We would like to acknowledge David O'Brien for his support with data analysis, and all the members of the Canto-Soler lab for their critical discussions. This work was supported by NIH grants EY004859 and EY022631 (MVCS), Core Grant EY1765, and an unrestricted departmental grant from Research to Prevent Blindness, Inc.

Alle in dieser Arbeit beschriebenen Verfahren wurden nach den von der Animal Care und Verwenden Ausschuss empfahl der Johns Hopkins University Richtlinien durchgeführt. 1. Ahead of Time: Herstellung von Instrumenten, Reagenzien und Geschirr <ol><li> Herstellung der Eier: Inkubieren befruchteten White Leghorn Hühnereiern bei 37,5 ° C und 60% relativer Luftfeuchtigkeit für 5 Tage in einem Brutkasten. HINWEIS: Ein Inkubator mit Schaukelkapazität kann hilfreich für die Standardisierung und die Synchronisierung der Embryonalentwicklung, aber Schaukel ist für den Ausgang dieses Protokoll unbedingt erforderlich. </li><li> Plasmidpräparation: <ol><li> Design Plasmidkonstrukt zur Gen-Überexpression oder Herunterregulierung (zum Beispiel durch die Verwendung von RNAi-Technik 1,34) je nach Bedarf. Fügen Sie ein Reporter-Gen (wie EGFP, RFP oder LacZ) in dem Konstrukt, wann immer möglich. HINWEIS: Einige häufig verwendete Promotoren, wie CMV, kann nach der Zeit zum Schweigen gebracht zu werden. Die CAG promotER (Huhn-beta-Aktin-Promotor CMV-Enhancer) ist eine sehr gute Alternative, eine effiziente langfristige Expression des Gens von Interesse 35-37. </li><li> Vorbereitung einer Plasmid-Lösung bei einer Konzentration von 1,5 ug / ul in sterilem PBS. OPTIONAL: Bereiten Plasmid-Lösung vor der Zeit und bewahren Sie sie eingefroren, bis gebrauchsfertig. HINWEIS: Höhere Plasmid-Konzentrationen nicht zu einer signifikanten Steigerung der Effizienz führen, wohingegen mit niedrigeren Plasmid-Konzentrationen senken die Transfektionseffizienz 1. </li></ol></li><li> Elektroden: <ol><li> Für die Anode Verwendung einer dicken Gold gekippt Elektrode (siehe Tabelle spezifischer Materialien / Ausrüstung). Wischen Sie mit 70% Ethanol. HINWEIS: Isolierung des Gold gekippt Elektrode, so dass etwa 0,5 mm ausgesetzt sind, ist es ratsam, um ein Auslaufen des elektrischen Stroms zu minimieren. Informationen für Isoliermaterial kann in Tabelle spezifischer Materialien gefunden werden. </li><li> Machen Sie eine Kathode aus einem 2,5 mm Quadrat box Filament, 4,5 mm breit (in der Regel in der Mikropipette Abzieher verwendet). Mit Hilfe einer Pinzette, lösen Sie die quadratische Box und strecken Filaments in einem langen Streifen. Klicken Sie dann unter Dissektionsmikroskop und mit einem Lineal als Führung, biegen Sie das Filament in eine quadratische U-Form 3 mm lang an der Basis und 2 mm hoch auf einer Seite, mit der anderen Seite die verbleibende Länge des Fadens (Abbildung 1 , C - D). Mit einer Pinzette, tauchen Sie die Elektrode in 96% Ethanol und Flamm zu sterilisieren. Bringen Sie dann eine elektrische Leitung mit einer kleinen Krokodilklemme mit dem längeren Arm der Elektrode. </li></ol></li><li> Elektroporationskammer Setup: <ol><li> Vorbereitung der Elektroporation Kammer durch Schneiden des Deckels einer sterilen 1,5-ml-Mikrozentrifugenröhrchen und Befestigen darauf flachen Seite nach unten auf eine 100 mm Petrischale mit Klebeband (1E). Hinweis: andere Behälter in Form eines