Diese Arbeit wurde vom RIKEN Brain Science Institute (BSI), Human Frontier Science Program (HFSP) (verliehen TS) und RIKEN Junior Research Associate Program (JRA, verliehen an MK) RIKEN Brain Science Institute (BSI), Human Frontier Science Program gefördert (HFSP) (verliehen TS) und RIKEN Junior Research Associate Program (JRA, verliehen an MK) gefördert dieser Arbeit.

1. Vorbereitung der Kapillar-und DNA-Lösung zur Injektion <ol><li> Purify Plasmid-DNA mit einem Maxi-Prep oder gleichwertiges Verfahren, um zu einer endgültigen Konzentration von 2-3 ug / ul. Aliquot 100 ug DNA, fügen Sie 10 &amp;mgr; l der schnellen grünen Farbstoff (1% Aktien) und TE (10 mM Tris Base, 1mM EDTA-Lösung, pH 8,0) und stellen ein Volumen von 100 ul die endgültige DNA-Konzentration zur Injektion Mix 1 g / machen ul. Die Konzentration von Plasmid-DNA verändert werden kann, hängt von der Größe des Plasmids, und auch ihre Transfektionseffizienz, die einzeln geprüft werden muss. </li><li> Drehen Sie die DNA-Lösung für 5 Minuten bei 14.000 RPM bei Raumtemperatur und sammeln Überstand, um alle Rückstände zu entfernen. </li><li> Ziehen Sie eine Glaskapillare tubea Glaskapillaren mit einer Mikropipette Abzieher. Pinch off der Spitze mit einer Pinzette, um die Spitze hat einen Durchmesser von 20-30 um. Messen Sie 800 um-1 mm von der Spitze und überprüfen Sie die Außendurchmesser von weniger als 60 pm (Abb. 1A). </li><li> Schließen Sie die Kapillare zu den Mikromanipulator, und füllen Sie mit Mineralöl. Um die Kapillare mit der DNA-Lösung, tauchen die Spitze in die Lösung zu füllen und zu saugen bis die DNA-Lösung. </li></ol> 2. Stick-Platin-Elektroden Hier haben wir Stick Platinelektroden (Nepe Gen, CUY611P2-1) in oberflächennahen Bereich des Gehirns, wie der Cortex (Abb. 1F) elektroporieren. 3. Vorbereitung von Mikroelektroden Verwenden Sie Mikro-Elektroden in tiefen Teilen des Gehirns (dh, Thalamus und Hypothalamus) (Abb. 2B-E) elektroporiert. <ol><li> Ein Ende des Wolfram (für negative Elektroden) und Platin (für positive Elektroden) Drähte auf vergoldeten gewickelt und fixiert mit Parafilm. Führen Sie den Draht um den Stamm ein Wattestäbchen und wickeln Sie beide Enden mit Parafilm (Abb. 1B). </li><li> Gleichmäßig schärfen die Spitze des Drahtes mit Sandpapier, bis sie 20-30 um Außendurchmesser und 60 pm von 800 um-1 mm von der Spitze (Abb. 1C und D) erreicht. </li><li> Tragen Sie eine dünne Schicht Nagellack auf den Draht für die Isolierung. Nach der Nagellack getrocknet ist, verwenden Sie ein Aceton getränkten Wattestäbchen, um es von der Spitze (etwa 200 um von der Spitze) zu entfernen. Wichtiger Hinweis: Um Schäden an der Maus die Gebärmutter zu verhindern, ist der Anteil innerhalb von 800 um-1 mm von der Spitze sollte nicht mehr als 70 um im Durchmesser (Abb. 1E) werden. </li></ol> 4. Vorbereitung der Operation <ol><li> Anesthetize eine schwangere Maus (E9.5-E15.5). Wir zeigen, Betäubung mit pharmazeutischer Qualität Natrium-Pentobarbital (Tabelle II; 50 ug pro Gramm Körpergewicht, intraperitoneal injiziert, und warten Sie 5 min). Alternativen zum Natrium-Pentobarbital als Narkosemittel beinhalten Ketamin / Xylazin Injektion oder Gas Anästhetika (Gas ist bevorzugt kurzer Dauer Verfahren). Die Injektion mit Aufmerksamkeit sollte getan werden, um alle Organe zu vermeiden, sonst wird es dazu führen, dass die Gebärmutter schädigen oder töten Tier. Als erfolgreiche IP geschehen ist, ist die Überlebensrate