Manejo de huevo <ol><li> Los huevos pueden mantenerse a 13 ° C durante hasta una semana antes de la incubación sin pérdida significativa de la viabilidad (un enfriador de vino pequeño es ideal para este propósito). </li><li> Antes de la incubación, los huevos permiten calentar a la temperatura ambiente, luego coloque el pollo en una incubadora humidificada conjunto a 37,8 ° C (100 ° F). </li><li> Por lo general, electroporar embriones de aproximadamente 48 horas desde el inicio de la incubación, cuando el embrión ha llegado a Hamburger y Hamilton etapa 10. </li><li> Tiempo de incubación puede variar de acuerdo a hamburguesa deseada y la etapa Hamilton. La temperatura es crítica y la desviación de la temperatura de incubación óptimo va a alterar el tiempo de incubación y la disminución de la viabilidad del embrión. </li><li> Durante la incubación, los huevos deben mantenerse en sus lados para que el embrión debe estar correctamente posicionado para la electroporación. El embrión flotar en la superficie de la yema y es útil para marcar la parte superior del huevo con un lápiz antes de ventanas para que usted sepa dónde cortar. </li></ol> Preparación <ol><li> Caliente Leibovitz L-15 medios de comunicación a 37 ° C. Tire de capilares de vidrio en las agujas. Electrodos posición y micromanipulador y conectar los electrodos al generador de pulsos. </li></ol> Ventanas <ol><li> Utilizando una jeringa con aguja de gran calibre, perforar la cáscara en el extremo más pequeño del huevo. </li><li> Apuntando la aguja hacia abajo con cuidado para evitar la interrupción de la yema de huevo, remover unos 5 ml de albúmina. </li><li> Selle el orificio con un pequeño trozo de cinta. </li><li> Cubra la parte superior del huevo con otro pedazo de cinta adhesiva (de 4x4 cm). </li><li> Con tijeras curvas, y teniendo cuidado de no perturbar el embrión, un agujero lo suficientemente grande como para proporcionar una ventana en la que trabajo. </li></ol> Opcional: Para visualizar el tubo neural, inyectar la solución de tinta con aguja de calibre 26 debajo del embrión (insertar la aguja en el embrión desde el exterior del anillo de la sangre). Tinta china se deben diluir 1:5 con Hanks o comparable solución estéril de buffer. Inyección y electroporación <ol><li> Romper la punta capilar de diámetro deseado con unas pinzas </li><li> Use la pipeta de boca de carga del capilar con un tinte verde plásmido rápido /. </li><li> La posición del embrión con la cabeza hacia usted e inserte la aguja en el tubo neural en un ángulo bajo. </li><li> Se inyecta la solución del plásmido en el lumen del tubo neural hasta el medio de contraste llena todo el espacio. </li></ol> Nota: Tenga cuidado de no tener la punta más grande que el diámetro del tubo neural. En general, usted será capaz de saber si el tamaño de la punta óptima se ha conseguido por lo fácil o difícil que es para sacar el líquido en el capilar. Si hay una resistencia extrema, la apertura es probablemente demasiado pequeño y la punta debe ser roto de nuevo más arriba. Si hay poca o ninguna resistencia, la apertura es demasiado grande y una aguja nueva debe ser utilizado. <ol><li> Coloque una a dos gotas de agua estéril 15 L-medios de comunicación sobre el embrión. </li><li> Inmediatamente colocar los electrodos a ambos lados del embrión, paralela a la del tubo neural, y los tiempos de pulso de 5 a 10 a 24 voltios durante 50 milisegundos a intervalos de 1 segundo. Verá burbujas cerca de los electrodos si ha funcionado correctamente. </li><li> Retire con cuidado los electrodos, colocar 5 gotas de L-15 en la parte superior de los embriones y sellar el huevo con cinta adhesiva. Asegúrese de que el huevo esté bien cerrado, como el secado es un importante contribuyente a la pérdida de embriones después de la electroporación. </li><li> Coloque los huevos de nuevo en la incubadora, la ventana lateral, y se incuban hasta que se alcance deseado H &amp; H etapa. </li></ol>

<ol>
	<li>Itasaki, N., Bel-Vialar, S., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&apos;Shocking&apos;+developments+in+chick+embryology:+electroporation+and+in+ovo+gene+expression.">&apos;Shocking&apos; developments in chick embryology: electroporation and in ovo gene expression.</a> Nat Cell Biol. 1, 203-207 (1999).</li></ol>

The authors have nothing to disclose.