gut ähnlicher Größe funktionieren würde; ist jedoch ein Mikrozentrifugenröhrchen Deckel leicht zugänglich und autoklavierbar. Der Brunnenmuss groß genug sein, um ein Küken Auge und Kathodenelektrode Haus, aber nicht so groß sein, daß sie zu einer Verschwendung von Plasmid-Lösung führen. </li></ol></li></ol> 2. Ex ovo Elektroporation Ordnung <ol><li> Vorbereitung des Instruments: <ol><li> Sterilisieren Mikrodissektion Werkzeuge (2 Paar große gebogene Moloney Zange zum Öffnen von Eierschalen und Aushöhlungen Embryonen, 2 Paar Feinspitze gekrümmt Dumont Pinzetten zum Entfernen von RPE, ein Paar Bonn gezahnten Pinzette zur Entfernung von Linse und Glas) durch Eintauchen des Tipps in 96% Ethanol und flammenden. Saubere Dissektion Umfang mit Tüchern in 70% Ethanol getaucht. Warm eine Flasche sterile Calcium-Magnesium-freien Hanks Balanced Salt Solution (CMF-HBSS) in einem 37 ° C Wasserbad. Füllen 100 mm sterile Petrischalen bis zu 3/4 mit warmen CMF-HBSS. </li><li> Zeigen Elektroporationskammer unter einem anderen sauberen Dissektionsmikroskop füllen und mit 120 ul von Plasmid-Lösung. Verbinden Elektroden Elektroporationseinrichtung, so dasssicher maßgeschneiderte Elektrode ist mit dem negativen Pol verbunden und Gold gekippt Elektrode an den positiven Pol. </li></ol></li><li> Embryo-Kollektion: <ol><li> Reinigen Sie die Eier durch Abwischen Muscheln mit Tüchern in 70% Ethanol benetzt. Pflegen sterile Verfahren für den Rest des Protokolls zu vermeiden, tragen Verschmutzung in die Kulturen. Verwenden von großen gekrümmten Moloney Zange ein Loch in der Eierschale vorsichtig öffnen durch leichtes Antippen auf der abgerundeten Spitze von einem Ei (über der Luftkammer) und dann Greifen und Herausziehen Schalenstücke. </li><li> Mit einer anderen Pinzette entfernen, Membranen und sammeln Embryos durch Schöpfen sie aus dem Ei (darauf achten, daß der Embryo schädigen). Legen Sie sie in eine Schüssel mit CMF-HBSS. HINWEIS: Sammeln Sie so viele Embryonen wie nötig, um die gewünschte Anzahl von Netzhaut-Tassen für Elektroporation zu erhalten, wie in 2.4.4 festgelegt </li><li> Entscheidender Schritt: Stufe werden die Embryonen nach Hamburger und Hamilton 38. WICHTIG: Um eine optimale Effizienz Embryonen zu erhaltenmuss im Stadium 27 (2A) zu sein. </li><li> Euthanize die Embryonen durch Enthauptung mit Moloney Pinzette. </li></ol></li><li> Beziehen Retinal Cups: <ol><li> Mit feinen Dumont Pinzetten vorsichtig entkernen die Augen und Transfer zu einem neuen CMF-HBSS Gericht. </li><li> Ausgehend von dem hinteren Teil jedes Auge, in der Nähe des Sehnervenkopfes und unter Verwendung von zwei feinen Dumont Pinzetten, präsentieren eine Spitze jedes Pinzette zwischen der neuronalen Netzhaut und RPE und sorgfältig sezieren die RPE vorsichtig zu sein, um nicht die Nervenschäden Netzhaut (2B). HINWEIS: Der Mangel an Ca 2+ und Mg 2+ in der Lösung wird die RPE Herauslösen aus dem neuronalen Netzhaut leichter. </li></ol></li><li> Elektroporation: <ol><li> Parameter Elektroporator eingestellt bis 5 Rechteckimpulse von 15 Volt, 50 ms Dauer und 950 ms-Intervall zu liefern. Platz U-förmige negative Elektrode im Inneren Elektroporationskammer (Mikrozentrifugendeckel filled mit 120 ul von Plasmid-enthaltenden Lösung). </li><li> Verwendung gekrümmt Dumont Pinzetten Übertragung einer Netzhautbecher an