Wir danken Herrn Maaz Enver für die Nutzung seiner HD-Kamera (Cannon VIXIA HFS100) zu danken. Diese Arbeit wurde zum Teil durch Zuschüsse von NIH (EY018738), New Jersey Kommission über die Spinal Cord Research (08 bis 3074-SCR-E-0 und 10-3091-SCR-E-0), und Busch Biomedical Research Award unterstützt.

Da einige Teile des Protokolls durchgeführt werden als zuvor in anderen JoVE Videos 2 und 3 Papiere mit geringen Modifikationen beschrieben werden wir nicht zu diskutieren, die hier im Detail. 1. Egg Handhabung und Zubereitung Needle <ol><li> Eier können in einem Wein gelagert-Kühler werden bei etwa 13 ° C für bis zu 1 Gespräch. Wenn die Temperatur zu hoch ist, werden Embryonen anfangen, abnormal entwickeln, während niedrigere Temperatur bewirkt eine hohe Mortalität. Sobald Sie bereit sind zu bebrüten nehmen Sie die Eier aus dem Weinkühler und legen die Eier senkrecht mit dem größeren Ende auf. Halten Sie die Eier in der Raumtemperatur für mindestens 2 Stunden, bevor sie sie in der 37,5 ° C Inkubator. </li><li> Bebrüten die Eier für 96 bis 100 Stunden, die über embryonalen Tag 4 ist zu Embryonen, die in der Hamburger und Hamilton Stufe 22 sind 5.4 zu erhalten. </li><li> Bereiten Mikropipette Nadeln aus gezogen Glaskapillarrohre und brechen Sie die Spitze unter einem Präpariermikroskop MikroUmfang mit einer Pinzette, um eine Spitze Eröffnung etwa 0,1 &amp;mgr; m im Durchmesser und 20 mm Aufnahme zu bekommen. Nadeln mit größeren Schwierigkeiten haben, Tipps Durchstechen der Dotterhaut, während kleinere Spitzen Schwierigkeiten haben, das Be-und Bereitstellung der DNA-Lösung. </li><li> Befestigen Sie die Nadel auf eine 0,1 ml Hamilton gasdichten Spritze auf einem Mikromanipulator montiert. Verwenden Sie ein kleines Stück Masterplex Silikonschlauch zur Befestigung der Nadel auf die Spritze. Da die Verbindungen dicht sind nicht nötig, Mineralöl hinzufügen, um diese Bindung zu versiegeln. </li><li> Mischen Sie 2 ul-Reporter-Plasmid-DNA-Lösung mit einer Konzentration im Bereich 3-6 ug / ul und 0,2 ul schnell grün (0,025%) auf einem Stück Parafilm. Schnelle grünen Farbstoff wird helfen, die Injektion zu visualisieren. Langsam, laden Sie die Nadel mit der Mischung. </li><li> Um die Dotterhaut von der inneren Membran frei drehen Sie das Ei vorsichtig um 180 ° und warten Sie einige Minuten und drehen Sie ihn zurück in die ursprüngliche Position und stellen Sie ihn für die Elektroporation. </li><li> Wischen Sie die FORCeps und Eierschale mit 70% Ethanol auf eine Infektion des Embryos zu vermeiden. </li><li> Machen Sie ein kleines Loch auf dem Ei unmittelbar über dem Luft-Zelle mit einem Paar der Größe AA Pinzette. Achten Sie darauf, die Eischale zu knacken. Entfernen Sie kleine Stücke ein zu einer Zeit um ein kleines Fenster zu machen. Entfernen Sie vorsichtig die innere Membran mit Hilfe der Pinzette ohne Berührung der Dotterhaut. </li></ol> 2. Injektion und Elektroporation <ol><li> Um eine Beschädigung des Gehirns oder des Herzens, die Position der Nadel kontralateralen der wichtigsten Bündel von Blutgefäßen in das Auge eintritt und weisenden Schnabel zu verhindern. </li><li> Pierce durch die Dotterhaut, Lederhaut, Netzhaut und des Glaskörpers durch einen plötzlichen milden Druck auf die Nadel. Wenn die Nadel sticht durch das andere Ende des Auges sollte es in Ordnung sein, wenn es keine größeren Vene Blut schädigt. </li><li> Langsam nach hinten ziehen Sie die Nadel an der Kante der Öffnung und legen Sie es fast Tangente an der Außenwand des Augapfels. </li><li> Insert die Nadel in den Sub retinalen Raum zwischen der Lederhaut und