Préparation de la cellule 1) <ol><li> Lors de l&#39;utilisation des cellules adhérentes, il est nécessaire de trypsiniser et de recueillir les cellules avant l&#39;électroporation. </li><li> Pour comparer la transfection parmi les quatre fibroblastes embryonnaires de souris (MEF) les cultures, ce qui représente trois chiffres autre passage, effectuez les opérations suivantes sur chaque flacon. </li><li> Aspirer milieux de culture cellulaire. </li><li> Ajouter PBS à laver les cellules. </li><li> Retirer du PBS, ajouter la trypsine suffisant pour couvrir les cellules, et d&#39;attendre quelques minutes pour permettre à la trypsine pour détacher les cellules. </li><li> Vérifiez flacons avec un microscope afin de vérifier l&#39;état des cellules, au beau flacon pour détacher les cellules, puis vérifiez à nouveau flacon pour fabriquer des cellules que sont tous détachés. </li><li> Temps d&#39;attente supplémentaires et répéter si nécessaire. </li><li> Une fois que toutes les cellules sont détachées, ajouter de sérum contenant des médias pour neutraliser la trypsine. </li><li> Transfert des cellules dans un tube de centrifugeuse, et les cellules par centrifugation (RCF = 300 xg). </li><li> Retirer les cellules surnageant et remettre dans un volume connu de PBS. </li><li> Compter les cellules. </li><li> Transfert à un nouveau tube le volume approprié de suspension cellulaire de fournir le nombre requis de cellules pour des expériences (vous aurez besoin de 150 ul de cellules par puits à une densité de 1 x 10 6 cellules / ml). </li><li> Centrifuger les cellules. </li><li> Retirer les cellules surnageant et remettre dans le volume approprié de tampon Gene Pulser électroporation pour atteindre une densité cellulaire de 1 x 10 6 cellules / ml. </li><li> Ajouter 20 pg de plasmide par ml de suspension cellulaire et mélanger doucement. </li></ol> 2) l&#39;installation cuve d&#39;électroporation et l&#39;électroporation Configuration de la plaque et l&#39;électroporation <ol><li> Chambre de la plaque Branchez le module d&#39;alimentation du système d&#39;électroporation Gene Pulser MXcell. </li><li> Pipeter 150 uL mélange de cellules ou de tampon dans le puits d&#39;une plaque de 96 puits électroporation. </li><li> Mettez la plaque dans la chambre de la plaque et le pouls. </li><li> Retirer la plaque de la chambre. </li><li> Mélanger le contenu bien par aspiration et refoulement dans chaque puits. </li><li> Transférer les cellules de chaque puits de pré-chauffée tampon en plaques de 12 puits. </li><li> Tap plaque à distribuer des cellules et le mettre dans l&#39;incubateur. </li><li> Laissez les cellules récupérer pendant 24 heures. </li></ol> Configuration Cuvette et l&#39;électroporation <ol start="9"><li> Débranchez chambre de la plaque du module de puissance du système d&#39;électroporation Gene Pulser MXcell et de brancher le ShockPod ™ cuvette de chambre. </li><li> Pipeter 600 pi de suspension cellulaire dans une cuvette d&#39;électroporation de 0,4 cm fossé. </li><li> Mettez la cuvette dans la chambre ShockPod et livrer impulsion électrique. </li><li> Retirer la cuvette de la chambre. </li><li> Mélanger le contenu cuvette par pipetage de haut en bas dans la cuvette. </li><li> Transférer les cellules de chaque puits de pré-chauffée tampon en plaques de 12 puits. </li><li> Tap plaque à distribuer des cellules et le mettre dans l&#39;incubateur. </li><li> Laissez les cellules récupérer pendant 24 heures. </li></ol> Résultats Représentant 3) Après la transfection de cellules et de leur permettre de récupérer, d&#39;analyser l&#39;efficacité de transfection qualitativement, en utilisant la microscopie à épifluorescence, et quantitativement, en utilisant la cytométrie de flux. <img src="/files/ftp_upload/1662/1662fig1.jpg" alt="Figure 1" /> Cellules