1) preparación de células <ol><li> Cuando se utilizan las células adherentes, es necesario trypsinize y recoger las células antes de la electroporación. </li><li> Para comparar la transfección de las cuatro de fibroblastos embrionarios de ratón (MEF) los cultivos, lo que representa tres números diferentes paso, haga lo siguiente en cada frasco. </li><li> Aspirar de medios de cultivo celular. </li><li> Añadir PBS para lavar las células. </li><li> Quitar PBS, añadir tripsina suficiente para cubrir las células, y esperar unos minutos para permitir que la tripsina para separar las células. </li><li> Compruebe frascos con un microscopio para verificar el estado de las células, justo frasco para separar las células, y luego verifique frasco de nuevo para hacer que las células que están separadas. </li><li> Esperar más tiempo y repita si es necesario. </li><li> Una vez que todas las células se separan, añadir el suero que contiene los medios para neutralizar la tripsina. </li><li> La transferencia de células a un tubo de centrífuga, y precipitar las células por centrifugación (FCR = 300 xg). </li><li> Eliminar las células sobrenadante y resuspender en un volumen conocido de PBS. </li><li> Recuento de células. </li><li> Transferir a un tubo nuevo el volumen apropiado de la suspensión celular para proporcionar la cantidad necesaria de células para los experimentos (se necesita 150 ml de células por pocillo en una densidad de 1 x 10 6 células / ml). </li><li> Centrifugue las células. </li><li> Eliminar las células sobrenadante y resuspender en la cantidad adecuada de tampón de electroporación Gene generador de impulsos para alcanzar una densidad celular de 1 x 10 6 células / ml. </li><li> Agregar 20 g de plásmido por mL de suspensión celular y mezclar suavemente. </li></ol> 2) La electroporación configuración de buques y la electroporación Placa de instalación y electroporación <ol><li> Conecte la cámara de placas en el módulo de potencia del sistema de electroporación Gene Emisores MXcell. </li><li> Pipetear 150 l mezcla de células o tampón en los pocillos de una placa de electroporación de 96 pozos. </li><li> Poner la placa en la cámara de la placa y el pulso. </li><li> Retire la placa de la cámara. </li><li> Mezclar el contenido y pipeteando arriba y abajo en cada pozo. </li><li> Transferencia de las células de cada pocillo a pre-calentado de búfer en placas de 12 pocillos. </li><li> Toque en la placa para distribuir las células y lo puso en la incubadora. </li><li> Vamos a recuperar las células durante 24 horas. </li></ol> Cubeta de configuración y electroporación <ol start="9"><li> Desconecte la cámara de la placa del módulo de potencia del sistema de electroporación Gene Emisores MXcell y conectar el ShockPod ™ cubeta de la cámara. </li><li> Pipetear 600 l de suspensión celular en una cubeta de electroporación de 0,4 cm vacío. </li><li> Ponga la cubeta en la cámara de ShockPod y entregar pulsos eléctricos. </li><li> Retire la cubeta de la cámara. </li><li> Mezclar el contenido de la cubeta con la pipeta hacia arriba y abajo en la cubeta. </li><li> Transferencia de las células de cada pocillo a pre-calentado de búfer en placas de 12 pocillos. </li><li> Toque en la placa para distribuir las células y lo puso en la incubadora. </li><li> Vamos a recuperar las células durante 24 horas. </li></ol> 3) Resultados de Representante Después de la transfección de células y que les permita recuperar, analizar la eficiencia de transfección cualitativamente, mediante microscopía de epifluorescencia, y cuantitativamente, mediante citometría de flujo. <img src="/files/ftp_upload/1662/1662fig1.jpg" alt="Figura 1" /> Las células Figura 1. Que han sido exitosamente electroporación y ahora que expresan el gen GFP aparecen bajo el microscopio de epifluorescencia. <img src="/files/ftp_upload/1662/1662fig2.jpg" alt="Figura 2" /> Figura 2. Visualización de las células en contraste de fase permite la