Damos las gracias a todos los miembros de nuestro laboratorio útil para los debates. Damos las gracias a Joanna Lisztwan y Gewinner Christina para la lectura crítica del manuscrito. Este trabajo fue apoyado por el BBSRC BB/I021248/1 subvención y el Wellcome Trust subvención 090090/Z/09/ZAH es un Wellcome Research Trust Carrera compañero de Desarrollo en el Instituto UCL Cancer.

1. Clonación de pcDNA6_TetR_IRES_blast <ol><li> Como se ilustra en la Figura 3, realizar una digestión parcial del plásmido pcDNA6/TR (V1025-20, Invitrogen) con las enzimas de restricción Xba I y Nco I para eliminar el gen TetR y el promotor de la resistencia a blasticidina (BlastR) de genes. Aislar el fragmento de 4,6 kb del vector. </li><li> Para eliminar la secuencia de poliadenilación del gen TetR, introducir por PCR un sitio Xho I inmediatamente después del codón de parada de la TetR cDNA mientras se conserva el sitio Xba I en el extremo 5 &#39;del ADNc. Subclonar el fragmento resultante en pMigR1 17 usando las enzimas de restricción Xba I y Xho I para generar el plásmido pMigR1_TetR. </li><li> Se digiere el vector pMigR1_TetR con las enzimas de restricción Xba I y Nco I para liberar el fragmento TetR_IRES. Aislar el fragmento de 1,25 kb de inserción. </li><li> Se liga el 4,6 kb pcDNA6/TR y los fragmentos de 1,25 kb TetR_IRES. Transform el producto de ligamiento en E. coli competente e identificar el constructo pcDNA6_TetR_IRES_blast deseada utilizando técnicas estándar de biología molecular. </li></ol> 2. Generación de líneas celulares que expresan establemente TetR <ol><li> En el día 1, Semillas 1x10 6 células por 10-cm placa de cultivo de tejidos utilizando el medio de crecimiento estándar (por ejemplo, DMEM suplementado con 10% FCS). Semilla dos placas, una para la transfección con pcDNA6_TetR_IRES_blast, y el otro como control negativo para la selección blasticidina. Asegúrese de utilizar la menor posible paso y el medio de crecimiento recomendada. </li><li> En el día 2, transfectar una placa con 2 g de pcDNA6_TetR_IRES_blast utilizando Fugene 6 (E2691, Promega) siguiendo las instrucciones del fabricante. No transfectar la segunda placa. </li><li> El día 3, se añade medio de crecimiento estándar que contiene 5 mg / ml blasticidin a ambas placas. Tenga en cuenta que la concentración óptima selección puede variar de una línea celular determinada y podría need a ser determinado de antemano. </li><li> El día 4, dividir las células en 1/5, 1/25, 1/50 y 1/100 utilizando placas de 10 cm y el medio de cultivo estándar que contiene 5 mg / ml de blasticidina. Asegúrese de etiquetar correctamente las placas que contienen las células no transfectadas o transfectadas. Prepare dos planchas por el paso de dilución. </li><li> Compruebe diariamente las células, lo que garantiza que todas las células en las placas no transfectadas murieron en la primera semana. Cambie medio de selección cada 2-3 días. </li><li> Después de 1-2 semanas, las colonias resistentes a blasticidina deben comenzar a aparecer. Seleccione placas que contienen 5 a 50 colonias para asegurar que las colonias individuales se pueden recoger. </li><li> Cuando las colonias han alcanzado un diámetro de aproximadamente 5 mm (o mayor), aislar al menos 12 clones independientes usando cilindros de clonación para recoger colonias individuales. Transferir cada clon en un pocillo separado de una placa de cultivo tisular de 24 pocillos. Cuando confluente, dividir las células de cada pocillo en un solo pocillo de una placa de cultivo tisular de 6 pocillos. </li><li> Siga cl culturaen los medios de selección. Cuando las células confluentes, se separó de cada pocillo en dos pocillos individuales de una placa de cultivo tisular de 6 pocillos. </li><li> Para cada clon a analizar, congelar un pocillo de células confluentes y las células de la cosecha en el bien, para un control posterior por inmunotransferencia. </li><li> Detectar TetR por transferencia Western utilizando el anticuerpo monoclonal de ratón anti-TET02 anticuerpo (MoBiTec GmbH). Recuerde incluir un lisado de la línea celular parental como un control negativo. </li><li> Seleccione 3 clones con la más alta expresión TetR y ampliar cada cultivo clonal. Congelar las existencias de cada clon prometedor tan pronto como sea posible. Finalmente, examine los parámetros de biología molecular y celular de interés mediante la comparación de las líneas celulares de sus padres con los clones que expresan TetR. Seleccione el &quot;mejor&quot; clon con la más