Wir danken Ekaterina Karelina für die Hilfe bei der Aufnahme Soundtrack für das Video, Ivan Molotkov für 3D-Animation und Dr. Peter Blaesse für CAG-EGFP Plasmid-Präparation. Die Arbeit wurde durch Zuschüsse aus dem Zentrum für Internationale Mobilität von Finnland, Finnish Cultural Foundation und der Academy of Finland unterstützt.

1. Einführung Erstellung von transgenen Tieren ist eine leistungsfähige Methode zur Untersuchung von Genfunktionen in lebenden Tieren 1 und zur Enträtselung Krankheitsmechanismus 2,3 sowie für die Manipulation von Eigenschaften der Zellen 4. Allerdings ist das Verfahren ziemlich aufwendig, extrem zeitaufwendig und teuer, so rechtfertigt die Verwendung von alternativen Gentransfer-Methoden wie virale Injektion 5, neonatalen ventrikulären Injektion mit Elektroporation 6 und in utero Elektroporation 7,8. Die Methode der postnatalen nicht ventrikuläre Injektion und Elektroporation hat eine einzigartige Reihe von Vorteilen: Nutzung von nicht-viralen Gen-Konstrukte, die Möglichkeit zu einem lokalen Expressionsmuster in den Bereich von Interesse zu schaffen; in situ Transfektion von nicht-wandernde Zelltypen, wie zB kortikale Astrozyten. In diesem Video zeigen wir die Einzelheiten des Verfahrens der postnatalen Gentransfer auf die Neugeborenen Rattenhirn mit einem Plasmid kodiert für das Enhanced Green Fluorescent Protein unter chicken beta-Aktin-Promotor mit CMV-Enhancer (pCAG-EGFP) 9. Wir erläutern die Herstellung der Plasmid-haltigen Injektionslösung, die Herstellung von dünnen Glas-Pipetten und Montage der Mikroinjektor auf einem stereotaktischen Instrument. Dann reden wir über betäubende Rattenjungen mit Isofluran, über die Durchführung der Operation, über das Injektionsverfahren und über die Elektroporation mit einer Pinzette Elektroden und die in vivo porator. Schließlich diskutieren wir kurz die zu erwartenden Ergebnisse, Perspektiven und Schwierigkeiten, die während dieser Experimente entstehen können. 2. Plasmid-Präparation für die Elektroporation <ol><li> Wir bereiten meistens 10 ul der Plasmid-Injektionslösung in einer 200 ul dünnwandige Rohr. Diese Lösung ist ausreichend für mehrere Experimente. </li><li> Wir mischen 8 ul der Plasmid-Lösung (siehe Materialien und Geräte) mit 1 ul des 10X PBS (siehe Materialien und Ausrüstung) und 1 ul 0,1% Fast Green-Wasser-Lösung (siehe Materialien und Ausrüstung). </li><li> Die Endkonzentration des Plasmids in der Injektionslösung sollte zwischen 1 und 3 ug / ul werden. Niedrigere Konzentrationen von Plasmid-DNA verringert die Transfektionseffizienz, während die höheren DNA-Konzentration wird zu viskos für die Injektion durch die dünne Spitze der Glaskapillare. </li></ol> 3. Glasnadel Vorbereitung <ol><li> Für die Glaspipette Vorbereitung, verwenden wir Borosilikatglas Kapillare mit einem Filament (siehe Materialien und Ausrüstung) und eine vertikale Elektrode Glas Abzieher (siehe Materialien und Geräte) auf maximale Zugkraft und Heizung Parameter. </li><li> Wir brechen dann die Spitze der Pipette mit einem Stück Papier, um einen Tipp 10-20 um im Durchmesser erreichen. </li><li> Wir überprüfen die Spitze Durchmesser und Form unter dem Mikroskop-Objektiv. </li></ol> 4. Mikroinjektor Assembling <ol><li> Mit einer dünnen Polymerschicht Kapillare (siehe Materialien und Ausrüstung) füllen wir ca. 1 / 3 des Volumens einer 10 ul Hamilton-Spritze (siehe Materialien und Ausrüstung) mit Mineralöl (siehe Materialien und Ausrüstung). Vermeiden Sie Luftblasen! </li><li> Dann füllen wir die Glaspipette mit Mineralöl und legen Sie die Pipette in die Hamilton-Spritze. Vermeiden Sie Luftblasen! </li><li> Die Spritze mit Glaspipette wird dann auf dem Mikroinjektor verbunden mit einem Festwert (siehe Materialien und Ausrüstung). </li><li> Die Mikroinjektion Setup auf einem stereotaktischen Instrument (siehe Materialien und Ausrüstung) fixiert ermöglicht es uns, gefahrlos tauchen die Spitze der Glaspipette in das Rohr mit dem Plasmid Injektionslösung. </li><li> Wenn die Spitze die Oberfläche der Lösung berührt, tauchen sie durch 1 oder 2 mm und füllen Sie die Pipette mit etwa 1 ul der Injektion Mix mit dem Mikro-Injektor-Controller. </li></ol> 5. Narkotisierende Tier Alle Verfahren vorgestellt wurden hier entsprechend der University of Helsinki Regelungen für Tierversuche durchgeführt. <ol><li> Für jedes Experiment, füllen wir eine gasdichte Spritze (siehe Materialien und Geräte) mit ca. 2 ml von Isofluran (siehe Materialien und Ausrüstung). </li><li> Als die Spritze in der Anästhesie-Einheit (siehe Materialien und Ausrüstung) verbunden, um den Luftstrom Quelle festgelegt ist, das Tier-Box und die Maske auf stereotaktische Setup fixiert. </li><li> Auf dem Narkosegerät, passen wir den Luftstrom auf etwa 250 ml / min und die Isofluran-Ebene, um 