die Elektroporation Kammer (mit einer Schöpfkelle, nicht drücken), und legen Sie sie im Inneren des U-förmigen Elektrode, mit dem hinteren Teil der Netzhaut in Richtung der Unterseite der Elektrode und dem Objektiv nach oben ( 2D). </li><li> Legen Sie die Anode, so dass sie den vorderen Teil des Auges berührt, neben dem Objektiv, und mit Hilfe der Elektroporator die Fußpedal den gewünschten elektrischen Impulsen. </li><li> Übertragen elektroporiert retinalen Bechers an eine neue Petrischale mit CMF-HBSS und wiederholen Elektroporationsverfahren mit jedem der übrigen Netzhautschalen. HINWEIS: Bis zu 6 retinalen Becher können mit dem gleichen Plasmid Lösung ohne signifikante Einbußen der Transfektionseffizienz elektroporiert werden. </li><li> Entfernen Sie die Linse und Glaskörper jeder Elektroporation Netzhautbecher durch Greifen sie mit Bonn Zahnzange und leichtes Ziehen durch ter Öffnung Schüler weitergegeben, während das Auge an Ort und Stelle mit der Rückseite der gekrümmten Dumont Pinzetten halten. </li><li> Fahren Sie mit der Dissoziation und Kultur der Elektroporation Netzhautzellen nach Standardverfahren. HINWEIS: Für eine detaillierte Protokoll auf der Dissoziation und Kultur der Küken Netzhautzellen beziehen sich auf Vergara und Canto-Soler, 2012 Weitere Datei 4 21 In diesem Protokoll werden die Zellen bei geringer Dichte auf Polyornithin beschichteten 6-Well-Platten oder 35 ausgesät. -mm Gerichte (6 x 10 5 Zellen / 35 mm-Durchmesser gut oder Schale). Das verwendete Medium ist Medium 199 mit Penicillin / Glutamin, 10% fötalem Kälberserum (FCS) und Linolsäure-BSA bei 100 &amp; mgr; g / ml. Die Kulturen werden in einem befeuchteten Inkubator bei 37ºC mit 5% CO 2 gehalten. Mit dieser Vorgehensweise 5 - kann 6 x 10 6 Zellen in der Regel pro jeder Stufe 27 elektroporiert Auge erhalten werden. HINWEIS: Im Idealfall sollte nicht mehr als 3 Stunden zwischen Embryo-Entnahmeeinheiten und Zellbeschichtung übergeben, um eine gute Zelle Überleben zu sichernund minimieren Stress. Ein typisches Timing für Beobachtung und weiteren Analyse ist nach 4 Tagen in Kultur, da an dieser Stelle die Zellen guten morphologischen und molekularen Differenzierung erreicht und das Überleben ist immer noch hoch. </li></ol></li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+gene+promoters+for+liver+expression+by+hydrodynamic+gene+transfer.">Evaluation of gene promoters for liver expression by hydrodynamic gene transfer.</a> J Surg Res. 148 (1), 60-66 (2008).</li><li>Xu, L., 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=CMV-beta-actin+promoter+directs+higher+expression+from+an+adeno-associated+viral+vector+in+the+liver+than+the+cytomegalovirus+or+elongation+factor+1+alpha+promoter+and+results+in+therapeutic+levels+of+human+factor+X+in+mice.">CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice.</a> Hum Gene Ther. 12 (5), 563-573 (2001).</li><li>You, L. M., 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=A+hybrid+promoter-containing+vector+for+direct+cloning+and+enhanced+expression+of+PCR-amplified+ORFs+in+mammalian+cells.">A hybrid promoter-containing vector for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells.</a> Mol Biol Rep. 37 (6), 2757-2765 (2009).</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> Dev Dyn. 195 (4), 231-272 (1992).</li></ol>

