Diese Arbeit wurde durch ein Stipendium der Deutschen Forschungsgemeinschaft (; LU1455/1-1 DFG) gefördert.

1. Ex ovo Kultur <ol><li> Frische befruchtete Eier werden auf ihrer langen Seite in einem Zwangs-Entwurf Inkubator (BSS160, Ehret, Deutschland) bei 37,5 ° C mit 60% Luftfeuchtigkeit für die Inkubation gelegt. Eier bei 12 ° C für länger als eine Woche gelagert sollten nicht verwendet werden. </li><li> Nach einer Inkubationszeit von 2,5 Tage (ca. Stadium HH17), nehmen Sie die Eier und beschriften Sie die oben mit einem Bleistift die Richtung für das Knacken geben. </li><li> Vor dem Brechen, gießen Sie etwa 20 ml sterilem destilliertem Wasser in einem sauberen großen Petrischale (Durchmesser von 145 mm; Greiner Bio-One GmbH, Deutschland). </li><li> Legen Sie einen weiteren kleinen sterilen Petrischale (Durchmesser 94 mm; Greiner Bio-One GmbH) in die große Petrischale. </li><li> Die Eier auf dem Boden gegen scharfe Kanten, öffnen Sie vorsichtig die Eier und den gesamten Inhalt der einzelnen Eier in den kleinen Petrischale. </li><li> Decken Sie die große Petrischale mit einem Deckel und steckte es in einen anderen Inkubator (BINDER GmbH, Tuttlinge, Deutschland)für die weitere Kultur bei 37,5 ° C mit etwa 60% Luftfeuchtigkeit. Nach der Inkubation für die gewünschte Anzahl von Tagen, können die Embryonen für die Elektroporation verwendet werden. </li></ol> 2. Vorbereitung für die ex ovo Elektroporation <ol><li> Ziehen von Glaskapillaren mit einer Mikroelektrode Abzieher (PUL-100 Feinpipettenziehvorrichtung, World Precision Instruments, Berlin, Deutschland) mit Glasröhren von 1,0 mm Durchmesser (TW100F-4; World Precision Instruments) und brechen Sie die Spitze der Glaskapillare in eine geeignete Durchmesser. </li><li> Einrichten geeigneter Parameter (dh unterschiedliche Spannungen für die verschiedenen Gewebe und hat eine Größe von Embryonen) zur Elektroporation und eine Verbindung Elektroden mit einer Elektroporator (CUY21-Korrektur, Nepa Gene, Chiba, Japan) gemäß dem Zielgewebe und eine Größe von Embryonen. </li><li> Vorbereiten einer Lösung, die Plasmid pCAGGS Plasmide in Vektor, der ein Zielgen, zB cadherin7 (Cad7; pCAGGS-Cad7 mit einer Konzentration von 2,0 ug / ul) und ein Markergen,z. B. grün fluoreszierendes Protein (GFP; pCAGGS-GFP mit einer Konzentration von 0,25 ug / ul). Fügen Fast Green mit einer Endkonzentration von 0,1% (Sigma)-Lösung, um das Plasmid mit der grünen Farbe zu markieren. </li><li> Laden Sie die Glaskapillare mit dem Plasmid-Lösung mit einer Pipette Mund. </li></ol> 3. Gentransfer in Huhn Tectum opticum von ex ovo Elektroporation <ol><li> Nach ex ovo Kultur, zB durch Inkubation Tag (E) 6 werden die Hühnerembryonen für ex ovo Elektroporation verwendet. </li><li> Reißen Sie die Vitellin und Amnion-Membranen über die optische Tectum mit feinen Pinzetten und 3 Tropfen steriler 0,9% Natriumchlorid-Lösung auf der Oberfläche des Tectum. </li><li> Injizieren der Plasmid-Lösung in den Hohlraum des Tectum durch Glaskapillare mit dem Mund Pipette. </li><li> Platzieren Sie die Elektroden mit einem Wolfram-Nadel Kathode und einer Anode Rechteck Platte (CUY 661-3x7, Nepa Gene) neben dem Tectum undunmittelbar gelten elektrischen Impulsen (sechs Impulse, 25 V, 60 ms Impulslänge, 100 ms Intervallen in jedem Fall) durch die Elektroporator der Tectum hergestellt. </li><li> Decken Sie die große Petrischale mit dem Deckel und senden Sie es in den Inkubator. Nach Inkubation für geeignete Tagen (z. B. 2 oder 3 Tage nach der Elektroporation) werden die Tectum gesammelt und für die Immunfärbung (Abbildung 1) und biologische Detektion befestigt. </li></ol> 4. Repräsentative Ergebnisse Erfolgreiche Überexpression der exogenen Proteine ​​Cad7 und GFP durch ex ovo Elektroporation ist in Abbildung 1 als Beispiel gezeigt. Wenn Cad7 Plasmid zusammen mit GFP-Plasmid in die Tectum war bei E6 elektroporiert, zwei oder drei Tage nach der Elektroporation wurden die Embryonen wurden und fixiert. Bei E8 wird das GFP-Protein stark im Tectum in whole mount Bilder (Abbildung 1a-c) zum Ausdruck gebracht. Ferner wird in den Abschnitten des Tectum bei E9,GFP-Protein (grün in 1D) und Cad7 Protein (rot in 1E) sind coexprimierten (gelb in 1F), was darauf hindeutet, dass ex ovo Elektroporation ein erfolgreiches Verfahren für den Gentransfer in den älteren Hühnerembryonen in vivo ist. <img src="/files/ftp_upload/4078/4078fig1.jpg" alt="1"/> 1. Wenn die Plasmide GFP-codierende und Cad7 in die Hühner Tectum sind bei E6 cotransfiziert, zwei oder drei Tage nach ex ovo Elektroporation wird GFP-Protein (grün) stark exprimiert, wie in wholemount Bilder (AC) bei E8 gezeigt. In den Abschnitten des Tectum bei E9, sind GFP-Protein (grün in D) und Cad7 Protein (rot E) coexprimierten (gelb F). BF, Hellfeldbild. Maßstabsbalken: 200 um in A für AC; 100 um in D für DF.