4%. </li><li> Wir legen Jungtier in das Tier-Box für 2-5 Minuten. </li><li> Wenn der Welpe nicht mehr bewegt, legen Sie es auf dem Heizkissen (siehe Materialien und Geräte) an den stereotaktischen Setup. </li><li> Legen Sie eine rostralen Teil der Welpe den Kopf in die betäubende Maske. </li><li> Es dauert 5 bis 10 Minuten für dieWelpen, die tiefe Narkose geben. </li><li> Überprüfen Narkose Tiefe mit einem Schwanz kneifen und eine Verringerung der Isofluran Zufluss über 1,5 bis 2,0%. </li></ol> 6. Chirurgie, Mikroinjektion und Elektroporation <ol><li> Behandeln Sie die Haut an den Welpen den Kopf mit dem 70% Ethanol. </li><li> Mit einer kleinen Schere (siehe Materialien und Ausrüstung) und dünnen Pinzette (siehe Materialien und Ausrüstung), schneiden Sie die Haut von einem scruff der Welpe seine Stirn. </li><li> Beugen Hautstücke seitwärts und ziehen Ohr Bars leicht in klanglicher Öffnungen des Schädels für Kopf und Haut-Fixierung. </li><li> Mit einem Binokular, suchen wir die Bregma Punkt auf dem Schädel. </li><li> Identifizieren Sie die Region von Interesse mit der stereotaktischen Koordinaten. </li><li> Positionieren Sie den Injektionspipette über diesem Bereich, und markieren Sie durch Fallenlassen 25-50 nl der Injektionslösung auf der Kopfoberfläche. </li><li> Drill des Schädels sanft und vorsichtig an der markierten Stelle mit einem High-Speed-chirurgische Bohrer (siehe Materialien und Geräte) unter dem Mikroskop, bis die Flüssigkeit wird in die gebohrten Gebiet. </li><li> Vereinbaren Sie einen electropotation Pinzette Elektroden (siehe Materialien und Geräte) an den Seiten des Schädels mit aktuellen leitfähiges Gel (siehe Materialien und Ausrüstung), um eine bessere Leitfähigkeit zu erreichen. </li><li> Entfernen Sie die Flüssigkeit Drop aus dem Bohrloch mit kleinen Tampons (siehe Materialien und Ausrüstung) und tauchen Sie das Glas Nadel in das Loch nach den Koordinaten der Injektionsstelle. </li><li> Infuse von 25 bis 100 nl der Injektionslösung in Höhe von 5 bis 20 nl / sec. </li><li> Schnelles Entfernen der Pipette und elektroporieren sofort. Elektroporation ist durch die Anwendung fünf Rechteck-Impulsen mit der Dauer von 50 ms und Amplitude von 99 V bei einer Frequenz von 1 Hz durchgeführt. </li><li> Entfernen Sie Elektroden und Ohr Bars. </li><li> Nähen Sie den Schnitt der Haut durch eine Kombination eines stummen (siehe Materialien und Ausrüstung) und scharfen Zangen und chirurgische Fäden (siehe Materialien und Ausrüstung). </li><li> Platzieren Sie den Welpen in die warme Kammer für 15-30 Minuten, um ihre Erholung nach der Narkose zu unterstützen. </li><li> Dann legen Sie den Welpen zurück zu seiner Mutter in den Käfig. </li></ol> 7. Repräsentative Ergebnisse Die Expression des Transgens in den erfolgreich transfizierten Zellen erscheinen etwa 10 Stunden nach der Elektroporation, und es bleibt für mehrere Wochen stabil. Wir mikroinjiziert das Plasmid entweder in tiefen kortikalen Schichten oder Striatum Region postnatalen Tag 2 (P2) Wistar Rattenjungen und durchgeführt in vivo Elektroporation wie oben beschrieben. Hirnschnitten (400 um Dicke) wurden nach 2 bis 6 Tage verkürzt (P4-P8) mit einem vibrotome in kaltes Schneiden Medium (Hepes gepuffert Earle Balanced Salt Solution) 10. Bildaufnahme erfolgte mit Zeiss Axioplan 2 LSM5 Pascal konfokalen Mikroskop (Zeiss, Deutschland) in der Slicing-Medium bei Raumtemperatur. Die Injektionsstellen in tiefen kortikalen Schichten und Striatum enthielt viele transfizierten Zellen, die EGFP, dass in einem kompakten Region von etwa 200-300 &amp;mgr; m im Durchmesser (Abbildung 1A, B) befanden. Innerhalb transfizierten Zellen, die EGFP Expression ausreichend hoch, um Bildgebung von dünnen zellulären Prozessen (Abbildung 1C) mit Dendriten mit Stacheln in verschiedenen Formen (Abbildung 1D) und Axon-Bundles (Abb. 1E) zu ermöglichen. Die robuste Stabilität des EGFP als auch die Lebensfähigkeit der Zellen nach der Elektroporation erlaubt Tracing distalen Teile der Axone (bis 1,5-2 mm vom Zellkörper) wie kortikale Terminals (Abbildung 1F). <img src="/files/ftp_upload/2244/2244fig1.jpg" alt="Abbildung 1" /> Abbildung 1. (A) erfolgreich Zellen in das Striatum Region P4 Rattenhirn Abgrenzung der Plasmid Injektionsstelle transfiziert. (B) transfizierten Zellen in tiefen kortikalen Schichten (ca. 1000 um unten kortikalen Oberfläche) P5 Rattenhirn. (C) A transfizierten Zelle in das Striatum Region P8 Rattenhirn. (D) Dendritische Prozesse einer transfizierten Zelle in das Striatum Region P8 Rattenhirn; Pfeile zeigen verschiedene Arten von dendritischen Dornen und Filopodien. (E) Axone von transfizierten Zellen innerhalb Kollateralweg. (F) Axone enden an der Pia-Oberfläche P8 Rattencortex (Pfeilspitze). Scale-Bars sind 100 um auf A, B, E und F; 10 um auf C und D.

