Wir erkennen K. Mikic, der auf die Anfangsphase des Films und M. Nicolescu für die miR-Bild beigetragen. C. Huber wurde von einer Gemeinschaft des IZKF der Universtitätsklinikum Tübingen, A. Alwin Prem Anand von der Fortune-Programm der Universtitätsklinikum Tübingen unterstützt.

1. Voraussetzungen für die In-ovo Elektroporation <ol><li> Vorbereiten des Vektorkonstrukt: miR Sense und Antisense-Primer wurden nach den Anweisungen des Ambion entworfen, geglüht und in ligiert ApaI / EcoRI verdaut pSilencer U6.1 Vektor. Nach einer erfolgreichen Transformation der erhaltenen Klone wurde angebaut und pSilencer Plasmid wurde durch alkalische Lyse isoliert mit einem Quiagen Midi Vorbereitung Kit. </li><li> Elektroden: Die Elektroden können gekauft werden oder einfach gebaut 13 werden. Wir verwenden selbstgemachte Elektroden aus Platindraht (Φ = 0,2, 0,25 oder 0,5 mm Durchmesser) oder in einigen Fällen eine noch kleinere Wolframdrahtelektrode (Φ = 0,1 mm) 8 je nachdem, welchen Bereich wir elektroporieren möchten. Ein Versengen des Gewebes zu vermeiden, in der Regel verwendet man je näher an das Gewebe je dünner die Elektroden. Für breite Elektroporation von Mittelhirn, verwenden wir 0,5 mm Platindrähte mit einem Messing / Edelstahl-Rohr gelötetWelle und einen Elektrodenhalter mit einem Abstand von 3-5 mm (Fig. 1) befestigt. Bestimmte Bereiche Elektroporation, verwenden wir 0,25 mm Platindraht Anoden und 0,2 mm Platindraht oder 0,1 mm Wolfram für die Kathode mit einem ungefähren Abstand von 1 mm oder weniger. </li><li> Lösungen: Sind die folgenden Lösungen bereit: <ol><li> Autoklavierte Huhn Ringer, pH 7,5 (9,0 g NaCl, 0,42 g KCl, 0,24 g CaCl2, lösen sich in 1 L destilliertem Wasser). </li><li> PBS (8 g NaCl, 0,2 g KCl, 0,24 g KH 2 PO 4, 1,44 g Na 2 HPO 4, lösen sich in 1 Liter destilliertem H 2 O). </li><li> Fast Green: Stammlösung 10 mg / ml destilliertem H 2 O </li><li> Langsam trocknende Tinte (weniger Schellack und damit weniger Vergiftung für den Embryo). Hinweis: Eine blaue dichroitische Filter auf ein Glasfaserlampe befestigt, wie in Canto-Soler und Adler 14 beschrieben wird der Kontrast und die Notwendigkeit beseitigen, um Tinte zu injizieren. </li></ol></li></ol><img src="/files/ftp_upload/50024/50024fig1.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50024/50024fig1highres.jpg"/> Fig. 1 ist. Schematische Darstellung der Elektroden und Elektrodenhalter. (A, B, C) ​​Platindraht wurde auf eine Seite eines Edelstahlrohrwelle verlötet. Ein Loch wurde in die andere Seite der Rohrwelle gebohrt so dass Pin Stecker können fit in. (A) Rot und Blau farbigen Drähte wurden an die Pin-Stecker verbunden. Am anderen Ende wurde der Draht zu Bananenstecker, die mit den Steckdosen der Elektroporator arbeiten ausgestattet. (A, C) Der Platindraht wurde in einen Engel von 90 ° gebogen. Eine Schicht Nagellack isoliert die Welle über der Kurve. 2. Vorbereitungen unmittelbar vor In-ovo Elektroporation <ol><li> Bereiten Sie das folgende Material: <ol><li> Verdünnte Tinte mit Huhn Ringer 01.06, fügen Antibiotika-Antimykotika-Lösung (100x von GIBCo) 1:100. </li><li> Bereiten Vektorlösung: 1-2 ug / ul pro spezifische DNA in H 2 O mit Fast Green (1:20) ergänzt. </li><li> Sterilisieren Zangen, Scheren, Nadelwolframelektroden und mit 70% Ethanol. </li><li> Ziehen Mikropipetten für die Nukleinsäure-Injektion aus Borosilikatglas Kapillaren (Länge zu Außendurchmesser: 100 mm x 0,9 mm). Wir verwenden eine einfache Abzieher PUL-1 (WPI, Berlin) mit den in Tabelle 1 gezeigten Einstellungen. </li><li> Die Parameter des Elektroporators wie in Tabelle 2 gezeigt. </li></ol></li></ol><table border="1" ><tbody><tr><td fo:width="1in"> Parameter </td><td fo:width="1in"> Werte </td></tr><tr><td> Wärme </td><td> 350 </td></tr><tr><td> Ziehen </td><td> 100 </td></tr><tr><td> Geschwindigkeit </td&gt; <td> 50 </td></tr><tr><td> Verzögerung </td><td> 200 </td></tr></tbody></table> Tabelle 1. Einstellungen für die PUL-1 <table border="1"><tbody><tr><td fo:width="1.5in"> Parameter </td><td fo:width="1.5in"> Werte </td></tr><tr><td> Frequenz </td><td> 50 