Netzhaut. </li><li> Spritzen Sie die DNA, bis Sie die grüne Lösung Füllen der Seite des Augapfels und Schieben der Netzhaut nach innen durch die Schaffung einer Ausbuchtung visualisieren können. </li><li> Wenn Ihre Nadel Platzierung nicht korrekt ist dann wirst du sehen die DNA-Lösung Ausbreitung im Inneren des Glaskörpers Auffüllen der Mitte des Augapfels. Auch, wenn Sie die Netzhaut beschädigen zu viel dann werden Sie sehen die DNA-Lösung kommt aus dem Augapfel. </li><li> Langsam entfernen Sie die Nadel und sofort platzieren die Elektroden parallel im Ei nach dem Einweichen in PBS. Drücken Sie die Elektroden zu versenken in das Fruchtwasser in einer Weise, so dass das Auge injiziert zwischen den Elektroden befindet. Berühren Sie keine größeren Blutgefäße oder das Herz mit den Elektroden beim Platzieren sie. Die negative Elektrode sollte an der Injektionsstelle Seite zu sein so dass die DNA von sub retinalen Raum in der Netzhaut in Richtung positive Elektrode transportiert werden kann. ElectroCorporate die Netzhaut mit 5 Impulsen von 15V für 50 ms mit 950 ms Intervallen. </li><li> Entfernen Sie vorsichtig die Elektroden und dichten die Fenster des Eies mit Stücken von klaren Tesafilm. </li><li> Label-und datieren Sie das Ei injiziert, bevor es wieder in den Inkubator. Normalerweise dauert es etwa 3 bis 5 Minuten bis die ganze Elektroporation Vorgang abzuschließen. </li><li> GFP-Expression kann so früh wie 8 Stunden nach der Elektroporation gesehen werden. Allerdings können Sie warten, bis der Embryo auf die gewünschte Stufe vor der Ernte. </li></ol> 3. Repräsentative Ergebnisse In unserer Studie verwenden wir verschiedene Plasmid-Konstrukte, um die Regulation der Genexpression, dass retinalen Zell-Entwicklung beteiligt zu studieren. In diesem Video pCAG-GFP (Transfektion Kontrolle) wurde verwendet, um eine erfolgreiche Injektion und Elektroporation folgen. Es kann jedoch jedes Plasmid Konstrukt mit Reportergen (GFP, RFP usw.) verwendet werden. Auch wenn GFP-Expression kann so früh wie 8 Stunden nach der Ele gesehen werdenctroporation wir in der Regel beginnen die Ernte das Ei am Tag 6 (E6) und weiter. Elektroporiertem Netzhäute wurden aus dem Embryo seziert und analysiert unter fluoreszierendem Dissektionsmikroskop vor Einbetten und Schneiden. Typischerweise kann die Expression des Reportergens zumindest in einem Quartal der Netzhaut nach einer erfolgreichen Elektroporation (Abb. 1) gesehen werden. Die transfizierten retinalen Gewebe wurden durch weitere Schnitte für eine klare Visualisierung von Zellmorphologien analysiert. Immunhistochemie mit Zelltyp-spezifische Marker (Brn3a, Pax6 etc.) erlaubt die Charakterisierung von Zell-spezifischen GFP-Expression (Abb. 2). <img src="/files/ftp_upload/3792/3792fig1.jpg" align="Figure 1" /> Abbildung 1. Erfolgreiche Elektroporation von Reporterplasmid Ergebnisse positiv GFP-Expression. Huhn embryonalen Netzhaut injiziert wurden und an embryonalen Tag 4 elektroporiert und geerntet an embryonalen Tag 6. Mindestens 25% der Netzhaut wurde erfolgreich transfiziert (A = Ansicht von oben, B= Ansicht von unten). Maßstab = 1 mm. <img src="/files/ftp_upload/3792/3792fig2.jpg" align="Figure 2" /> Abbildung 2. Charakterisierung von GFP-exprimierenden Zellen der Netzhaut mittels Immunhistochemie. GFP exprimierenden Netzhautgewebe bei E7 Bühne waren fester und geschnitten. Diese Schnitte wurden dann mit verschiedenen Zell-spezifische Marker gefärbt. Brn3a wurde verwendet, um Ganglienzellen (A) zu bestimmen, während Pax6 wurde verwendet, um horizontale, Amacrin und Ganglienzellen (B) zu bestimmen. Balken = 50 um.