<ol>
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F., Holmes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ascent+of+mouse:+advances+in+modeling+human+depression+and+anxiety.">The ascent of mouse: advances in modeling human depression and anxiety.</a> Nat. Rev. Drug Discov. 4, 775-790 (2005).</li><li>Wells, T., Carter, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+neural+function+in+transgenic+rodents:+towards+a+comprehensive+strategy.">Genetic engineering of neural function in transgenic rodents: towards a comprehensive strategy.</a> J. Neurosci. Methods. 108, 111-130 (2001).</li><li>Pilpel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=reproducible+transduction+of+select+forebrain+regions+by+targeted+recombinant+virus+injection+into+the+neonatal+mouse+brain.">reproducible transduction of select forebrain regions by targeted recombinant virus injection into the neonatal mouse brain.</a> J. Neurosci. Methods. 182, 55-63 (2009).</li><li>Boutin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+vivo+electroporation+of+the+postnatal+rodent+forebrain.">Efficient in vivo electroporation of the postnatal rodent forebrain.</a> PLoS One. 3, e1883-e1883 (2008).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev. Biol. 240, 237-246 (2001).</li><li>Saito, T. <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+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat. Protoc. 1, 1552-1558 (2006).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proc. Natl. Acad. Sci. U S A. 101, 16-22 (2004).</li><li>De Simoni, A., Yu, L. 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Methoden der Gentransfer in lebende Nagetier Gehirn sind gut für die in utero Elektroporation 7,8,11,12 gegründet und seit kurzem auch für die postnatale Elektroporation 6. Allerdings sind diese Methoden auf intraventrikuläre Injektion der Plasmid-DNA, die für mehrere Anwendungen kann die Begrenzung basiert. Zum Beispiel, haben diese Methoden nicht zulassen, dass Targeting-Zellen in bestimmten Bereichen des Gehirns wie Hippocampus, noch die Transfektion von solchen nicht-wandernde Zel...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2A-sa dumb Tweezers, 115mm</td>
<td>equipment</td>
<td>XYtronic</td>
<td>XY-2A-SA</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Biological Temperature Controller with stainless steel heating pad</td>
<td>equipment</td>
<td>Supertech</td>
<td>TMP-5b</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate tube with filament</td>
<td>material</td>
<td>Sutter Instruments</td>
<td>BF120-69-10</td>
<td>Glass needle</td>
</tr>
<tr>
<td>Disposable drills</td>
<td>material</td>
<td>Meisinger</td>
<td>HP 310 104 001 001 008</td>
<td>&nbsp;</td>
<tr>
<td>Dulbeco's PBS 10X</td>
<td>reagent</td>
<td>Sigma</td>
<td>D1408</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps, 110 mm</td>
<td>equipment</td>
<td>FST</td>
<td>91150-20</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Ealing microelectrode puller</td>
<td>equipment</td>
<td>Ealing</td>
<td>50-2013</td>
<td>Vertical electrode glass puller</td>
</tr>
<tr>
<td>Ethilon monofil polyamide 6-0 FS-3 16 mm 3/8c</td>
<td>material</td>
<td>Johnson &amp; Johnson Medical</td>
<td>EH7177H</td>
<td>Surgical threads</td>
</tr>
<tr>
<td>Exmire micro syringe 10.0 ml</td>
<td>equipment</td>
<td>Exmire</td>
<td>MS*GLLX00</td>
<td>Gas-tight syringe</td>
</tr>
<tr>
<td>Fast Green</td>
<td>reagent</td>
<td>Sigma</td>
<td>F7252</td>
<td>&nbsp;</td>
<tr>
<td>Forceps electrodes</td>
<td>equipment</td>
<td>BEX</td>
<td>LF650P3</td>
<td>Treat with 70% ethanol for disinfection prior to use</td>
</tr>
<tr>
<td>Foredom drill control</td>
<td>equipment</td>
<td>Foredom </td>
<td>FM3545</td>
<td>Surgical drill power supply and control. Currently available analogue is micromotor kit K.1070 (Foredom) </td>
</tr>
<tr>
<td>Foredom micro motor handpiece</td>
<td>equipment</td>
<td>Foredom</td>
<td>MH-145</td>
<td>Currently available analogue is micromotor kit K.1070 (Foredom)</td>
</tr>
<tr>
<td>Gas anesthesia platform for mice</td>
<td>equipment</td>
<td>Stoelting</td>
<td>50264</td>
<td>Assembled on stereotaxic instrument</td>
</tr>
<tr>
<td>Isoflurane</td>
<td>reagent</td>
<td>Baxter</td>
<td>FDG9623</td>
<td>&nbsp;</td>
<tr>
<td>Micro dressing forceps, 105 mm</td>
<td>equipment</td>
<td>Aesculap</td>
<td>BD302R</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Microfil</td>
<td>material</td>
<td>WPI</td>
<td>MF34G-5</td>
<td>Micro syringe filling capillaries</td>
</tr>
<tr>
<td>Mineral oil</td>
<td>reagent</td>
<td>Sigma</td>
<td>M8410</td>
<td>&nbsp;</td>
<tr>
<td>NanoFil Syringe 10 microliter</td>
<td>equipment</td>
<td>WPI</td>
<td>NANOFIL</td>
<td>Hamilton syringe</td>
</tr>
<tr>
<td>plasmid CAG-EGFP</td>
<td>reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Extracted and purified with EndoFree Plasmid Maxi Kit (Qiagen) and dissolved in nuclease free water to concentration 1.5 mg/ml</td>
</tr>
<tr>
<td>Pulse generator CUY21Vivo-SQ</td>
<td>equipment</td>
<td>BEX</td>
<td>CUY21Vivo-SQ</td>
<td>&nbsp;</td>
<tr>
<td>Schiller electrode gel</td>
<td>reagent</td>
<td>Schiller AG</td>
<td>2.158000</td>
<td>Conductive gel</td>
</tr>
<tr>
<td>Small animal stereotaxic instrument</td>
<td>equipment</td>
<td>David Kopf Instruments</td>
<td>900</td>
<td>&nbsp;</td>
<tr>
<td>Stoelting mouse and neonatal rat adaptor</td>
<td>equipment</td>
<td>Stoelting</td>
<td>51625</td>
<td>Assembled on stereotaxic instrument.
Treat earbars with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Student iris scissors, straight 11.5 cm</td>
<td>equipment</td>
<td>FST</td>
<td>91460-11</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Sugi absorbent swabs 17 x 8 mm</td>
<td>material</td>
<td>Kettenbach</td>
<td>31602</td>
<td>Surgical tampons</td>
</tr>
<tr>
<td>UMP3 microsyringe pump and Micro 4 microsyringe pump controller</td>
<td>equipment</td>
<td>WPI</td>
<td>UMP3-1</td>
<td>Microinjector and controller</td>
</tr>
<tr>
<td>Univentor 400 Anesthesia Unit</td>
<td>equipment</td>
<td>Univentor</td>
<td>8323001</td>
<td>&nbsp;</td>		</tbody>
		</table>
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gene delivery to postnatal rat brain by non-ventricular plasmid injection and electroporation