Hz </td></tr><tr><td> Verzögerung </td><td> 20-50 ms * </td></tr><tr><td> Breite </td><td> 20 msec </td></tr><tr><td> Spannung </td><td> 8-25 V * </td></tr><tr><td> Puls </td><td> 3-5 mal </td></tr><tr><td colspan="2"> * Je nach Elektrodendurchmesser und Entfernung. Bittemit berücksichtigen bei der Änderung einer der Parameter. </td></tr></tbody></table> Tabelle 2. Einstellungen für den Impuls Stimulator <ol start="2"><li> Bereiten Hühnerembryonen für die Elektroporation: <ol><li> Befruchteten Hühnereiern auf ihre längere Seite Inkubation bei 37 ° C mit 65% Luftfeuchtigkeit für 39 Stunden, Embryonen zwischen Hamburger &amp; Hamilton (HH) erwerben Stufen 15 9-11. Der Embryo setzt sich am obersten Teil des Eies, so sollten Sie es mit einem Bleistiftstrich markieren. </li><li> Desinfizieren Sie die Eier mit 70% Ethanol. </li><li> Durchbohren das Ei an der breiteren Seite, wo die Luft Hohlraum sein sollte. </li><li> Entfernen Sie 1-2 ml Eiweiß. Dadurch wird der Embryo aus der Eierschale lösen. </li><li> Cello-Band der Eierschale zu Granatsplitter fallen auf den Embryo zu vermeiden. Verwenden Sie eine Schere, um ein kleines Fenster in der Eierschale auf der Bleistiftlinie geschnitten. </li><li> Spritzen Sie ein bisschen Tinteunter dem Bereich opaca, um den Embryo zu visualisieren. </li><li> Machen Sie einen Schnitt in die Dotterhaut mit einem geschärften Wolfram-Nadel, um eine bessere Zugänglichkeit des Embryos für die Nukleinsäure-Injektion und Elektroporation zu erreichen. </li></ol></li></ol> 3. Elektroporation und Inkubation <ol><li> Fügen Sie ein paar Tropfen warmen Ringer-Lösung auf die Embryonen. Dadurch werden die Embryonen zu senken, eine Überhitzung zu vermeiden, Verkleben der Elektroden an das Gewebe und ein Medium für den Strom zwischen den beiden Elektroden. </li><li> Spritzen Sie die Vektorlösung in das Lumen des Neuralrohrs in Mittelhirn-Ebene. Wenn Ihr DNA-Expressionsvektor keine Reporter-Gen (zB EGFP) enthält, fügen Sie ein etwas niedriger konzentrierten Reportergenvektor (1 ug / ul miR-Expressionsvektor zu 0,9 ug / ul Reportergenvektor) zu der DNA-Lösung. Dadurch wird sichergestellt, dass Sie transfizierten Zellen später 8 zu erkennen. Wir nutzen die so genannte pCAX Vektor, eine Art Geschenk herm C 13 Krull. </li><li> Für ein breites Transfektion des Mittelhirns, legen Sie die links Elektroden und des Embryos auf der Höhe des Mittelhirns. Der Abstand zwischen den Elektroden sollte etwa 3-5 mm betragen. Die anderen Einstellungen sind 15V, 20 ms und 50 ms Impulse Verzögerung für HH 10. Ändern der Position der beiden Elektroden leicht entlang der dorso-ventralen (DV) Achse des Mittelhirns sichert eine eine breite Transfektion von Zellen der DV Achse Transfektion. </li><li> Für ventralen Transfektion, fügen Sie einige Ringer, um den Kopf aus der Dottermembran zu erhöhen und legen Sie die Anode unter ventralen Mittelhirn. Wenn der Kopf nicht steigen drücken Sie die Elektrode unter der Membran, die Dotter und Embryo trennt, um der sengenden Neuralrohr zu vermeiden. Die Kathode sollte über dem Mittelhirn dorso-lateral gegenüber der Anode angeordnet werden. Berühren Sie nicht das Neuralrohr. Wir Transfektion der linken ventralen Hälfte des Mittelhirns, Schwierigkeiten mit der Herzentwicklung zu vermeiden. Der Abstand zwischen den Elektrodenetwa 1-0,5 mm, der Rest der Einstellungen 10 V, 20 msec und 50 msec Breite Verzögerung. </li><li> Bewerben einen Tropfen Kochsalzlösung auf der Embryonen, um es abzukühlen und entfernen Luftblasen. </li><li> Verschließen Sie die Eier mit dem Band und Inkubation für mindestens 4 Stunden, die Mindestzeit für Vektorausdruck erforderlich. </li></ol> 4. Fixierung von Hühnerembryonen <ol><li> Entfernen Sie die Embryonen aus ihren Eiern. </li><li> Saubere Embryonen unter einem Stereomikroskop und inwieweit der Transfektion mit einem Fluoreszenzstereomikroskop. </li><li> Fix die Embryonen über Nacht in 4% PFA bei 4 ° C Weiter mit Schneiden und / oder Färbung (in-situ-Hybridisierung, Immunhistochemie), um Änderungen in der transfizierten Bereich zu analysieren. </li></ol>