mehr als 95%. Auch die Chirurgie kann in Open-Air-Umgebung durchgeführt werden. Bevor begann die Operation, Test für eine fehlende Reaktion durch das Tier zu kneifen ihre Fußspitze Betäubung bestätigen. </li><li> Shave das Haar aus dem Bauch mit einer Rasierklinge und 50% EtOH. Bereiten Sie die Haut mit wechselnden scrubs von betadine und Alkohol (70%). </li><li> Machen Sie einen Schnitt am Bauch-Mittellinie mit einer feinen Schere und ziehen Sie alle Uterushörner vorsichtig auf 37 ° C vorgewärmten Phosphat-gepufferter Salzlösung (PBS)-angefeuchtet Baumwollgaze, die um die Wunde gelegt wird. Halten Sie die Gebärmutter feucht mit PBS die ganze Zeit. </li><li> Halten Sie eine flexible Glasfaser-Kabel zwischen dem Zeige-und Mittelfinger und legen Sie es unter dem Uterushorn. Saubere Kabel mit 70% EtOH vor dem Gebrauch. Kein Mikroskop oder Lupe ist für die Visualisierung benötigt. Position Gebärmutter zwischen den Glasfaser-Licht-Optik-Kabel und den Daumen, und drücken Sie sanft nach oben zu drücken den Embryo näher an der Gebärmutterwand. </li></ol> 5. Die Injektion von DNA und Elektroporation <ol><li> Als Embryo positioniert ist, legen Glas sorgfältig Kapillare in Ziel Herzkammer und spritzen ca. 1 ul DNA-Lösung (Abb. 2A). </li><li> Für die Elektroporation auf oberflächliche Teil des brainIf Verwendung mit Stick Platin-Elektroden für electoporation, legen Sie die Elektroden außerhalb der Gebärmutter und gelten vorgeschlagen Rechtecksignale Stromimpulse nach Herstellung der Anweisung (Tabelle I). Wenn usingFor Elektroporation zu tieferen Teil des Gehirns, nmicro Elektroporation werden eedle Typ Elektroden verwendet. Iinsert einem feinen Wolfram negative Elektrode in die DNA injiziert Ventrikel und einer Platin-Elektrode in die Gebärmutter und Ort Zielregion zwischen zwei Elektroden, dann schlug Rechtecksignale Stromimpulse (Tabelle I) (Abb. 2B) gelten. </li></ol> 6. Beitrag Elektroporation <ol><li> Legen Sie die Uterushorn zurück in seine original Lage mit und fügen Sie 500 ul von 37 ° C vorgewärmten PBS. </li><li> Suture die innere Schicht mit chirurgischem Nahtmaterial und schließen äußere Schicht mit einem 9 mm autoclipauto Clip. </li><li> Legen Sie Tiere auf einem Heizkissen für 2 Stunden, damit aus der Narkose. Analgetika wie Carprofen oder Buprenorphin kann für postoperative Schmerzen verabreicht werden. </li></ol> 7. Repräsentative Ergebnisse: Einige Beispiele der kortikalen Elektroporation von pCAG-EYFP konstruieren mit Stick Platinelektroden zu verschiedenen Zeitpunkten sind in Abbildung Abbildung 32 dargestellt. Brains sind E13.5, E14.5 und E15.5 elektroporiert und geerntet postnatalen Tag (P) 6. ADie ntibody Färbung RORC zeigt Lage der Schicht 4 und die Position der EYFP transfizierten Zellen durch GFP Antikörper-Färbung werden aufgedeckt, und die beiden Bilder sind super verhängt (Abb. 3A und B). Labeled kortikalen Zellen Schicht der kortikalen Zellen elektroporiert unterscheidet sich deutlich in jedem Experiment (Abb. 32), die zeitabhängige Elektroporation macht Corticalis-spezifische GOF / LOF möglich suggeriert. Die Lebensfähigkeit von Embryonen und die Effizienz der Transfektion von Zellen variiert nach Alter des Embryos und die Lage der Elektroporation abhängig. Die Proben in Tabelle 1 aufgelistet sind, jedoch sollten die Bedingungen für jede Elektroporation je auf der Bühne und ein Teil des Gehirns optimiert werden. Elektroporation in eine tiefe Gewebe