alta expresión TetR que no muestra alteraciones en los parámetros de biología molecular y celular de interés. </li></ol> 3. Prueba piloto de PTER y pT-Rex Vectores DEST30 ExpreseING shRNA o ADNc, Respectivamente <ol><li> Generar PTER plásmido con insertos shRNA como se ha descrito 12. Construir pT-Rex DEST30 vector (12,301-016, Invitrogen) que contiene el cDNA de interés utilizando la tecnología de Gateway (Invitrogen). En caso de expresión de ADNc, la adición de una etiqueta con el ADNc más adelante facilita la detección de la proteína exógena expresada. </li><li> Transitoriamente transfectar la línea de blanco celular de sus padres o con PTER pT Rex-DEST30 plásmidos (que manifieste shRNAs o ADNc de interés) utilizando el reactivo de transfección de elección. Para el ensayo de plásmidos de expresión de shRNA, garantizar una alta eficiencia de transfección se puede lograr. </li><li> Cosecha de las células transfectadas y el proceso para la inmunotransferencia utilizando los anticuerpos apropiados, esperando para detectar un agotamiento o sobreexpresión de su gen de interés. </li></ol> 4. Generación de líneas celulares estables con inducible por tetraciclina (Tet-on) Expresión de shRNAs o cDNAs usando PTER o pT-Rex Vectores DEST30 </p&gt; <ol><li> El día 1, semillas 1x10 6 células de la &quot;mejor&quot; TetR que expresan clon por 10 cm de la placa utilizando el medio estándar de crecimiento suplementado con tetraciclina certificado libre de suero (FBS por ejemplo de Invitrogen; 16,000-014). Seed una placa para la transfección con PTER (o pT-Rex DEST30), y una placa como control negativo para selección doble. Asegúrese de utilizar el pase más bajo posible. </li><li> En el día 2, transfectar una placa con 2 g de PTER (o Pt-Rex DEST30) usando Fugene 6. No transfectar la segunda placa. </li><li> El día 3, se añade medio de crecimiento estándar que contiene 5 mg / ml blasticidin y 500 ug / ml de zeocina (o 1 mg / ml de G418). </li><li> El día 4, dividir las células en 1/5, 1/25, 1/50 y 1/100 utilizando placas de 10 cm y el medio de cultivo estándar que contiene 5 mg / ml y blasticidin 500 mg / ml de zeocina (o 1 mg / ml G418). Prepare dos planchas por el paso de dilución. </li><li> Ver las células diariamente, asegurándose de que dentro de la primera semana todas las células en las placas no transfectadas han muerto.Cambiar el medio de crecimiento estándar que contiene blasticidina y zeocina (o G418) cada 3-4 días. </li><li> Después de 2-3 semanas, blasticidin / zeocina (o blasticidin/G418) doble colonias resistentes deberían comenzar a aparecer. Seleccione placas que contienen 5 a 50 colonias a recoger colonias individuales. </li><li> Cuando las colonias han alcanzado un diámetro de aproximadamente 5 mm (o mayor), utilizar cilindros de clonación para recoger colonias individuales. Aislar al menos 24 clones individuales. Transferir cada clon en un pocillo separado de una placa de cultivo tisular de 24 pocillos. </li><li> Clones de cultivo en medio de mantenimiento que contienen concentraciones de blasticidina (5 mg / ml) y zeocina (250 ug / ml) [o G418 (0,5 mg / ml)]. Cuando confluente, dividir las células de cada pocillo en un solo pocillo de una placa de cultivo tisular de 6 pocillos. </li><li> Para cada clon a analizar, dividir un confluente pocillo de una placa de 6-así en tres pocillos individuales de una placa de 6-así. Cuando confluente, congelar las células de un pozo. Utilice los restantes dos pozos para probar la tetraciclinae-inducible de expresión. Añadir tetraciclina a una concentración de 1 mg / ml a un pocillo, mientras que deja el segundo tetraciclina bien libre. </li><li> Después de 24 a 96 horas de incubación con células de tetraciclina cosecha, y el proceso para inmunotransferencia utilizando anticuerpos apropiados. Reducción de los tiempos de incubación de tetraciclina se recomiendan cuando la detección de la inducción de la expresión de cDNA, mientras que los tratamientos más prolongados tetraciclina será necesario determinar shRNA mediada por el agotamiento de las proteínas endógenas. </li><li> Seleccione al menos dos clones con el agotamiento deseado / sobreexpresión y expandir cada cultura. Congelar las existencias de cada clon prometedor tan pronto como sea posible. Al crecer Tet-on clones para experimentos asegúrese siempre de utilizar medios de comunicación complementado con tetraciclina libre de suero y los antibióticos de selección correspondientes a las concentraciones de mantenimiento. </li></ol>