<ol>
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Cell Biol. 1, 203-207 (1999).</li><li>Momose, T., et al. <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>Nakamura, H., Watanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Misexpression+of+genes+in+brain+vesicles+by+in+ovo+electroporation.">Misexpression of genes in brain vesicles by in ovo electroporation.</a> Dev. Growth Differ. 42, 199-201 (2000).</li><li>Voiculescu, O., Papanayotou, C., Stern, C. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+on+using+in+ovo+electroporation+to+analyze+gene+function.">A primer on using in ovo electroporation to analyze gene function.</a> Dev. Dyn. 229, 433-439 (2004).</li><li>Canto-Soler, M. V., Adler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+cup+and+lens+development+requires+Pax6+expression+in+the+early+optic+vesicle+during+a+narrow+time+window.">Optic cup and lens development requires Pax6 expression in the early optic vesicle during a narrow time window.</a> Developmental biology. , 294-2119 (2006).</li><li>Hamburger, V., Hamilton, H. 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Wir haben nichts zu offenbaren.

Dieses Video zeigt eine effektive Methode, um das Plasmid in die Neuroepithelzellen von bestimmten Bereichen des Mittelhirns Küken transfizieren. Rechteckige elektrische Impulse von Niederspannungs können DNA in Zellen der Küken Neuralrohr in ovo 6,16 einzuführen. Jedoch ist die Genauigkeit der DNA-Targeting oft durch die breiten elektrischen Feldes, das durch den relativ großen Elektroden (Φ = 0,5 mm) steigt behindert. Wir haben versucht, dieses Problem durch Verwendung von Elektroden im Durch...