The authors declare that they have no competing financial interests or other conflicts of interest.

Der kritischste Schritt für den Erfolg dieses Protokolls ist die Auswahl des geeigneten Stadium der Embryonen. In früheren Veröffentlichungen wird ein Bereich von Embryonalstadien für diesen Kulturen gegeben, die typischerweise von den Tagen Inkubation oder embryonale Tage (ED) definiert sind; somit ist es in der Regel davon ausgegangen, dass die Verwendung von ED 5 bis ED 6 Embryonen zu vergleichbaren Ergebnissen führen. Wir haben jedoch gefunden, dass auf der Bühne 27 (ED &amp; sub5;), wird die Transfektionseffi...

Dissoziierten Zellkulturen von embryonalen Hühnernetzhaut wurden allgemein verwendet, um verschiedene Aspekte der Photorezeptor Zellbiologie, einschließlich deren Überleben 2-9, Differenzierung 10-12, Neuritenwachstum 13 und mehr zu studieren. Die Vorteile dieses Systems, in den 1980er Jahren von Ruben Adler und Mitarbeiter entwickelt und von seinem und anderen Gruppen 14-20, perfektioniert liegen in den intrinsischen Eigenschaften des Kükens als Tiermodell 21. Die Größe des Hühnerauge, auch bei embryonalen Stadien, bietet große Mengen an Material für Kulturen. Außerdem, wenn Kulturen mit embryonalen Tag (ED) durchgeführt 5 - 6 Retinae, 55 - 80% ihrer Vorläuferzellen zu differenzieren, wie Photorezeptoren, 14,15,18,22,23, und da etwa 86% der Photorezeptoren in diesem Tier sind Kegel 24 sind diese Kulturen besonders geeignet für die Studien, die sich auf dieser Zelltyp. Wir haben vor kurzem developed und dadurch eine einfache Technik, die für hocheffiziente Plasmid Transfektion der Zellen in diesen Kulturen ermöglicht, wodurch die Erweiterung der Nützlichkeit dieses Systems durch Erleichterung genetische Studien missexpression 1. Die Entwicklung dieser Technik ergab sich aus einem Hohlraum in der wissenschaftlichen Literatur von Methoden, die ein akzeptables Niveau der Transfektion bereitstellen würde, um für die Untersuchung der Gen-Funktion in einer Zelle autonom zu ermöglichen. Dies ist zum Teil, weil primären neuronalen Kulturen sind notorisch schwer zu transfizieren 25,26. Einige der am häufigsten verwendeten Techniken, die zuvor für diesen Zweck zur Verfügung enthalten chemische Transfektionsverfahren wie Lipofektion oder Calciumphosphat-vermittelte Transfektion, die in Wirkungsgrade in der Grßenordnung von 3-5% führen und kann beträchtliche Toxizität 27-32 auszuüben. Obwohl die Verwendung von Plasmiden mit einem enzymatischen Reporter-Systems kann das Problem der schlechten Transfektionseffizienz durch Verstärken des Signals zu umgehen, sie nichtdiskriminieren zellspezifische Wirkungen, und ihre Ergebnisse werden auf einer kleinen Zellpopulation, die nicht repräsentativ für die gesamte erweisen. Eine andere weit verbreitete Methode in der Küken, RCAS Virus-Infektion, ist nur auf proliferierende Zellen anwendbar und somit nicht für diese primäre Netzhautkultursystem 33 geeignet ist. Im aktuellen Protokoll embryonalen Hühneraugen werden in Stadium 27 (ED &amp; sub5;), der retinalen Pigmentepithel (RPE) wird entfernt, und das Netzhautbecher in einer Elektroporationsküvette Kammer mit einem Plasmid enthaltenden Lösung gefüllt war enukleiert und Elektroporation mit maßgeschneiderten Elektroden, gefolgt von retinalen Dissoziation und Kultur unter Verwendung von Standardtechniken 21. Nach der Optimierung dieses Verfahrens waren wir, ohne die Überlebensrate und Differenzierungsmerkmale der Lage zu Transfektionseffizienzen erzielen durchweg in der Größenordnung von 22% der Gesamtzahl der Zellen in der Kultur und 25% innerhalb der Photorezeptorpopulation AlleinKulturen 1. Hier bieten wir Ihnen ein detailliertes Protokoll umreißt alle wichtigen Schritte dieses Verfahrens, um den Erfolg und die Reproduzierbarkeit dieser Technik zu gewährleisten.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ECM 830 Electro Square Porator</td>
			<td>BTX/ Harvard Apparatus</td>
			<td>45-0052</td>
			<td></td>
		</tr>
		<tr>
			<td>Genetrode, L-Shaped, 5 mm Gold Tip</td>
			<td>BTX/ Harvard Apparatus</td>
			<td>45-0115 model 512</td>
			<td>Gold tipped electrode used as anode</td>
		</tr>
		<tr>
			<td>Polyimide Tubing</td>
			<td>Vention Medical</td>
			<td>custom made</td>
			<td>Internal Diameter: 0.5mm / wall thickness: 0.15-0.2mm. Used for insulating gold tiped electrode</td>
		</tr>
		<tr>
			<td>2.5mm square box filament, 4.5mm wide</td>
			<td>Sutter Instrument Company</td>
			<td>FB245B</td>
			<td>Used to make cathode electrode</td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red</td>
			<td>Gibco - Life Technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Moloney forceps</td>
			<td>Roboz</td>
			<td>RS-8254</td>
			<td>Serrated; Slight Curve; 4.5&#34; Length</td>
		</tr>
		<tr>
			<td>Dumont tweezers 5/45</td>
			<td>Roboz</td>
			<td>RS-5058</td>
			<td>Pattern #5, 45 Degree Angle; .10 X .06mm Tip Size; 109mm Length</td>
		</tr>
		<tr>
			<td>Bonn micro forceps, 1x2 teeth</td>
			<td>Roboz</td>
			<td>RS-5172</td>
			<td>Tying Platform; 1X2 Teeth, 0.12mm Teeth; 3.75&#34; Length; .3mm Tip Width</td>
		</tr>
	</tbody>
</table>

