Nos gustaría dar las gracias al Sr. Enver Maaz para el uso de su cámara de alta definición (Cannon VIXIA HFS100). Este trabajo fue apoyado en parte por subvenciones de NIH (EY018738), Nueva Jersey Comisión de Investigación de la Médula Espinal (08-3074-SCR-E-0 y 10-3091-SCR-0-E), y Busch Premio de Investigación Biomédica.

Dado que algunas partes del protocolo se realizó tal como se ha descrito previamente en otros vídeos JOVE 2 y 3 papeles con modificación menor no vamos a discutir aquellos en detalle aquí. 1. Manejo de los huevos y preparación de la aguja <ol><li> Los huevos pueden ser almacenados en un enfriador de vino menos aproximadamente 13 ° C hasta por 1 semana. Si la temperatura es demasiado alta, los embriones comienzan a desarrollarse anormalmente, mientras que una temperatura más baja provoca una alta mortalidad. Una vez que esté listo para incubar sacar los huevos del refrigerador para vinos y establecer los huevos en posición vertical con el extremo más arriba. Mantenga los huevos en la temperatura ambiente durante al menos 2 horas antes de ponerlos en la incubadora de 37,5 ° C. </li><li> Se incuban los huevos para 96-100 horas, que es alrededor del día 4 de embriones para obtener embriones que se encuentran en el Hamburger y Hamilton 22 etapa de 4-5. </li><li> Preparar agujas micropipeta de tubos de vidrio tirados capilar y romper la punta de disección bajo un microalcance con unas pinzas para obtener una apertura de la punta alrededor de 0,1 m de diámetro y un cono de 20 mm. Agujas con puntas más grandes tienen dificultades para atravesar la membrana vitelina mientras que los más consejos tienen dificultades para la carga y la entrega de la solución de ADN. </li><li> Coloque la aguja en un 0,1 ml jeringa Hamilton estanca montado en un micromanipulador. Use un pequeño trozo de tubo de silicona Masterplex para fijar la aguja a la jeringa. Dado que las conexiones estén bien ajustadas sin necesidad de añadir aceite mineral para sellar esta unión. </li><li> Mezclar 2 l de solución de ADN plásmido reportero con una concentración que oscila 3-6 ug / l y 0,2 l de verde rápido (0,025%) en un trozo de Parafilm. Fast tinte verde le ayudará a visualizar la inyección. Poco a poco, la carga de la aguja con la mezcla. </li><li> Para liberar la membrana vitelina de la membrana interna del huevo gire suavemente alrededor de 180 ° y espere algunos minutos y lo gira a la posición original y lo puso para la electroporación. </li><li> Limpie el forzamientoeps y la cáscara de huevo con el 70% de etanol para evitar la infección del embrión. </li><li> Hacer un pequeño agujero en el huevo inmediatamente por encima de la celda de aire con un par de fórceps de tamaño AA. Tenga cuidado de no romper la cáscara del huevo. Retire los trozos pequeños de uno en uno para hacer una pequeña ventana. Retire con cuidado la membrana interna utilizando las pinzas sin tocar la membrana vitelina. </li></ol> 2. Inyección y electroporación <ol><li> Para evitar daños en el cerebro o el corazón, la posición de la contraindicado aguja lateral al paquete principal de los vasos sanguíneos que entran en el ojo y apuntando hacia el pico. </li><li> Pierce a través de la membrana vitelina, la esclera, la retina y el humor vítreo por un súbito empujón suave de la aguja. Si la aguja perfora a través del otro extremo del ojo debe estar bien a menos que daña cualquier vena sanguíneo importante. </li><li> Lentamente tire hacia atrás la aguja en el borde de la abertura y colocarlo casi tangente a la pared externa del globo ocular. </li><li> Insert de la aguja en el espacio sub retiniana entre la esclerótica y la retina. </li><li> Inyectar el ADN hasta que se pueda visualizar la solución