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No hay conflictos de interés declarado.

Creemos que Tet-On sistemas facilitan en gran medida el análisis de la función génica, particularmente en sistemas de células que son difíciles de manipular y / o cuando el gen manipulado es esencial para la supervivencia celular. Además, la capacidad de controlar la expresión de genes altamente específicos por Tet-On sistemas ofrece la oportunidad de estudiar las funciones de genes en diferentes etapas (por ejemplo durante la progresión del ciclo celular o bien definidos los procesos de diferenciación). El m?...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Suero bovino fetal (FBS) </td><td> Invitrogen </td><td> 16000-044 </td><td> Probado Tet-libre </td></tr><tr><td> Blasticidina </td><td> Invivogen </td><td> ant-bl-1 </td><td></td></tr><tr><td> Zeocina </td><td> Invivogen </td><td> ant-zn-5 </td><td></td></tr><tr><td> G418 </td><td> PAA laboratorios </td><td> P31-011 </td><td> 100 mg / ml en los medios de comunicación </td></tr><tr><td> anti-TET02 </td><td> MoBiTec GmbH </td><td> TET02 </td><td> Use a 1/1000 a 1/2000 para WB </td></tr><tr><td> pcDNA6/TR </td><td> Invitrogen </td><td> V1025-20 </td><td></td></tr><tr><td> pT-Rex DEST30 </td><td> Invitrogen </td><td> 12301-016 </td><td></td&gt; </tr><tr><td> La tetraciclina </td><td> Sigma </td><td> 87128 </td><td> 2 mg / ml en etanol </td></tr><tr><td> Doxycycline </td><td> Sigma </td><td> D9891 </td><td> 2 mg / ml en agua </td></tr><tr><td> Cilindros de clonación </td><td> Bellco Glass Inc. </td><td> 2090-00808 </td><td> reutilizable </td></tr></tbody></table>

generation of stable human cell lines with tetracycline-inducible (tet-on) shrna or cdna expression