wie Thalamus und Hypothalamus mit der Verwendung von Platin-Elektroden-Stick geben oft geringe Effizienz (Tabelle 1). Dieses Problem kann durch Verwendung von Mikro-Elektroden whichelectrodes, die tiefen Teile des Gehirns erreichen wird gelöst werden. Abbildung 3 4 zeigt beispielsweise der pCAG-EYFP Elektroporation in den Entwicklungsländern Zwischenhirn. Plasmid-DNA-Lösung wird in der 3. Ventrikel bei E11.5 injiziert, gefolgt von Mikro-Elektroporation (Abb. 3C4C). Die Elektroporation Bereich ist in den Thalamus beschränkt, wo die meisten Axone zum Kortex (Abb. 3B4B). Axon-Projektion der Schicht 4 des Kortex kann auch visualisiert EYFP, die bis fillesfills das gesamte Neuron. (Abb. 3A4A). <img src="/files/ftp_upload/3024/3024fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Cartoon schematische demonstriert die richtige Form der Kapillare. (A) A Glaskapillare gezogen wird mit einer Mikropipette puller bestimmte Form zu machen. Ein Tipp ist abgeschnürt durch Zangengeburt zu 20-30 um zu machen. Messen Sie 800 um-1mm von der Spitze (rote Klammer) und stellen Sie sicher, dass externe Durchmesser von weniger als 60 pm (grüne Klammer) ist. (B) Ein Ende des Wolfram-oder Platin-Drähte auf vergoldeten gewickelt und fixiert mit Parafilm. Dann nutzen Sie Schleifpapier, ihre Spitze stellen, um 20-30 um Außendurchmesser und 60 um 1 mm von der Spitze zu werden. (C) Bild von der Wolfram-Draht vor der Formgebung. (D) Bild von der Wolframdraht nach der Formgebung. (E) Bild von der Wolframdraht nach dem Auftragen dünn Nagellack. Maßstabsbalken in E ist 10 pm (kleinste Skala) für CE. (F) Stick Platinelektroden (Nepa Gen. CUY611P2-1). Balken in F ist 2 mm. <img src="/files/ftp_upload/3024/3024fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Cartoon-Schema des chirurgischen Eingriffs. (A) Schematische Darstellung der Injektion in Seitenventrikel oder großen Embryonen (links) und in 3 rd Ventrikels oder kleine Embryonen (rechts). (B) Schematische Darstellung der electrporation durch Platin Stabelektrode (links) und Nadel Elektrode (rechts). <img src="/files/ftp_upload/3024/3024fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Entwickeln Maus Telencephalon wurde mit pCAG-EYFP bei E13.5 (A), E14.5 (B) und E15.5 (C) elektroporiert und geerntet P6. Die Gehirne wurden geschnitten koronalen und gefärbt mit GFP-Antikörper oder Antikörper RORC getrennt und Bilder zusammengeführt werden. (A) Elektroporation von pCAG-EYFP Plasmid bei E13.5 transfizieren vielen Schicht 4 Neuronen. die Überschneidung mit Schicht 4 Marker RORC. (B) Elektroporation von pCAG-EYFP Plasmid bei E14.5 Transfektion von Zellen, die mehr oberflächlich als Schicht 4 Neuronen. (C) E15.5 Elektroporation label viele oberflächliche Neuronen wie Layer 2 / 3. Maßstabsbalken 200 um. <img src="/files/ftp_upload/3024/3024fig4.jpg" alt="Abbildung 4"/> Abbildung 4. Transfektion von EYFP in thalamo-kortikalen Axone (TCA). (A) TCA Endstation in kortikalen Schicht 4 wird durch GFP-Antikörper-Färbung zeigte. RORC Antikörper-Färbung wird verwendet, um eine Position der Schicht 4 zu enthüllen. Beide Bilder stammen aus demselben Abschnitt übernommen und zusammengeführt. (B) Position der Elektroporation in den Thalamus wird auch von GFP Antikörper-Färbung und RORC Färbung, die ihren Ausdruck in den Thalamus 11 begrenzt wird angezeigt. (C) Cartoon Schaltplan für thalamischen Elektroporation und TCA-Projektion gezeigt. Maßstabsbalken 200 um.