de verde de llenado del lado del globo ocular y empujar hacia adentro la retina mediante la creación de un bulto. </li><li> Si su colocación de la aguja no es correcta, entonces podrás ver la solución de ADN propagación en el interior del humor vítreo de llenado por el centro del globo ocular. Además, si se daña la retina demasiado, entonces podrás ver la solución de ADN sale del globo ocular. </li><li> Lentamente retire la aguja y de inmediato colocar los electrodos en paralelo dentro del huevo después de la inmersión en PBS. Empuje hacia abajo los electrodos a sumergirse en el líquido amniótico de una manera para que el ojo inyectado se encuentra entre los electrodos. Evite tocar cualquier vaso sanguíneo principal o el corazón con los electrodos mientras que colocarlas. El electrodo negativo debe estar en el lado de la inyección para que el ADN puede ser transportado desde el espacio sub retinal en la retina hacia el electrodo positivo. Electroporado la retina con 5 pulsos de 15 V para 50 ms, con intervalos de 950 ms. </li><li> Retire con cuidado los electrodos y sellar la ventana del huevo con trozos de cinta adhesiva transparente. </li><li> Etiqueta y la fecha del huevo inyectada antes de volver a la incubadora. Normalmente, se tarda alrededor de 3 a 5 minutos para completar el proceso de electroporación conjunto. </li><li> La expresión de GFP puede ser visto como temprano como 8 horas después de la electroporación. Sin embargo, usted puede esperar hasta que el embrión alcanza el punto deseado antes de la cosecha. </li></ol> 3. Los resultados representativos En nuestro estudio, utilizamos diversas construcciones de plásmidos para estudiar la regulación de la expresión de genes que participan el desarrollo de células de la retina. En este vídeo pCAG-GFP (control de transfección) se utilizó para seguir una inyección exitosa y electroporación. Sin embargo, cualquier constructo plásmido con el gen reportero (GFP, RFP etc) se puede utilizar. A pesar de que la expresión de GFP puede ser visto como a las 8 horas después del elementoctroporation, que suelen comenzar la recolección del huevo en el día 6 (E6) y en adelante. Electroporated retinas fueron disecados del embrión y se analizaron bajo el microscopio de disección fluorescente antes de inclusión y corte. Típicamente, reportero de la expresión génica puede ser visto por lo menos en una cuarta parte de la retina después de una exitosa electroporación (Fig. 1). Los tejidos de la retina transfectadas fueron analizados a través de seccionamiento para una visualización clara de la morfología celular. La inmunohistoquímica con marcadores de células específicas del tipo (Brn3a, Pax6 etc) permite la caracterización de la expresión de GFP específico de las células (Fig. 2). <img src="/files/ftp_upload/3792/3792fig1.jpg" align="Figure 1" /> Figura 1. El éxito de la electroporación de expresión reportero GFP resultados positivos plásmido. Embriones de pollo retinas fueron inyectados y electroporated en el día embrionario 4 y cosechadas en el día embrionario 6. Al menos el 25% de la retina se transfectadas con éxito (Un punto de vista = Arriba, B= Vista desde abajo). Barra de escala = 1 mm. <img src="/files/ftp_upload/3792/3792fig2.jpg" align="Figure 2" /> Figura 2. Caracterización de las buenas prácticas agrarias expresando células de la retina mediante inmunohistoquímica. GFP expresando tejidos retina en la etapa de E7 se fija y se secciona. Estas secciones fueron teñidas con marcadores específicos de células diferentes. Brn3a se utilizó para determinar las células ganglionares (A) mientras Pax6 se utilizó para determinar amacrinas horizontal, y las células ganglionares (B). Barra de escala = 50 micras.