<ol>
	<li>Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K., Yasuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+gene+expression+in+chick+embryos+by+microelectroporation.">Efficient targeting of gene expression in chick embryos by microelectroporation.</a> Dev. Growth Differ. 41, 335-344 (1999).</li><li>Luo, J., Ju, M. J., Lin, J., Yan, X., Markus, A., Mix, E., Rolfs, A., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin-20+expression+by+motor+neurons+is+regulated+by+Sonic+hedgehog+during+spinal+cord+development.">Cadherin-20 expression by motor neurons is regulated by Sonic hedgehog during spinal cord development.</a> NeuroReport. 20, 365-370 (2009).</li><li>Luo, J., Ju, M. J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regionalized+cadherin-7+expression+by+radial+glia+is+regulated+by+Shh+and+Pax7+during+chicken+spinal+cord+development.">Regionalized cadherin-7 expression by radial glia is regulated by Shh and Pax7 during chicken spinal cord development.</a> Neuroscience. 142, 1133-1143 (2006).</li><li>Nakamura, H., Katahira, T., Sato, T., Watanabe, Y., 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=Gain-+and+loss-of-function+in+chick+embryos+by+electroporation.">Gain- and loss-of-function in chick embryos by electroporation.</a> Mech. Dev. 121, 1137-1143 (2004).</li><li>Dai, F., Yusuf, F., Farjah, G. H., Brand-Saberi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-induced+targeted+silencing+of+developmental+control+genes+during+chicken+embryogenesis.">RNAi-induced targeted silencing of developmental control genes during chicken embryogenesis.</a> Dev. Biol. 258, 80-90 (2005).</li><li>Rao, M., Baraban, J. H., Rajaii, F., Sockanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+comparative+study+of+RNAi+methodologies+by+in+ovo+electroporation+in+the+chick+embryo.">In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo.</a> Dev. Dyn. 231, 592-600 (2004).</li><li>Kos, R., Tucker, R. P., Hall, R., Duong, T. D., Erickson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+introducing+morpholinos+into+the+chicken+embryo.">Methods for introducing morpholinos into the chicken embryo.</a> Dev. Dyn. 226, 470-477 (2003).</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.">A series of normal stages in the development of the chick embryo.</a> J. Morphol. 88, 49-92 (1951).</li><li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev. Biol. 10, 391-394 (1974).</li><li>Luo, J., Treubert-Zimmermann, U., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherins+guide+migrating+Purkinje+cells+to+specific+parasagittal+domains+during+cerebellar+development.">Cadherins guide migrating Purkinje cells to specific parasagittal domains during cerebellar development.</a> Mol. Cell. Neurosci. 25, 138-152 (2004).</li><li>Luo, J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+ovo+electroporation+for+gene+transfer+into+older+chicken+embryos.">Ex ovo electroporation for gene transfer into older chicken embryos.</a> Dev. Dyn. 233, 1470-1477 (2005).</li><li>Treubert-Zimmermann, U., Heyers, D., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+axons+to+specific+fiber+tracts+in+vivo+by+altering+cadherin+expression.">Targeting axons to specific fiber tracts in vivo by altering cadherin expression.</a> J. Neurosci. 22, 7617-7626 (2002).</li></ol>

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Dieses Protokoll stellt einen Leitfaden für den Gentransfer in älteren Hühnerembryonen (z. B. in das Tectum opticum bei E6) von ex ovo Elektroporation. Diese Methode erweitert die Verwendung von Elektroporation, um älteren Hühnerembryonen und kann leicht an vielen Labors zur Untersuchung von Genfunktionen in vivo in späteren Stadien angewendet. Embryonen aus der E4 bis E7 zumindest kann erfolgreich mit dieser Methode elektroporiert werden, und die Embryonen können elektroporierte Überleben sein...