efficient gene transfer in chick retinas for primary cell culture studies: an ex-ovo electroporation approach

Die Kegelphotorezeptor-angereicherten Kulturen von embryonalen Hühnernetzhaut abgeleitet sind zu einem unverzichtbaren Werkzeug für Forscher auf der ganzen Welt das Studium der Biologie der Netzhautneuronen, insbesondere Photorezeptoren. Die Anwendungen dieses Systems gehen über die Grundlagenforschung, da sie leicht zu Hochdurchsatztechnologien zur Wirkstoffentwicklung angepasst werden. Jedoch genetische Manipulation von retinalen Photorezeptoren in diesen Kulturen hat sich als sehr schwierig, sie eine erhebliche Einschränkung für die Nützlichkeit des Systems. Wir haben vor kurzem entwickelt und validiert eine ex ovo Plasmid Elektroporationstechnik die die Rate der Transfektion von Retinazellen in diesen Kulturen erhöht durch das Fünffache im Vergleich zu anderen gegenwärtig verfügbaren Protokolle 1. Bei diesem Verfahren embryonalen Hühneraugen an Stufe 27 ausgeschält, die RPE wird entfernt, und das Netzhautbecher wird in einem Plasmid-haltigen Lösung gegeben und elektroporiert Verwendung einfach aufgebaut Kundenhergestellten Elektroden. Die Netzhaut werden dann dissoziiert und kultiviert Verwendung von Standardverfahren. Diese Technik kann durch die Verwendung von Plasmid-driven RNAi-Technologie zu Expressionsstudien sowie zu der Herunterregulierung der Genexpression verwendet werden, zum Beispiel, allgemein erzielen die Transgen-Expression in 25% der Photorezeptorpopulation. Das Videoformat der vorliegenden Veröffentlichung wird diese Technologie leicht zugänglich für Forscher auf dem Gebiet zu machen, so dass die Untersuchung von Gen-Funktion in der primären Netzhautkulturen. Wir haben auch detaillierte Erläuterungen der kritischen Schritte dieses Verfahrens für ein erfolgreiches Ergebnis und Reproduzierbarkeit.