<ol>
	<li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+Visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: Visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</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> Developmental Biology. 240, 237-246 (2001).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294, 1071-1074 (2001).</li><li>Kataoka, A., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf8+controls+regional+identity+in+the+developing+thalamus.">Fgf8 controls regional identity in the developing thalamus.</a> Development. 135, 2873-2881 (2008).</li><li>Imayoshi, I., Shimogori, T., Ohtsuka, T., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hes+genes+and+neurogenin+regulate+non-neural+versus+neural+fate+specification+in+the+dorsal+telencephalic+midline.">Hes genes and neurogenin regulate non-neural versus neural fate specification in the dorsal telencephalic midline.</a> Development. 135, 2531-2541 (2008).</li><li>Shimogori, T., Banuchi, V., Ng, H. Y., Strauss, J. B., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+signaling+centers+expressing+BMP,+WNT+and+FGF+proteins+interact+to+pattern+the+cerebral+cortex.">Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex.</a> Development. 131, 5639-5647 (2004).</li><li>Shimogori, T., Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+application+with+in+utero+electroporation+in+mouse+embryonic+brain.">Gene application with in utero electroporation in mouse embryonic brain.</a> Development Growth &#38; Differentiation. 50, 499-506 (2008).</li><li>Takahashi, Y., Watanabe, T., Nakagawa, S., Kawakami, K., Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+Embryology.">Avian Embryology.</a> Methods in Cell Biology. Vol 87, 271-271 (2008).</li><li>Feil, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-activated+site-specific+recombination+in+mice.">Ligand-activated site-specific recombination in mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 10887-10890 (1996).</li><li>Indra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally-controlled+site-specific+mutagenesis+in+the+basal+layer+of+the+epidermis:+comparison+of+the+recombinase+activity+of+the+tamoxifen-inducible+Cre-ERT+and+Cre-ERT2+recombinases.">Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases.</a> Nucleic Acids Research. 27, 4324-4327 (1999).</li><li>Nakagawa, Y., O'Leary, D. D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+patterned+expression+of+orphan+nuclear+receptor+genes+ROR+alpha+and+ROR+beta+in+developing+mouse+forebrain.">Dynamic patterned expression of orphan nuclear receptor genes ROR alpha and ROR beta in developing mouse forebrain.</a> Developmental Neuroscience. 25, 234-244 (2003).</li></ol>