<ol>
	<li>Doh, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+retinal+cell+development+in+chick+embryo+by+immunohistochemistry+and+in+ovo+electroporation+techniques.">Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.</a> BMC. Dev. Biol. 10, (2010).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Ovo+Electroporations+of+HH+Stage+10+Chicken+Embryos.">In Ovo Electroporations of HH Stage 10 Chicken Embryos.</a> J. Vis. Exp. (9), e408-e408 (2007).</li><li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.+1951.">A series of normal stages in the development of the chick embryo. 1951.</a> Dev. Dyn.. 195, 231-272 (1992).</li><li>Hamburger, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stage+series+of+the+chick+embryo.">The stage series of the chick embryo.</a> Dev. Dyn. 195, 273-275 (1992).</li></ol>

Todos los experimentos con animales fueron aprobados por el animal de Atención Institucional y el Comité de Servicios de la Universidad Rutgers.

Dirigida por inyección en la retina y la electroporación in ovo en la retina de embrión en la etapa E4 pueden dirigirse específicamente a las células retinianas progenitoras derivadas de la capacidad de visualizar todos los seis tipos principales de células de retina en el nivel de células individuales. En la electroporación in ovo en HH10 (~ E1.5) centradas en la vesícula óptica es capaz de transfectar células que se desarrollan para formar el ojo. Sin embargo, estas célu...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Fertilizados libres de patógenos (SPF) White Leghorn huevos de gallina </td><td> Granjas de Sunrise (Catskill, Nueva York) </td><td></td><td></td></tr><tr><td> Incubadora de huevo de gallina </td><td> MAC es la fabricación, Savannah, GA </td><td> Modelo-1550 </td><td> Se establece en 60% de humedad y 37.5 ° C. </td></tr><tr><td> Vidrio capilares tubos </td><td> World Precision Instrument Inc. </td><td> TW150F-4 </td><td></td></tr><tr><td> 0,1 ml jeringa </td><td> Hamilton Co. Reno, Nevada </td><td> Estanca entre 1710 </td><td> Alternativamente jeringa 1ml puede ser utilizado como así </td></tr><tr><td> Masterflex de silicona (peróxido) Tubería </td><td> Cole-Parmer </td><td> HV-96400-13 </td&gt; <td> Corte un pedazo pequeño (1 cm) para fijar la aguja de cristal a la jeringa </td></tr><tr><td> Pinzas </td><td> Dumont </td><td> AA </td><td></td></tr><tr><td> Micromanipulador </td><td> World Precision Instrument Inc. </td><td> M3301-M3 </td><td></td></tr><tr><td> El ADN del plásmido </td><td></td><td></td><td> pCAG-GFP fue tomado de la Dra. Connie Cepko </td></tr><tr><td> Fast Green FCF </td><td> Sigma-Aldrich </td><td> F7252 </td><td> Diluir a 0,025% con PBS </td></tr><tr><td> Generador de impulsos </td><td> Aparato de Harvard, MA </td><td> BTX ECM 830 </td><td> Generador de onda cuadrada </td></tr><tr><td> Electrodos </td><td> Aparato de Harvard, MA </td><td> BTX Modelo 514 </td><td> Nuestros electrodos estaban separados 3-5 mm de distancia </td></tr><tr><td> Cámara digital blanco y negro </td><td> Zeiss, Alemania </td><td> Axiocam MRM </td><td></td></tr><tr><td> Microscopio de disección fluorescente </td><td> Leica Microsystems, Alemania </td><td> Leica MZ16FA </td><td></td></tr><tr><td> Microscopio de fluorescencia vertical </td><td> Zeiss, Alemania </td><td> Zeiss Axio Imager A1 </td><td></td></tr></tbody></table>

in ovo electroporation in embryonic chick retina

La retina embrionaria de pollo es una excelente herramienta para estudiar el desarrollo de la retina en los vertebrados superiores. Debido al enorme tamaño y desarrollo externo, es comparativamente muy fácil de manipular la retina de embriones de pollo con ADN recombinante / tecnología de ARN. La electroporación de ADN / ARN construcciones dentro de la retina embrionaria tienen una gran ventaja para estudiar la regulación de genes en la retina células madre / progenitoras durante el desarrollo de la retina. Diferentes tipos de ensayos tales como la prueba del gen, la sobre-expresión del gen, el gen tumbar (shRNA), etc se puede realizar utilizando la técnica de electroporación. Este video muestra la inyección dirigida la retina y en la electroporación in ovo en la retina de embriones de pollo en el Hamburger y Hamilton etapa de 22-23, que es aproximadamente el día embrionario 4 (E4). Aquí nos muestran un rápido y conveniente en la técnica de electroporación in ovo por el cual un plásmido de ADN que expresa la proteína verde fluorescente (GFP) como marcador se entregarán directamente en elde embriones de pollo subretiniano espacio y seguido por pulsos eléctricos para facilitar la absorción de ADN por la retina células madre / progenitoras. El nuevo método de inyección de retina y la electroporación en E4 permite la visualización de todos los tipos de células retinales, incluyendo la neuronas tarde-nacido en 1, que ha sido difícil con el método convencional de inyección y la electroporación en E1.5 2.

Watch this Scientific Journal Video about In Ovo Electroporation in Embryonic Chick Retina at JoVE.com

In Ovo Electroporation in Embryonic Chick Retina

El objetivo general de este video es mostrar cómo llevar a cabo la inyección específica de retina y<em&gt; In ovo</em&gt; Electroporación de ADN / ARN construcciones en la retina de embriones de pollo en el Hamburger y la etapa de Hamilton 22-23, que es aproximadamente el día embrionario 4 (E4). Esta técnica es muy útil para estudiar la expresión génica, la regulación de genes, y el cambio morfológico en el desarrollo de retina de pollo.

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 ...

in-ovo-electroporation-in-embryonic-chick-retina

Research

JoVE Journal

Biology

In Ovo Electroporation in Embryonic Chick Retina

14.3K vistas.  University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School. El objetivo general de este video es mostrar cómo llevar a cabo la inyección específica de retina y In ovo Electroporación de ADN / ARN construcciones en la retina de embriones de pollo en el Hamburger y la etapa de Hamilton 22-23, que es aproximadamente el día embrionario 4 (E4). Esta técnica es muy útil para estudiar la expresión génica, la regulación de genes, y el cambio morfológico en el desarrollo de retina de pollo.

Video: In Ovo Electroporation in Embryonic Chick Retina

In Ovo Electroporations of HH Stage 10 Chicken Embryos

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 molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

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 ...

Investigación

Biología

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Método para la tinción de anticuerpos enteros en el monte del polluelo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Ensayo de inducción neural en el embrión de pollo

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 ...

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 ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

Descifrar Caminos axonal de los grupos definidos genéticamente las neuronas en el tubo neural del polluelo Utilizando In ovo La electroporación

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Vídeo Bioinformática Análisis de Crecimiento Humano Colonia células madre embrionarias

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

Transfección de células individuales en embriones de pollo

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

En electroporación in vivo de Morpholinos en la retina de pez cebra adultos

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...