visualización de las células, tanto transfectadas y transfectadas. Estas son las células que fueron expuestos al pulso de voltaje más bajo de la electroporación en 200V. Las células son en gran parte debido a la confluencia de alta densidad celular. <img src="/files/ftp_upload/1662/1662fig3.jpg" alt="Figura 3" /> Figura 3. El mismo campo de visión bajo epifluorescencia muestra una serie de células que expresan el marcador GFP, pero estos son sólo un pequeño porcentaje de las celdas visibles en la imagen anterior. <img src="/files/ftp_upload/1662/1662fig4.jpg" alt="Figura 4" /> Figura 4. En 250, el número total de células vivas visto en contraste de fase disminuye ligeramente. <img src="/files/ftp_upload/1662/1662fig5.jpg" alt="Figura 5" /> Figura 5. Bajo epifluorescencia, se puede ver que el número de células que expresan GFP se ha incrementado. <img src="/files/ftp_upload/1662/1662fig6.jpg" alt="Figura 6" /> Figura 6. En la mayor tensión aplicada, 375V, hay menos células visibles en vivo. <img src="/files/ftp_upload/1662/1662fig7.jpg" alt="Figura 7" /> Figura 7. Sin embargo, un gran porcentaje de las células restantes se expresan GFP. ¿Qué condición es óptima depende del diseño experimental. En algunos experimentos con el mayor número de células transfectadas ser óptimo, en otros experimentos de la transfección porcentaje más alto puede ser mejor. Estamos interesados ​​en el porcentaje de células que son positivas las buenas prácticas agrarias en cada condición y cómo los porcentajes varían con la edad de la célula. La citometría de flujo puede proporcionar información cuantitativa sobre los resultados de la transfección en cada una de las condiciones de electroporación diferentes. <img src="/files/ftp_upload/1662/1662fig8.jpg" alt="Figura 8" /> Figura 8. Aquí el porcentaje de células que son positivas las buenas prácticas agrarias en el paso 5 células en cada una de las 12 condiciones de electroporación se muestran. El porcentaje máximo de transfección fue aproximadamente del 80% bajo el impulso de la decadencia máxima tensión exponencial, el estado 6, y 70% en el pulso más fuerte onda cuadrada a prueba, el estado 12. <img src="/files/ftp_upload/1662/1662fig9.jpg" alt="Figura 9" /> Figura 9. Con las células pasa nueve horas antes de la electroporación, el patrón general del porcentaje de transfección es casi idéntica, pero con una disminución muy ligera en los porcentajes de transfección. <img src="/files/ftp_upload/1662/1662fig10.jpg" alt="Figura 10" /> Figura 10. Aquí se presentan los porcentajes de células GFP en el pasaje 13 células que muestran una marcada disminución en el porcentaje de transfección en relación con las células más jóvenes. Los mayores porcentajes de transfección fueron aproximadamente la mitad de lo que se logró con las células más jóvenes que demuestran la importancia de utilizar las células sanas tan pronto como sea posible el aislamiento. <table border="1"><tbody><tr><td colspan="3"> Las condiciones de electroporación utiliza para la transfección de células MEF utilizando el Gene Pulser MXcell </td></tr><tr><td> Condición (1-6) Pulsos exponenciales Decay, todos con más de 350 uF, 1000ohm </td><td> (V) </td><td></td></tr><tr><td> 1 </td><td> 200 </td><td></td></tr><tr><td> 2 </td><td> 250 </td><td></td></tr><tr><td> 3 </td><td> 300 </td><td></td></tr><tr><td> 4 </td><td> 326 </td><td></td></tr><tr><td> 5 </td><td> 350 </td><td></td></tr><tr><td> 6 </td><td> 376 </td><td></td></tr><tr><td> Condición (7-12) Plaza de los pulsos de onda, todas ellas con 2.000 uF, 1000 ohm, y el pulso de una </td><td> (V) </td><td> Duración del pulso (ms) </td></tr><tr><td> 7 </td><td> 200 </td><td> 10 </td></tr><tr><td> 8 </td><td> 250 </td><td> 10 </td></tr><tr><td> 9 </td><td> 300 </td><td> 10 </td></tr><tr><td> 10 </td><td> 200 </td><td> 20 </td></tr><tr><td> 11 </td><td> 250 </td><td> 20 </td></tr><tr><td> 12 </td><td> 300 </td><td> 20 </td></tr></tbody></table>