Keine Interessenskonflikte erklärt.

In diesem Protokoll haben wir nur beschrieben Vorteile der Technik der pCAG-EYFP Transfektion in den geschützten Bereich einer kleinen embryonalen Gehirn mit verschieden geformten Elektroden. Zum Vergleich der Transfektionseffizienz von anderen Promotor zuvor beschrieben wird, zeigt, welche Zelltypspezifität 1. Allerdings gibt es Einschränkungen für die Technik, wie zum Beispiel die Transfektion ist nur vorübergehend und variiert abhängig von der Plasmid, es muss nicht jede Zelle Spezifität und es ist schwer, die Zahl der transfizierten Zellen zu steuern. ThereforeHowever, wird die Kombination dieses Protokoll mit anderen Techniken weiter bieten Möglichkeiten für verschiedene Methoden zur Manipulation. Zunächst wird Einbeziehung zellspezifischen Promotors in Plasmid-DNA ermöglichen zelltypspezifisch Transfektion, wie Neuronen, Gliazellen und Astrozyten. Zweitens, da die Transfektion transient ist, ist es nicht geeignet für Wissenschaftler, die die Funktion des Gens im späteren Leben analysieren möchten. Daher wird die Kombination mit einer Transposase, um Plasmid-DNA in das Genom integrieren sie in eine stabile Transfektion 8 zu konvertieren. Als nächstes Annahme des Tet-On-oder Tet-Off-System wird es möglich, den Zeitpunkt oder Zelltyp-spezifische Gen-Expression in neuronalen Zellen 8 manipulieren. Alternativ kann man dieses Protokoll mit Tamoxifen-induzierbare Cre-ER (T) Rekombinasen 9 und Reporter-Mäuse 10 zu kombinieren. Diese breite Zugänglichkeit von Geweben wird sich drastisch verändern experimentellen Designs in den Neurowissenschaften eingesetzt.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Bare Wolfram Drahtdurchmesser 125 pm </td><td> AM-Systeme </td><td> 797600 </td></tr><tr><td> blanken Platindraht Durchmesser 125 pm </td><td> AM-Systeme </td><td> 767000 </td></tr><tr><td> Stabelektroden </td><td> Nepa-Gen </td><td> CUY610P4-1 </td></tr><tr><td> Glaskapillare </td><td> Stölting </td><td> 50611 </td></tr><tr><td> Mikromanipulator </td><td> KD Scientific </td><td> KDS310 </td></tr><tr><td> Schere </td><td> Roboz </td><td> RS-5865 </td></tr><tr><td> Impulsgeber </td><td> AM-Systeme </td><td> Modell 2100 </td></tr><tr><td> 9 mm autoclip </td><td> Roboz </td><td> RS-9260 </td></tr><tr><td> mit vergoldeten </td><td> WPI </td><td> 5482 </td></tr><tr><td> RORC Antikörper </td><td> Perseus Proteomics </td><td> H3925 </td></tr></tbody></table>