Un enfoque importante en el campo de la biología de células de mamíferos es la manipulación de la expresión de genes de interés en líneas celulares seleccionadas, con el fin de revelar una o varias de la función del gen (s) mediante la sobreexpresión transitoria / estable o caída del gen de interés. Desafortunadamente, para las investigaciones de células biológicas diferentes este enfoque no es adecuado cuando las manipulaciones del resultado de la expresión génica en células de crecimiento / proliferación de defectos o la diferenciación celular, no deseada. Por lo tanto, los investigadores han adaptado la proteína represora de tetraciclina (TetR), tomada de la E. coli resistencia a la tetraciclina operón 1, para generar sistemas de regulación eficientes y muy estrecho para expresar ADNc en células de mamífero 2,3. En resumen, TetR se ha modificado para cualquiera de iniciación bloque (1) de la transcripción mediante la unión al operador Tet-(A) en la región del promotor tras la adición de tetraciclina (Tet-off denomina sistema) o bind (2) a la A en tél ausencia de tetraciclina (Tet-on denomina sistema) (Figura 1). Teniendo en cuenta el inconveniente de que el sistema Tet-off requiere la presencia continua de tetraciclina (que tiene una vida media de aproximadamente 24 horas en medio de cultivo de tejido de células) el sistema Tet-on ha sido más ampliamente optimizada, lo que resulta en el desarrollo de muy apretado y sistemas eficientes de vector para la expresión de ADNc tal como se usa aquí. Poco después del establecimiento de la interferencia de ARN (RNAi) para knockdown gen en células de mamífero 4, vectores que expresan corto horquilla RNAs (shRNAs) se describe que la función muy similar a siRNAs 5-11. Sin embargo, estos enfoques shRNA mediada desmontables tienen la misma limitación como estrategias knockout convencionales, ya que el agotamiento estable no es factible cuando los objetivos de genes son esenciales para la supervivencia celular. Para superar esta limitación, van de Wetering et al. 12 modificó la expresión shRNA vector pSUPER 5 </sup&gt; mediante la inserción de una A en la región promotora, lo que les permitió generar líneas celulares estables con depleción inducible por tetraciclina de sus genes diana de interés. Aquí se describe un método para generar eficientemente humano estable Tet-en líneas celulares que conducen fiable sobreexpresión inducible o el agotamiento del gen de interés. Usando este método, se han generado con éxito Tet-en líneas celulares que facilita considerablemente el análisis de la cascada de MST / hMOB / NDR en centrosoma 13,14 y 15,16 apoptosis de señalización. En este reporte se describe nuestros vectores de elección, además de describir los dos pasos consecutivos de manipulación que son necesarios para generar eficientemente humano Tet-en líneas celulares (Figura 2). Por otra parte, además de plantear un protocolo para la generación de recursos humanos Tet-en líneas celulares, vamos a discutir los aspectos críticos relacionados con los procedimientos técnicos y la caracterización de Tet-en las células.

Un ejemplo para la caracterización inicial de RPE-1 líneas celulares que expresan establemente TetR se muestra en la Figura 4. Tenga en cuenta que todos los clones de RPE-1 expresan niveles variables de TetR (compárense las calles 2 y 5), mientras que la línea celular parental (que sirve como control negativo) no expresa la proteína TetR exógeno (Figura 4, carril 1). Esta variación en la expresión entre TetR RPE-1 Tet-on clones de células que se espera, puesto que la expresión...

Watch this Scientific Journal Video about Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression at JoVE.com

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

Una manera rápida y sencilla para generar líneas celulares humanas con sobreexpresión inducible y reversible de ADNc o shRNA mediada derribar-del gen de interés. Este método permite a los investigadores a las líneas celulares altamente fiable y reproducible manipular que son difíciles de modificar por métodos de transfección transitoria o estrategias convencionales desmontables / knockout.

Generación de líneas celulares estables Humanos (Tet-on) inducible por tetraciclina o shRNA expresión cDNA

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

generation-stable-human-cell-lines-with-tetracycline-inducible-tet-on

Research

JoVE Journal

Biology

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

35.0K vistas.  UCL Cancer Institute. Una manera rápida y sencilla para generar líneas celulares humanas con sobreexpresión inducible y reversible de ADNc o shRNA mediada derribar-del gen de interés. Este método permite a los investigadores a las líneas celulares altamente fiable y reproducible manipular que son difíciles de modificar por métodos de transfección transitoria o estrategias convencionales desmontables / knockout.

Video: Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

HUES pases madre de embriones humanos líneas celulares con tripsina

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Investigación

Biología

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Generación de transgénicos estable C. elegans con microinyección

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

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 ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Generación de humanos CD40 activado por células B

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Aislamiento del ARN de síntesis embrionarias pez cebra y el cDNA de análisis de expresión génica

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

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 ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Transducción de las células humanas con polímeros complejos Lentivirus ecotropic de Mayor Seguridad de la Biotecnología

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

Preparación de líneas celulares de Derribo condicional de la expresión génica y la medición de los efectos desmontables en la muerte celular inducida por E4orf4