Figure 1. Qui ont été avec succès et sont maintenant électroporées exprimant le gène de la GFP en microscopie à épifluorescence apparaissent. <img src="/files/ftp_upload/1662/1662fig2.jpg" alt="Figure 2" /> Figure 2. Affichage des cellules sous contraste de phase permet la visualisation des cellules transfectées et non transfectées fois. Ce sont les cellules qui ont été exposés à l&#39;impulsion de tension au plus bas électroporation 200V. Les cellules confluentes sont largement due à la haute densité cellulaire. <img src="/files/ftp_upload/1662/1662fig3.jpg" alt="Figure 3" /> Figure 3. Le même champ de vue sous épifluorescence montre un certain nombre de cellules exprimant le marqueur sont GFP, mais ils sont seulement un petit pourcentage de cellules visibles dans l&#39;image précédente. <img src="/files/ftp_upload/1662/1662fig4.jpg" alt="Figure 4" /> Figure 4. A 250V, le nombre total de cellules vivantes vu sous contraste de phase diminue légèrement. <img src="/files/ftp_upload/1662/1662fig5.jpg" alt="Figure 5" /> Figure 5. Sous épifluorescence, on peut voir que le nombre de cellules GFP exprimant a augmenté. <img src="/files/ftp_upload/1662/1662fig6.jpg" alt="Figure 6" /> Figure 6. Au plus haut voltage appliqué, 375V, il ya moins de cellules vivantes visibles. <img src="/files/ftp_upload/1662/1662fig7.jpg" alt="Figure 7" /> Figure 7. Toutefois, un pourcentage important des cellules restantes sont exprimant la GFP. Quelle condition est optimale dépend de la conception expérimentale. Dans certaines expériences le plus grand nombre de cellules transfectées pourrait être optimale, dans d&#39;autres expériences de transfection pourcentage le plus élevé pourrait être meilleure. Nous sommes intéressés par le pourcentage de cellules GFP-positives qui sont dans chaque condition et comment les pourcentages varient selon l&#39;âge des cellules. La cytométrie en flux peuvent fournir des informations quantitatives sur les résultats de transfection sous chacune des conditions d&#39;électroporation différents. <img src="/files/ftp_upload/1662/1662fig8.jpg" alt="Figure 8" /> Figure 8. Voici le pourcentage de cellules GFP-positives qui sont dans le passage 5 cellules dans chacune des 12 conditions d&#39;électroporation sont affichés. Le pourcentage de transfection maximale était d&#39;environ 80% sous l&#39;impulsion de la carie tension la plus élevée exponentielle, la condition 6, et 70% sous l&#39;onde de pouls la plus forte testée carrés, l&#39;état 12. <img src="/files/ftp_upload/1662/1662fig9.jpg" alt="Figure 9" /> Figure 9. Avec les cellules passé 9 fois avant l&#39;électroporation, le schéma global de pourcentage de transfection est presque identique, mais avec une très légère diminution dans les pourcentages de transfection. <img src="/files/ftp_upload/1662/1662fig10.jpg" alt="Figure 10" /> Figure 10. Montré ici sont les pourcentages de cellules GFP dans le passage 13 cellules qui montrent une nette diminution en pourcentage par rapport à la transfection des cellules jeunes. Les pourcentages plus élevés de transfection ont été environ la moitié ce qui fut réalisé avec les cellules les plus jeunes démontrant l&#39;importance d&#39;utiliser des cellules saines dès que possible après l&#39;isolement que possible. <table border="1"><tbody><tr><td colspan="3"> Conditions d&#39;électroporation utilisé pour les cellules MEF transfection utilisant le Gene Pulser MXcell </td></tr><tr><td> Condition (1-6) Impulsions de décroissance exponentielle, le tout avec 350 uF et 1000ohm </td><td> Tension (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> Condition (7-12) Impulsions d&#39;ondes carrées, le tout avec 2000 uF, 1000 ohms, et une impulsion </td><td> Tension (V) </td><td> Durée d&#39;impulsion (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>