<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>

Alle Tierversuche wurden von der Institutional Animal Care und Einrichtungen Ausschuss an der Rutgers University zugelassen.

Gezielte Injektion Netzhaut und in ovo Elektroporation in embryonale Retina bei E4 Bühne können gezielt retinalen Vorläuferzellen was in der Fähigkeit, alle sechs großen Netzhaut Zelltypen auf der Ebene einzelner Zellen sichtbar zu machen. In ovo Elektroporation bei HH10 (~ E1.5), deren Zielgruppe die Augenblase ist in der Lage, Zellen, die um das Auge bilden entwickeln transfizieren. Jedoch haben diese Zellen eine sehr hohe Fluktuation in dieser Zeit und dieses Verfahren ist nicht spezifisch für...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Befruchteten pathogen-freien (SPF) White Leghorn Hühnereiern </td><td> Sonnenaufgang Bauernhöfe (Catskill, NY) </td><td></td><td></td></tr><tr><td> Hühnerei Inkubator </td><td> DQR Fertigung, Savannah, GA </td><td> Modell-1550 </td><td> Set zu 60% Luftfeuchtigkeit und 37,5 ° C. </td></tr><tr><td> Glaskapillarrohre </td><td> World Precision Instruments Inc. </td><td> TW150F-4 </td><td></td></tr><tr><td> 0,1-ml-Spritze </td><td> Hamilton Co. Reno, Nevada </td><td> Gasdichte 1710 </td><td> Alternativ 1 ml-Spritze kann ebenso verwendet werden </td></tr><tr><td> Masterflex Silikon (Peroxid) Tubing </td><td> Cole-Parmer </td><td> HV-96400-13 </td&gt; <td> Schneiden Sie ein kleines Stück (1 cm) zum Befestigen das Glas Nadel an der Spritze </td></tr><tr><td> Pinzette </td><td> Dumont </td><td> AA </td><td></td></tr><tr><td> Mikromanipulator </td><td> World Precision Instruments Inc. </td><td> M3301-M3 </td><td></td></tr><tr><td> Plasmid-DNA </td><td></td><td></td><td> pCAG-GFP wurde von Dr. Connie Cepko ausgeliehen </td></tr><tr><td> Fast Green FCF </td><td> Sigma-Aldrich </td><td> F7252 </td><td> Verdünnen Sie es zu 0,025% mit PBS </td></tr><tr><td> Impulsgeber </td><td> Harvard Apparatus, MA </td><td> BTX ECM 830 </td><td> Rechteckgenerator </td></tr><tr><td> Elektroden </td><td> Harvard Apparatus, MA </td><td> BTX-Modell 514 </td><td> Unsere Elektroden wurden 3-5 mm voneinander beabstandet </td></tr><tr><td> Monochrome Digitalkamera </td><td> Zeiss, Deutschland </td><td> Einxiocam MRM </td><td></td></tr><tr><td> Fluoreszierende Dissektionsmikroskop </td><td> Leica Microsystems, Deutschland </td><td> Leica MZ16FA </td><td></td></tr><tr><td> Aufrechtes Fluoreszenzmikroskop </td><td> Zeiss, Deutschland </td><td> Zeiss Axio Imager A1 </td><td></td></tr></tbody></table>