Die Anerkennung der kleine nicht-kodierende RNAs als zusätzliche Spieler für die Genexpression eine neue Komplexität, um genomische Programmierung / Genregulation. Verschiedene Arten von nicht-kodierenden RNAs funktionelle Bedeutung in neuronalen Zellen, einschließlich der kleinen nicht-kodierenden RNAs 1-4. MicroRNAs (miR-oder miRNA) zeigen beispielsweise verschiedene und wechselnde Expressionsprofile in die Entwicklung des Gehirns 5. In ovo Elektroporation von Hühnerembryonen Gezielte bietet eine einzigartige Gelegenheit für zeitliche und räumliche Steuerung der Genexpression und der Silencing während der Entwicklung. Dieses Video zeigt die verschiedenen Schritte der Durchführung ektopische Expression von miRs in bestimmten Bereichen des Mittelhirns mit Küken in ovo Elektroporation 10.06. Um eine dauerhafte Wirkung dieser kleinen nicht-kodierenden RNAs in den Zellen zu gewährleisten, wurden die DNA-Sequenz miRs in mono-oder bi-cistronischen Vektoren kloniert. Für die in-ovo Elektroporation, miR enthält vector in das Mittelhirn Neuralrohr, indem die Embryos nach einem kleinen Fenster in der Eierschale injiziert. Bestimmte Bereiche des Mittelhirns kleine Plus (Anode) und minus (Kathode) zu transfizieren Platinelektroden werden an bestimmten Positionen angeordnet. Zur ventralen Mittelhirn Transfektion wird die Anode unter dem ventralen Mittelhirn linken und der Kathode über der rechten Hälfte des Mittelhirns vor dem Anlegen eines Stroms angeordnet. Die Öffnung in der Eierschale ist mit Klebeband geschlossen und Embryonen werden, solange für jede Analyse erforderlich inkubiert. Dieses Verfahren wurde ursprünglich von Muramatsu et al. 6 beschrieben und Momose et al. 8 zum spezifischen Bereich der Transfektion verbessert. <img src="/files/ftp_upload/50024/50024schematic1.jpg" fo:content-width-="6in" fo:src="/files/ftp_upload/50024/50024schematic1highres.jpg"/> Schematische Übersicht. <ol><li> Der Embryo im Ei wird durch Schneiden ein kleines Fenster in th ausgesetzte Eierschale. </li><li> Das gelöste Vektor (en) in das Mittelhirn unter Verwendung einer Mikrokapillare injiziert. </li><li> Zwei Elektroden - parallel oder unter und über dem Embryo gelegt - erzeugen einen gepulsten elektrischen Feld. </li><li> Das elektrische Feld zeitlich erzeugt Poren in der Zellmembran, die den Eintritt in die Zelle durch die negativ geladene DNA (oder RNA) zu erleichtern zogen Anode 11,12. </li></ol>

<table border="1">
<tbody>
 <tr>
 <td>Name </td>
 <td>Company</td>
 <td></td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Borosillicate glass capillaries </td>
 <td>Hartenstein</td>
 <td></td>
 <td>Model: 0.9 mm</td>
 </tr>
 <tr>
 <td>Microcapillary puller</td>
 <td>WPI, Berlin</td>
 <td></td>
 <td>Model: Pul1-E</td>
 </tr>
 <tr>
 <td>Electroporator</td>
 <td>Intracel</td>
 <td></td>
 <td>Model: TSSIC</td>
 </tr>
 <tr>
 <td>Stereomicroscope - fluorescence </td>
 <td>LEICA</td>
 <td></td>
 <td>Model: MZFLIII</td>
 </tr>
 <tr>
 <td>Stereomicroscope</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Stemi</td>
 </tr>
 <tr>
 <td>Camera and software</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Axiocam MRc/ Axiovision Re. 4.8</td>
 </tr>
 </tbody>
</table>

