gota a gota en el tubo Fugene 6/DMEM-containing, mezclar suavemente tocando los dedos y se incuba durante 20 min. </li><li> Añadir el ADN / Fugene 6 gota a gota complejo en el plato de 293, y se incuban durante la noche a 37 ° C, 5% de CO 2. </li><li> A la mañana siguiente: Aspirar el reactivo de transfección medio que contiene, añadir 5 ml de medio fresco de células ES, y volver a las células a la incubadora. </li><li> A las 48 horas y 72 horas después de la transfección, recoge el medio de la 293 mediante el uso de un niño de 10 ml jeringa desechable estéril, filtrándola a través de un filtro de poro de 0,45 micras de tamaño de acetato de celulosa, y la transferencia en un tubo de 15 ml. </li><li> Concentrar el sobrenadante del virus por ultracentrifugación. Resuspender el precipitado virus en el volumen deseado y hacer amixture de partes iguales del soporte de Oct-3/4-, Sox2, Klf4 y c-Myc-lentivirus. </li><li> Al tiempo que el virus, o preparar Oct4-neo/rtTA MEFs Oct4-GFP/rtTA (paso &lt;3) y el 90% de confluencia en los platos de 10 cm (2x10 6 células por placa) </li><li> Aspirar el medio de cultivo y lavar con 10 ml de PBS. </li><li> Deseche PBS, añadir 1 ml por placa de 0,05% trypsin/0.53 mM EDTA y se incuba a 37 ° C durante 10 min. </li><li> Añadir 9 ml del medio de cultivo, suspender las células en una sola célula, y transferir a un tubo de 15 ml. </li><li> Cuente el número de células, y ajustar la concentración de 8x10 4 células por ml. Transferir 10 ml de suspensión celular (8x10 5 células) a un plato de 10 cm recubiertas con gelatina. Incubar el plato de la noche a 37 ° C, 5% de CO 2. </li><li> Aspirar el medio de un plato de fibroblastos, y agregar 10 ml de medio que contiene el virus. Se incuban las células a partir de 4 h de la noche a 37 ° C, 5% de CO 2. </li><li> Después de 24 aspirar el medio de un plato de fibroblastos, y agregar 10 ml de medio fresco de células ES. </li><li> Vuelva a colocar medio normal de células ES con un medio que contiene doxiciclina para iniciar la expresión de los cuatro genes, es decir, iniciar la reprogramación. </li><li> Si se utiliza un reportero Oct4-neo, a continuación, iniciar la sección de drogas en cualquier momento entre 6 y 9 días. </li><li> Cambiar el medio de todos los días hasta que las colonias se lo suficientemente grande como para ser recogidos. Primeras colonias que se hacen visibles aproximadamente una semana después del inicio de la reprogramación. Deben ser lo suficientemente grande como para ser recogidos alrededor del día 20. </li></ol> Paso 3: Recogiendo las colonias iPS El día antes de contestar: <ol><li> Semilla de la cantidad necesaria de gelatina con cubierta 24 y placas con γ-irradiados DR4 MEFs </li></ol> Recogiendo clones en el Día 1: <ol><li> Alimentar a las colonias en las placas de 1-2 horas antes de la cosecha. </li><li> Añadir 15 l de PBS en los pocillos de una placa de fondo en V de 96 pozos. </li><li> Lave el recipiente que contenga las colonias para ser recogidos una vez con PBS y añadir 10 ml de PBS para el plato. </li><li> Elige las colonias con las puntas de pipeta pequeña 5μl (establecer el Pipetman P20, el 4 de l). La transferencia de colonies a un pozo en el plato de 96 pozos. </li><li> Añadir 20 l de tripsina a cada pocillo utilizando el multi-canal (12) pipetor. Pipeta de arriba a abajo 3 veces. Se incuba a 37 ° C durante 4 minutos. </li><li> Añadir 100 ml de medio de ES a las colonias con tripsina. Pipeta de arriba y abajo 10 veces. La transferencia de 6 clones en un momento en el plato de 96 pozos a un plato de 24 pozos con una punta en cualquier otra posición en el pipetor lugar 12. </li><li> Crecer las células en la placa de 24 pocillos en un 37 ° C, 5% de CO 2 incubadora hasta que las células llegar a 80-90% de confluencia, la alimentación diaria con el medio de células ES. Las células probablemente estará lista para expandirse en 3-7 días. </li></ol> Paso 4 <ol><li> Expansión de las células iPS <ol><li> Aspirar el medio, y se lavan las células con 1 ml de PBS. </li><li> Eliminar por completo PBS, añadir 0,1 ml de tripsina 0,25% / 1 mM EDTA y se incuba a 37 ° C durante 10 min. </li><li> Añadir 0,4 ml del medio de ES y suspender las células pipeteando arriba y abajo a la suspensión de células individuales. </li><li> La transferencia de la suspensión celular a un pocillo de placa de 6 pocillos, agregar 1,5 ml de medio de células ES, y se incuba en un 37 ° C, 5% de CO 2 incubadora hasta que las células alcanzan el 80-90% de confluencia en placas de 6 pocillos. En este punto, preparar acciones congeladas de las células, de la siguiente manera. </li></ol></li><li> Preparación de las acciones congelar <ol><li> Aspirar el medio, y se lavan las células con 2 ml de PBS. </li><li> Eliminar por completo PBS, se añaden 0,5 ml de tripsina 0,25% / 1 mM EDTA y se incuba a 37 ° C durante 10 min. </li><li> Añadir 2 ml del medio de ES y suspender las células pipeteando arriba y abajo a la suspensión de células individuales. </li><li> La transferencia de la suspensión de células a un tubo de 15 ml, cuente el número de células y centrifugar las células. </li><li> Descartar el sobrenadante, resuspender las células con medio ES a la concentración en 2x10 6 células por ml. </li><li> Prepare dos células ES medio de congelación (20% DMSO en medio ES) y la alícuota es de 0,5 ml por vial. </li><li> La transferencia de 0,5 ml de la suspensión celular para congelar los viales y mezcle suavemente. </li><li> Ponga los viales en un contenedor de congelación de células y mantener a -80 ° C durante la noche, la transferencia a nitrógeno líquido al día siguiente para almacenamiento a largo plazo. </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>