in ovo electroporation in embryonic chick retina

Huhn embryonalen Netzhaut ist ein ausgezeichnetes Werkzeug zur Entwicklung der Retina bei höheren Wirbeltieren zu studieren. Aufgrund der Größe und externen Entwicklung ist es vergleichsweise sehr einfach, die Küken embryonalen Netzhaut mittels rekombinanter DNA / RNA-Technologie zu manipulieren. Elektroporation von DNA / RNA-Konstrukte in der embryonalen Retina haben einen großen Vorteil, um Genregulation in retinalen Stammzellen / Vorläuferzellen während der Entwicklung der Retina zu untersuchen. Verschiedene Arten von Assays wie Reportergenassay, Gen-Überexpression, Gen niederschlagen (shRNA) etc. kann mit Hilfe der Elektroporation Technik werden. Dieses Video demonstriert gezielte Einspritz-und Retina-in-ovo-Elektroporation in die embryonale Hühnchenretina in der Hamburger und Hamilton Stadium 22-23, die etwa embryonalen Tag 4 (E4) ist. Hier zeigen wir eines schnellen und komfortablen in-ovo-Elektroporation Technik, bei der eine Plasmid-DNA, die grün fluoreszierendes Protein (GFP) drückt als Marker wird direkt in die gelieferteKükenembryonen subretinalen Raum, gefolgt von elektrischen Impulsen zur DNA-Aufnahme durch Netzhaut-Stammzellen / Vorläuferzellen zu erleichtern. Die neue Methode der retinalen Injektion und Elektroporation auf E4 ermöglicht die Visualisierung der einzelnen retinalen Zelltypen, einschließlich der spätgeborene Neuronen 1, die schwierig gewesen ist mit dem herkömmlichen Verfahren der Injektion und Elektroporation bei 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

Das übergeordnete Ziel dieses Videos ist zu zeigen, wie die gezielte Injektion Netzhaut führen und<em&gt; In-ovo-</em&gt; Elektroporation von DNA / RNA-Konstrukten in das Küken embryonalen Netzhaut an der Hamburger und Hamilton Stadium 22-23, die etwa embryonalen Tag 4 (E4) ist. Diese Technik ist sehr nützlich, um die Genexpression, Genregulation und morphologische Veränderung bei der Entwicklung Hühnchenretina studieren.

In ovo Elektroporation in embryonalen Hühnchenretina

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 Aufrufe.  University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School. Das übergeordnete Ziel dieses Videos ist zu zeigen, wie die gezielte Injektion Netzhaut führen und In-ovo- Elektroporation von DNA / RNA-Konstrukten in das Küken embryonalen Netzhaut an der Hamburger und Hamilton Stadium 22-23, die etwa embryonalen Tag 4 (E4) ist. Diese Technik ist sehr nützlich, um die Genexpression, Genregulation und morphologische Veränderung bei der Entwicklung Hühnchenretina studieren.

Serie: 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.

In Ovo Electroporations von HH Stage 10 Hühnerembryonen

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 ...

Forschung

Biologie

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.

Verfahren zur Whole Mount Antikörper-Färbung in Küken

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.

Assay für Neural Induction im Hühnerembryo

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.

Küken Ex ovo Kultur und Ex ovo CAM-Assay: Wie es wirklich funktioniert

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.

Vorbereitung der Embryonen für Elektronenmikroskopie der Drosophila Embryonale Herz Rohr

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.

Primäre Kultur und Plasmid Elektroporation der Murine Corti-Organ.

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.

Die Entzifferung Axonal Pathways von genetisch definierte Gruppen von Neuronen in der Küken Neuralrohr Verwendung In ovo Elektroporation

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.

Video bioinformatischen Analyse von humanen embryonalen Stammzellen 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 ...

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.

Single Cell Transfektion in Hühnerembryonen

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.

In-vivo-Elektroporation von Morpholinos in die Adult Zebrafisch Retina

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