Los autores son empleados de Bio-Rad Laboratories, que produce los reactivos e instrumentos utilizados en este artículo

En este artículo de vídeo se muestra cómo utilizar el sistema de electroporación MXcell para identificar fácilmente las condiciones óptimas para la electroporación MEFs u otras líneas de células primarias. El formato de placa de 96 pocillos permite muchas repeticiones de las condiciones experimentales o de optimización para realizar de forma simultánea, lo cual puede eliminar la necesidad de muchos experimentos separados. Mientras lleva a cabo este procedimiento, hay que acordarse de utilizar las células sanas, tan pronto como sea posible después de su aislamiento y las condiciones de uso de la electroporación que coinciden con el buffer de electroporación.

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		<th>Material Name</th>
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<td>Gene Pulser&reg; Electroporation Buffer</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2676</td>
<td>&nbsp;</td>
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<td>Gene Pulser MXcell&trade; Electroporation System</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2670</td>
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<td>Gene Pulser MXcell&trade; ShockPod&trade; Cuvette Chamber</td>
<td>&nbsp;</td>
<td>Bio-Rad Laboratories, Inc.</td>
<td>165-2673</td>
<td>&nbsp;</td>
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<td>Gene Pulser MXcell&trade; Electroporation System With ShockPod&trade; Cuvette Chamber</td>
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using the gene pulser mxcell electroporation system to transfect primary cells with high efficiency

Cada vez es más evidente que la electroporación es la forma más eficaz para introducir ADN plásmido o siRNA en células primarias. El sistema de electroporación Gene Emisores MXcell y Gene tampón de electroporación de impulsos (Bio-Rad) se han desarrollado específicamente para transfectar con facilidad los ácidos nucleicos en células de mamíferos y difíciles de transfectar células, como células primarias y troncales. Vamos a demostrar cómo llevar a cabo un experimento simple para identificar rápidamente las mejores condiciones de electroporación. Vamos a demostrar cómo ejecutar varias muestras a través de una amplia gama de condiciones de electroporación para que un experimento puede llevarse a cabo al mismo tiempo, como la optimización se lleva a cabo. También vamos a mostrar cómo las condiciones óptimas identificados con placas de 96 pozos de electroporación se puede utilizar con cubetas de electroporación estándar, facilitando el cambio de placas de electroporación de cubetas de electroporación, manteniendo la eficiencia de electroporación mismo. En el video, también vamos a discutir algunos de los factores clave que pueden conducir al éxito o fracaso de los experimentos de electroporación.

Watch this Scientific Journal Video about Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency at JoVE.com

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

Este procedimiento muestra cómo utilizar el sistema de electroporación Gene Emisores MXcell para identificar rápida y fácilmente las mejores condiciones de electroporación de fibroblastos embrionarios de ratón (MEFs) o de otras células primarias. Consideraciones para la solución de problemas también se discuten en el vídeo asociado.

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

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

using-gene-pulser-mxcell-electroporation-system-to-transfect-primary

Research

JoVE Journal

Biology

23.9K vistas.  Bio-Rad Laboratories, Inc.. Este procedimiento muestra cómo utilizar el sistema de electroporación Gene Emisores MXcell para identificar rápida y fácilmente las mejores condiciones de electroporación de fibroblastos embrionarios de ratón (MEFs) o de otras células primarias. Consideraciones para la solución de problemas también se discuten en el vídeo asociado.

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

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

Investigación

Biología

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

La electroporación in vivo en el útero y el ex de la expresión génica en células de ratón ganglionares retinianas

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Cultura primaria y electroporación plásmido del órgano de Corti murino.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

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 ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

La cuantificación de ADN de doble cadena con el Hitachi F-7000 espectrofotómetro de fluorescencia y Dye PicoGreen

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

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 ...

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

Invierta genética Enfoque Morpholino Usando ventricular cardíaca inyección para transfectar múltiples tejidos difíciles de conectar por objetivo en el pez cebra Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

Alta Eficiencia Diferenciación de células madre pluripotentes humanas a cardiomiocitos y Caracterización por citometría de flujo

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

Transfectar células RAW264.7 con un gen informador de luciferasa

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

Análisis alta Content Screening para evaluar los efectos toxicológicos de nocivo Componentes y potencialmente perjudicial (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...