Hier präsentieren wir ein einfaches Protokoll für Plasmidtransfektion in entkernte Küken Netzhautbecher für nachfolgende dissoziierten Zellkultur. Transfektion durch Elektroporation unter Verwendung einfach individuelle Elektroden (- 2 Die Zahlen 1) Nutzen erzielt. Die in diesem Protokoll beschriebenen Parameter optimiert wurden Transfektionseffizienzen zwischen 20 und 27% (mit einem Durchschnitt von 22%) (Figur 3D) Bereich zu erhalten. Bemerken, dass bei den oben genannten Gründen...

Watch this Scientific Journal Video about Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach at JoVE.com

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonale Küken Netzhautzellkulturen stellen wertvolle Werkzeuge für das Studium der Photorezeptorbiologie. Wir haben ein effizientes Gentransfertechnik, die auf ex ovo Elektroporation von Plasmid der Netzhaut vor der Kultur entwickelt. Diese Technik wesentlich erhöht Transfektionseffizienzen über derzeit verfügbaren Protokolle, was die genetische Manipulation möglich für dieses System.

Effizienten Gentransfer in Küken Netzhäute für primäre Zellkulturstudien: Eine<em&gt; Ex-ovo</em&gt; Elektroporation Ansatz

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

efficient-gene-transfer-chick-retinas-for-primary-cell-culture

Research

JoVE Journal

Developmental Biology

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

8.1K Aufrufe.  The Johns Hopkins University School of Medicine.  Embryonale Küken Netzhautzellkulturen stellen wertvolle Werkzeuge für das Studium der Photorezeptorbiologie. Wir haben ein effizientes Gentransfertechnik, die auf ex ovo Elektroporation von Plasmid der Netzhaut vor der Kultur entwickelt. Diese Technik wesentlich erhöht Transfektionseffizienzen über derzeit verfügbaren Protokolle, was die genetische Manipulation möglich für dieses System. 

Serie: Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Optimierte<em&gt; Ex-ovo</em&gt; Züchtung von Hühnerembryonen zu fortgeschrittenen Entwicklungsstadium

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

Forschung

Entwicklungsbiologie

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Kultur von embryonalen Maus Cochlear Explantate und Gentransfer durch Elektroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Ein optogenetische Ansatz zur Abschätzung Bildung neuronaler Verbindungen in einer Co-Kultursystem

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &amp;#8212; A High-throughput Screen in ES Cells

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Vorwärts genetische Ansatz zur Stressresistenz-Gene in Mäuse Entdecken - Hochdurchsatz-Bildschirm in ES-Zellen

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

Ex-vivo-Kultur von Chick Kleinhirn-Slices und räumlich gezielte Elektroporation von Körnerzellvorstufen

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Gentransfer in das Huhn Hörorgan von<em&gt; In Ovo</em&gt; Micro-Elektroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

Ein Roman<em&gt; Ex Ovo</em&gt; Banding-Technik intrakardialer Hämodynamik in einem embryonalen Hühner System ändern

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Ein Abgetaucht Filterpapier Sandwich für Langzeit<em&gt; Ex Ovo</em&gt; Zeitraffer-Imaging von frühen Hühnerembryonen

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

CRISPR-vermittelten Verlust der Funktionsanalyse in zerebelläre Untermodul Zellen mit In Utero Elektroporation-basierte Gentransfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

In Ovo und Ex Ovo Methoden zur Untersuchung der Entwicklung des Vogelinnenohrs

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...