Este protocolo proporciona una guía paso a paso a la electroporación del tubo neural de embriones de pollo HH10. Además de mostrar la técnica, las directrices para el almacenamiento y manipulación de los huevos también se proporciona. Esta técnica tiene una amplia gama de aplicaciones para el análisis genético y la facilidad y bajo costo de los experimentos los hace factible para muchos laboratorios.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Chicken egg incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>humidified, set to 37.80&deg;C (100F). </td>
</tr>
<tr>
<td>Pulse generator </td>
<td>&nbsp;</td>
<td>Genetronics </td>
<td>BTX T820 </td>
<td>Square wave generator or comparable</td>
</tr>
<tr>
<td>Electrodes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>our electrodes were made from 0.25 mm diameter platinum wire and were 5 mm long and spaced 1 mm apart</td>
</tr>
<tr>
<td>Connecter cables</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting scope </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>with large working distance</td>
</tr>
<tr>
<td>Glass capillaries </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>and capillary puller or commercial glass needles</td>
</tr>
<tr>
<td>Leibovitz&#x2019;s L-15 media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>chicken eggs</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>fertilized</td>
</tr>
<tr>
<td>Curved scissors</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 ml syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Large bore needle </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>16-18G</td>
</tr>
<tr>
<td>Mouth pipette</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>India ink </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringe/syringe </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plasmid DNA </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&#x2265; 1&#181;g/&#181;l in sterile TE or 1X PBS containing 0.05% Fast Green dye</td>
</tr>		</tbody>
		</table>
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in ovo electroporations of hh stage 10 chicken embryos

Gran tamaño y externa para el desarrollo del embrión de pollo desde hace mucho tiempo convertido en una herramienta valiosa en el estudio de la biología del desarrollo. Con el advenimiento de técnicas de biología molecular, la chica se ha convertido en un sistema útil para estudiar la regulación de genes y su función. Por electroporating construcciones de ADN o ARN en el embrión de pollo en desarrollo, los genes pueden expresarse o derribado con el fin de analizar la función génica in vivo. Del mismo modo, reportero construcciones se pueden utilizar para la asignación de destino o para examinar los elementos de regulación de genes putativos. En comparación con experiencias similares en el ratón, la electroporación de pollo tiene la ventaja de ser rápido, fácil y barato. Este video muestra primero cómo hacer que una ventana en la cáscara del huevo para manipular el embrión. Luego, el embrión se visualiza con una solución diluida de tinta china inyectada por debajo del embrión. Una aguja de cristal y pipetas se utilizan para inyectar el ADN y el tinte verde rápido en el tubo neural en desarrollo, a continuación, los electrodos de platino se colocan en paralelo al embrión y cortos impulsos eléctricos se administran con un generador de pulsos. Por último, el huevo está sellada con cinta adhesiva y se coloca de nuevo en una incubadora para un mayor desarrollo. Además, el video muestra el almacenamiento de huevos y manejo adecuado y se analizan las posibles causas de la electroporación de embriones después de la pérdida.

Watch this Scientific Journal Video about In Ovo Electroporations of HH Stage 10 Chicken Embryos at JoVE.com

In Ovo Electroporations of HH Stage 10 Chicken Embryos

De pollo en la electroporación in ovo es una técnica que permite la manipulación genética del embrión aviar. Las aplicaciones más comunes de esta técnica incluyen el análisis funcional de los genes y la posible potenciador de los elementos. Este video muestra la electroporación del tubo neural en embriones de pollo HH 10. Técnica de inyección y la adecuada manipulación de los huevos se discuten.

En Electroporations Ovo de embriones de pollo HH Etapa 10

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

in-ovo-electroporations-of-hh-stage-10-chicken-embryos

Research

JoVE Journal

Biology

18.3K vistas.  University of Chicago. De pollo en la electroporación in ovo es una técnica que permite la manipulación genética del embrión aviar. Las aplicaciones más comunes de esta técnica incluyen el análisis funcional de los genes y la posible potenciador de los elementos. Este video muestra la electroporación del tubo neural en embriones de pollo HH 10. Técnica de inyección y la adecuada manipulación de los huevos se discuten.

Video: In Ovo Electroporations of HH Stage 10 Chicken Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Preparación de cultivos neuronales a partir de embriones de Drosophila Midgastrula Etapa

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Investigación

Biología

Windowing Chicken Eggs for Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

Los huevos de ventanas de pollo para Estudios del Desarrollo

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

La colocación de crecimiento recubierto Factor-bolas en los embriones tempranos de la etapa de pollo

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Pollo Ex ovo Cultura y Ex ovo CAM Ensayo: cómo funciona realmente

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparación de embriones para Microscopía Electrónica de la Drosophila Tubo cardíaco embrionario

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Un Ex-ovo</emPollo> Embrión de cultivo apropiado para aplicaciones de imagen y sistema de Microcirugía

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Celular imagen Cambiar forma de vida Drosophila Los embriones

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Ensayo de inmunoprecipitación de la cromatina de los genes específicos de tejido utilizando etapa temprana de embriones de ratón

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

En electroporación in ovo en la retina de embriones de pollo

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

Transferencia de genes en embriones de pollo por los mayores<em&gt; Ex ovo</em&gt; La electroporación

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

En Electroporations Ovo de embriones de pollo HH Etapa 10

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