in ovo expression of microrna in ventral chick midbrain

Nicht-kodierende RNAs sind weitere Spieler in der Regulation der Genexpression. In ovo Elektroporation von bestimmten Bereichen Gezielte bietet ein einzigartiges Werkzeug für die räumliche und zeitliche Kontrolle der Eileiter microRNA Expression. Allerdings sind ventralen Hirnstrukturen wie ventralen Mittelhirn ziemlich schwierig, für alle Manipulationen zu erreichen. Hier zeigen wir, eine effiziente Möglichkeit zum ventralen Mittelhirn miRNA in elektroporieren mit dünnen Platinelektroden. Dieses Verfahren bietet einen zuverlässigen Weg, um bestimmte Bereiche des Mittelhirns und nützliches Werkzeug für In-vivo-Studien zu transfizieren.

Der Umfang des Mittelhirns Bereich mit miR transfiziert ist durch die Expression von GFP aus einem Reportervektor zusammen mit der miR-Expressionsvektor (Abbildung 2) eingespritzt wird sichtbar. Verwendung von Platinelektroden mit einem Durchmesser von 0,5 mm und parallel zur Transfektion eines breiten Bereich entlang der DV-und AP-Achse, einschließlich Hinterhirn, Mittelhirn und Zwischenhirn manchmal (2A und B) angeordnet, um die AP-Achse des Mittelhirns führt normalerweise. Die Grö...

Watch this Scientific Journal Video about In ovo Expression of MicroRNA in Ventral Chick Midbrain at JoVE.com

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ektopische Expression ist eine Technik, um die microRNAs Rolle in der Entwicklung des Gehirns aufzuklären. , Für bestimmte Bereiche mit in ovo Elektroporation ist jedoch eine Herausforderung. Hier zeigen wir einen effizienten Weg, um selektiv elektroporieren ventrale und dorsale Mittelhirn Regionen.

In ovo Expression von microRNA in ventralen Mittelhirn-Küken

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

in-ovo-expression-of-microrna-in-ventral-chick-midbrain

Research

JoVE Journal

Neuroscience

In ovo Expression of MicroRNA in Ventral Chick Midbrain

10.4K Aufrufe.  University of Tübingen.  Ektopische Expression ist eine Technik, um die microRNAs Rolle in der Entwicklung des Gehirns aufzuklären. , Für bestimmte Bereiche mit in ovo Elektroporation ist jedoch eine Herausforderung. Hier zeigen wir einen effizienten Weg, um selektiv elektroporieren ventrale und dorsale Mittelhirn Regionen. 

Serie: In ovo Expression of MicroRNA in Ventral Chick Midbrain

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

Organotypischen Kulturen von embryonalen ventralen Mittelhirn: Ein System zur Dopaminerge Neuronale Development Study In-vitro-</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

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Neurowissenschaften

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

In ovo Elektroporation in Mittelhirn-Küken für Studien über Genfunktion in dopaminergen Neuronen Entwicklung

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Umfassende Profilierung der Verordnung Dopamin in der Substantia nigra und ventralen Tegmentum

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dual-Elektrophysiologische Recordings synaptisch-evozierte astrogliale und neuronale Antworten in Acute Hippocampusschnitte

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

RNA-Seq Analyse der differentiellen Genexpression in elektroporiertem Küken Embryonale Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Umwelt Modulationen der Anzahl der Mittelhirn Dopamin-Neuronen in erwachsenen Mäusen

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

Zuverlässige Identifikation von Wohn dopaminerge Neurone in Mittelhirns Kulturen unter Verwendung von RNA-Sequenzierung und TH-Promotor getriebene eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

Ein In Ovo -Modell zum Testen Insulin-mimetischen Verbindungen

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

Kombinierte in vivo anatomische und funktionelle Spurensuche von ventralen Tegmentalflächen-Glutamat-Terminals im Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Quantifizierung von Spontan-Ca2+-Fluxes und ihren nachgelagerten Auswirkungen in primären Maus-Midbrain-Neuronen

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...