Erstellung von transgenen Tieren ist ein Standard-Ansatz bei der Untersuchung von Funktionen eines Gens von Interesse in vivo. Allerdings sind viele Knockout oder transgenen Tieren, die nicht in den Fällen, wo das modifizierte Gen exprimiert oder gelöscht wird in den gesamten Organismus lebensfähig. Darüber hinaus eine Vielzahl von Kompensationsmechanismen machen es oft schwierig, die Ergebnisse zu interpretieren. Die kompensatorische Effekte können entweder durch Timing der Genexpression oder die Begrenzung der Höhe von transfizierten Zellen vermindert werden. Die Methode der postnatalen nicht ventrikuläre Mikroinjektion und in vivo Elektroporation ermöglicht die gezielte Verabreichung von Genen, siRNA oder Farbstoffmoleküle direkt auf eine kleine Region des Interesses an der neugeborenen Nagern Gehirn. Im Gegensatz zu herkömmlichen ventrikuläre Injektionstechnik ermöglicht diese Methode die Transfektion von nicht-wandernde Zelltypen. Tiere mit Hilfe der hier beschriebenen Methode transfiziert kann, zum Beispiel für Zwei-Photonen werden in-vivo-Bildgebung oder in elektrophysiologischen Experimenten an akuten Hirnschnitten.