mouse in utero electroporation: controlled spatiotemporal gene transfection

Um zu verstehen, die Funktion von Genen in bestimmten Regionen des sich entwickelnden Gehirns, einschließlich Signalmolekülen und Axon Führung Moleküle, lokale Gentransfer oder Knock-out ist erforderlich, zum Ausdruck gebracht. Gene targeting knock-in oder Knock-out in die lokalen Regionen möglich ist, mit Kombination mit einer bestimmten CRE Linie, die mühsam, kostspielig ist, und zeitaufwändig durchzuführen. Daher wird eine einfache Transfektion Methode, eine in utero Elektroporation Technik, die mit kurzen Zeit durchgeführt werden kann, sehr nützlich sein, um die mögliche Funktion der Kandidatengene vor der Erzeugung von transgenen Tieren 1,2 zu testen. Zusätzlich zu dieser, in utero Elektroporation Ziele Bereichen des Gehirns, wo keine besonderen CRE Linie existiert und wird begrenzt embryonalen Letalität 3,4. Hier präsentieren wir eine Methode, in utero Elektroporation Kombination von zwei verschiedenen Arten von Elektroden für die einfache und bequeme Gentransfer in Zielgebieten des sich entwickelnden Gehirns. Zunächst wird eine einmalige Beteiligung Methode von Embryonen mit Hilfe eines optischen Lichtleiter-Kabel machen kleine Embryonen (von E9.5) sichtbar für die gezielte DNA-Lösung Injektion in Ventrikel und Nadel-Typ Elektroden Einsetzen der gezielten Hirnareal 5,6. Die Strukturierung des Gehirns wie kortikalen Bereich auftreten in einem frühen embryonalen Stadium, daher sind diese frühen Elektroporation von E9.5 einen großen Beitrag zur gesamten Bereich Strukturierung Ereignis zu verstehen. Zweitens verhindert die genaue Form einer Kapillare uterine Schäden, indem sie Löcher durch Einsetzen der Kapillare. Darüber hinaus sind die genaue Form der Nadelelektroden mit Wolfram-und Platin-Draht geschaffen und geschärft mit Schleifpapier und isoliert mit Lack 7, eine Methode, die im Detail in diesem Protokoll beschrieben ist. Diese einzigartige Technik erlaubt die Transfektion von Plasmid-DNA in eingeschränkten Bereichen des Gehirns und ermöglicht es kleinen Embryonen elektroporiert werden. Dies wird dazu beitragen, ein neues Fenster öffnen für viele Wissenschaftler, die auf die Zelldifferenzierung, Zellwanderung, Axon Anleitung arbeiten in einem sehr frühen embryonalen Stadium. Darüber hinaus wird diese Technik den Wissenschaftlern ermöglichen, Plasmid-DNA in tiefen Teilen des sich entwickelnden Gehirns wie Thalamus und Hypothalamus, wo nicht viele regionsspezifische CRE Linien existieren für gain of function (GOF) oder Funktionsverlust (LOF) Analysen zu transfizieren.

Watch this Scientific Journal Video about Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection at JoVE.com

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Ein Gentransfer-Methode in den Entwicklungsländern Gehirn der Maus ist mit einer einzigartigen chirurgischen Verfahren und spezielle Form der Elektroden beschrieben. Diese einzigartige Technik erlaubt die Transfektion von Plasmid-DNA zeitlich und räumlich, was viele Neurowissenschaftler in das Studium der Entwicklung des Gehirns wird die Beihilfe.

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 ...

mouse-utero-electroporation-controlled-spatiotemporal-gene

Research

JoVE Journal

Neuroscience

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

42.7K Aufrufe.  RIKEN Brain Science Institute. Ein Gentransfer-Methode in den Entwicklungsländern Gehirn der Maus ist mit einer einzigartigen chirurgischen Verfahren und spezielle Form der Elektroden beschrieben. Diese einzigartige Technik erlaubt die Transfektion von Plasmid-DNA zeitlich und räumlich, was viele Neurowissenschaftler in das Studium der Entwicklung des Gehirns wird die Beihilfe.

Serie: Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Die Transfektion von Maus retinalen Ganglienzellen durch In vivo Elektroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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 ...

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualisierung und genetische Manipulation von Dendriten und Dornen in der Maus Hirnrinde und im Hippocampus mit<em&gt; In utero</em&gt; Die Elektroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

Erzeugung von Aktuell transgenen Ratten durch<em&gt; In utero</em&gt; Elektroporation und<em&gt; In-vivo-</em&gt; Biolumineszenz Screening

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Genetische Manipulation der Maus entwickeln Hypothalamus durch<em&gt; In utero</em&gt; Elektroporation

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex, Hippocampus-, Thalamus-, Hypothalamus, Lateral Septal Nucleus- und Striatum spezifische<em&gt; In Utero</em&gt; Electroporation in der C57BL / 6-Maus,

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

Zeitraffer-konfokale Bildgebung der Migration von Neuronen in der organotypischen Scheibe Kultur der embryonalen Maus Gehirn mit<em&gt; In Utero</em&gt; Elektroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

Induktion Cre Lox Rekombination in Maus Großhirnrinde durch Elektroporation In Utero

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...