En este video, que muestra cómo utilizar un inducible, un sistema para generar lentiviral GFP-positivo, las células iPS pluripotentes a partir de MEFs derivados de ratones Oct4-GFP/R26-M2rtTA y Nanog-GFP/R26-M2rtTA. Este procedimiento es un método útil para la generación de células iPS, ya que permite el control de la expresión del transgen durante el proceso de reprogramación. Aunque el uso de virus en la generación de células iPS impide el uso de estas células en un entorno clínico, este procedimiento nos permite responder a algunas de las principales cuestiones biológicas que subrayan el proceso de reprogramación aún no definida.

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

Pluripotencia puede ser inducida en murino diferenciados por la transducción viral de Oct4, Sox2, Klf4 y c-Myc (Takahashi y Yamanaka, 2006; Wernig, et al, 2007;. Okita, et al, 2007;.. Maherali, et al, 2007). Hemos diseñado una estrategia de reprogramación en el que estos cuatro factores de transcripción se expresan de doxiciclina (Dox)-inducible vectores lentivirus (Brambrink et al., 2008). El uso de estas construcciones inducible, se puede derivar células madre pluripotentes inducidas (iPS) a partir de células de fibroblastos embrionarios de ratón (MEFs). En este vídeo, que muestran el procedimiento para la generación de los lentivirus inducible que expresan los cuatro factores de transcripción y muestran cómo infectar MEFs con estos virus con el fin de producir células iPS. Mediante el uso de los lentivirus inducible, la expresión de los cuatro factores en el control de la adición de doxiciclina al medio de cultivo. La ventaja de este sistema a través de la infección retroviral tradicional es la capacidad de convertir los genes dentro y fuera de lo que la cinética de la reprogramación y las necesidades de expresión de genes puede ser analizado en detalle.

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 video muestra el procedimiento para generar células madre pluripotentes inducidas mediante lentivirus inducible que expresan Oct4, Sox2, c-Myc y Klf4.

La generación de células iPS a partir de MEFs través de la expresión forzada de Sox-2, Oct-4, c-Myc y 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

36.2K vistas.  Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology. Este video muestra el procedimiento para generar células madre pluripotentes inducidas mediante lentivirus inducible que expresan Oct4, Sox2, c-Myc y 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.

Desde que me MEFs Matrigel: hESCs pases en la presencia de MEFs

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

Investigación

Biología

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.

Desde MEFs a Matrigel 2: La división de hESCs MEFs en 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.

Desde MEFs a Matrigel 3: hESCs pases de Matrigel en 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 preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

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.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

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.

Utilizando el sistema de electroporación Gene Emisores MXcell para transfectar células primarias con alta eficiencia

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.

El uso de un contador de células automatizado para simplificar estudios de expresión génica: siRNA Derribo de IL-4 la expresión génica en células dependientes 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 ...

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.

Criopreservación y recuperación de células iPS humanas con anulación completa del alimentador de sustitución sin suero medio

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 ...

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 expresión adecuada y el general de la Plataforma para la producción de proteínas secretadas por las células humanas

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

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Screening siRNA para identificar ubiquitina y reguladores del sistema ubiquitina-como de las vías biológicas en células de mamífero en cultivo

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...