Watch this Scientific Journal Video about Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation at JoVE.com

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Dieses Protokoll beschreibt eine nicht-virale Methode der Lieferung von Gen-Konstrukte zu einem bestimmten Bereich des Gehirns lebende Nagetier. Die Methode besteht aus Plasmid-Präparation, Mikropipette Fertigung, neugeborenen Ratten pup Chirurgie, Mikroinjektion des Konstrukts und<em&gt; In vivo</em&gt; Elektroporation.

Gene Lieferung in Postnatale Rattengehirn von Non-ventrikuläre Plasmid Injektion und Elektroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

gene-delivery-to-postnatal-rat-brain-non-ventricular-plasmid

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JoVE Journal

Neuroscience

15.0K Aufrufe.  University of Helsinki. Dieses Protokoll beschreibt eine nicht-virale Methode der Lieferung von Gen-Konstrukte zu einem bestimmten Bereich des Gehirns lebende Nagetier. Die Methode besteht aus Plasmid-Präparation, Mikropipette Fertigung, neugeborenen Ratten pup Chirurgie, Mikroinjektion des Konstrukts und In vivo Elektroporation.

Serie: Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In utero Elektroporation gefolgt von primären neuronalen Kultur für ein Studium Gene Function in Subset von kortikalen Neuronen

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Forschung

Neurowissenschaften

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

Mosaic Analyse der Genfunktion in Postnatale Mäusehirnen mithilfe von Virus-basierten Cre Rekombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

Effiziente Gene Lieferung in mehrere ZNS Territories mit In Utero Elektroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

Maus in Utero Elektroporation: Kontrollierte Spatiotemporal Gene Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Gentransfer an sich entwickelnden Maus Innenohr<em&gt; In Vivo</em&gt; Die Elektroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Erweiterung der embryonalen und adulten neuralen Stammzellen<em&gt; In Utero</em&gt; Elektroporation oder Viral stereotaktische Injektion

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

In-vivo-Elektroporation und Postnatale Zeitraffer-Imaging Neuroblast von Migration in Maus Akute Hirnschnitten

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Subtyp-selektive Elektroporation kortikaler Inter

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Die Analyse der Genexpression Veränderungen im Hippocampus Ratte nach Tiefenhirnstimulation des Anterior Thalamuskern

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...