<table border="1"><tbody><tr><td> Name </td><td> Firma </td><td> Katalog-Zahl </td></tr><tr><td> Electroporator </td><td> Nepa Gene, Japan </td><td> CUY21-Edit </td></tr><tr><td> Elektroden </td><td> Nepa Gene, Japan </td><td> CUY661-3x7 </td></tr><tr><td> Egg Brutkasten </td><td> Ehret, Deutschland </td><td> BSS160 </td></tr><tr><td> Embryo Inkubator </td><td> BINDER GmbH, Deutschland </td><td></td></tr><tr><td> Fluoreszenzmikroskop </td><td> Keyence, Deutschland GmbH </td><td> BZ-8000 </td></tr><tr><td> Petrischale </td><td> Greiner Bio-One GmbH, Deutschland </td><td></td></tr><tr><td> Mikroelektroden-Abzieher </td><td> World Precision Instruments, Deutschland </td><td> PUL-100 </td></tr></tbody></table>

gene transfer into older chicken embryos by ex ovo electroporation

Der Hühnerembryo bietet ein exzellentes Modellsystem zur Untersuchung von Gen-Funktion und Regulation während der embryonalen Entwicklung. In ovo Elektroporation ist eine leistungsfähige Methode, um Over-Express exogenen Genen oder unten zu regulieren endogene Gene in vivo in Hühnerembryonen 1. Unterschiedliche Strukturen wie DNA-Plasmide kodierenden Gene 2-4, small interfering RNA (siRNA) Plasmide 5, kleine synthetische RNA Oligos 6 und 7 Morpholino Antisense-Oligonukleotide können problemlos in Hühnerembryonen werden durch Elektroporation transfiziert. Allerdings ist die Anwendung der in-ovo-Elektroporation von Embryonen in frühen Stadien Inkubation (jünger als Bühne HH20 - nach Hamburg und Hamilton) beschränkt 8 und gibt es einige Nachteile für die Anwendung in Embryonen in späteren Stadien (älter als Stadium HH22 - rund 3,5 Tag der Entwicklung). Zum Beispiel ist die Vitellin-Membran in einem späteren Stadium USUVerbündete hielt sich an die Membran shall und Öffnen eines Fensters in der Schale verursacht Bruch der Gefäße, was zu dem Tod der Embryonen; älteren Embryonen werden durch Dottergang und Allantois-Gefäße, in denen es schwierig ist, zugreifen und sie manipulieren die Embryonen fallen; älteren Embryonen bewegen kräftig und es ist schwierig, die Orientierung durch eine relativ kleine Fenster in der Schale zu kontrollieren. In diesem Protokoll zeigen wir eine ex ovo Elektroporation Methode für den Gentransfer in Hühnerembryonen in späten Stadien (älter als Stadium HH22). Für ex ovo Elektroporation werden Embryonen in Petrischalen kultiviert und 9 die Vitellin und Allantois-Gefässe sind weit verbreitet. Unter diesen Bedingungen werden die älteren Hühnerembryonen leicht zugänglich und manipulierbar. Daher überwindet dieses Verfahren die Nachteile der in-ovo-Elektroporation, die auf die älteren Hühnerembryonen. Mit dieser Methode können Plasmide leicht in verschiedenen Teilen transfiziert werdender älteren Hühnerembryonen 12.10.

Watch this Scientific Journal Video about Gene Transfer into Older Chicken Embryos by ex ovo Electroporation at JoVE.com

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

Verfahren zum Gentransfer in Hühnerembryos in späteren Stufen der Inkubation (älter als Hamburger und Hamilton Stufe (HH) 22) beschrieben. Dieses Verfahren überwindet die Nachteile der<em&gt; In-ovo-</em&gt; Elektroporation angewendet, um älteren Hühnerembryonen und ist eine nützliche Technik, um Gen-Funktion und Regulation bei älteren Entwicklungsstadien zu studieren.

Gentransfer in Hühnerembryonen durch Ältere<em&gt; Ex ovo</em&gt; Die Elektroporation

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

gene-transfer-into-older-chicken-embryos-by-ex-ovo-electroporation

Research

JoVE Journal

Biology

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

14.5K Aufrufe.  School of Medicine University of Rostock. Verfahren zum Gentransfer in Hühnerembryos in späteren Stufen der Inkubation (älter als Hamburger und Hamilton Stufe (HH) 22) beschrieben. Dieses Verfahren überwindet die Nachteile der In-ovo- Elektroporation angewendet, um älteren Hühnerembryonen und ist eine nützliche Technik, um Gen-Funktion und Regulation bei älteren Entwicklungsstadien zu studieren.

Serie: Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

Nuclear Transfer in Maus-Oozyten

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

Forschung

Biologie

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

In Utero intraventrikuläre Injektion und Elektroporation von E16 Rattenembryonen

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

In Utero intraventrikuläre Injektion und Elektroporation von E15 muriner Embryonen

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

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.

Platzieren Growth Factor-Coated Perlen auf Early Stage Hühnerembryonen

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

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 ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

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

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Mikroinjektion von Zebrafisch-Embryos, um Gene Function Analyze

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

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

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

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

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

In utero und ex vivo Elektroporation für die Genexpression in Maus retinalen Ganglienzellen

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

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 ...

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

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

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