Les auteurs sont employés par la société Bio-Rad Laboratories, qui produit des réactifs et des instruments utilisés dans cet article

Cet article vidéo montre comment utiliser le système d&#39;électroporation MXcell d&#39;identifier facilement les conditions optimales pour l&#39;électroporation FAE ou d&#39;autres lignées de cellules primaires. Le format de plaque de 96 puits permet de nombreuses répliques de l&#39;optimisation des conditions expérimentales ou à être effectuées simultanément, ce qui peut éliminer le besoin pour de nombreuses expériences distinctes. En effectuant cette procédure, il convient de rappeler d&#39;utiliser des cellules saines dès que possible après l&#39;isolement et à l&#39;utilisation conditions d&#39;électroporation qui sont appariés à la mémoire tampon d&#39;électroporation.

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<td>Gene Pulser MXcell&trade; Electroporation System</td>
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<td>Gene Pulser MXcell&trade; ShockPod&trade; Cuvette Chamber</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

Il est de plus en plus évident que l&#39;électroporation est le moyen le plus efficace pour introduire de l&#39;ADN plasmidique ou siRNA dans des cellules primaires. Le système d&#39;électroporation Gene Pulser MXcell et tampon d&#39;électroporation Gene Pulser (Bio-Rad) ont été spécifiquement développés pour facilement transfecter des acides nucléiques dans les cellules de mammifères et difficiles à transfecter des cellules, telles que des cellules primaires et des souches. Nous allons montrer comment réaliser une expérience simple pour identifier rapidement les meilleures conditions d&#39;électroporation. Nous allons démontrer comment exécuter plusieurs exemples à travers une gamme de conditions d&#39;électroporation de sorte qu&#39;une expérience peut être réalisée en même temps que l&#39;optimisation est effectuée. Nous allons aussi montrer comment les conditions optimales identifiées en utilisant des plaques 96 puits électroporation peut être utilisé avec des cuvettes d&#39;électroporation standard, facilitant le passage de plaques électroporation pour cuvettes d&#39;électroporation tout en conservant l&#39;efficacité d&#39;électroporation mêmes. Dans la vidéo, nous allons aussi discuter de certains des principaux facteurs qui peuvent conduire à la réussite ou l&#39;échec des expériences d&#39;électroporation.

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

Cette procédure montre comment utiliser le système d&#39;électroporation Gene Pulser MXcell rapidement et facilement d&#39;identifier les meilleures conditions d&#39;électroporation pour les fibroblastes embryonnaires de souris (MEF) ou d&#39;autres cellules primaires. Considérations pour le dépannage sont également abordés dans la vidéo associée.

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

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

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23.9K vues.  Bio-Rad Laboratories, Inc.. Cette procédure montre comment utiliser le système d'électroporation Gene Pulser MXcell rapidement et facilement d'identifier les meilleures conditions d'électroporation pour les fibroblastes embryonnaires de souris (MEF) ou d'autres cellules primaires. Considérations pour le dépannage sont également abordés dans la vidéo associée.

Vidéo: 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 préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

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Biologie

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.

En électroporation in vivo et ex utero pour l&#39;expression génique dans les cellules ganglionnaires de la rétine de la souris

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.

Culture primaire et l&#39;électroporation plasmidique de l&#39;Organe de Corti murin.

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.

Aide d&#39;un compteur de cellules automatisé pour simplifier les études d&#39;expression génique: Knockdown siRNA de l&#39;IL-4 dans l&#39;expression des gènes dépendant cellules 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.

Quantification des ADNdb utilisant le Hitachi F-7000 Spectrophotomètre de fluorescence et de PicoGreen Dye

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.

siRNA contrôle pour la ubiquitine et les régulateurs du système ubiquitine-comme des voies biologiques dans des cellules de mammifères en culture

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.

Inverser approche Morpholino génétique utilisant cardiaque ventriculaire injection à Transfecter tissus difficiles à cibles multiples dans le poisson zèbre Larve

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.

Haut différenciation de l&#39;efficacité de pluripotentes humaines des cellules souches à des cardiomyocytes et caractérisation par cytométrie en flux

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.

Transfecter des cellules RAW264.7 avec un gène rapporteur de la luciférase

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.

Haut Analyse de dépistage du contenu pour évaluer les effets toxicologiques des composants nocifs et potentiellement dangereux (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 ...