Name: cortex-, hippocampus-, thalamus-, hypothalamus-, lateral septal nucleus- and striatum-specific in utero electroporation in the c57bl/6 mouse
Uploaded: 2016-01-18T14:00:00.000+00:00
Duration: 6 min
Description: Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

Technical supported by Melanie Pfeifer and Nikolai Schmarowski (Institute for Microscopic Anatomy and Neurobiology, University Medical Center Mainz).

Ethics Statement: Die Handhabung der Mäuse und die experimentellen Verfahren wurden in Übereinstimmung mit europäischen, nationalen und institutionellen Richtlinien für die Tierpflege durchgeführt. 1. In Utero Elektroporation Hinweis: In utero Elektroporation wurde wie zuvor veröffentlichten 12,14. Daher ist das Verfahren nur kurz beschrieben in den folgenden (1). <ol><li> Vorbereitungen <ol><li> Bereiten Fast Green farbigen endotoxinfreiem advanced Transfektion-Grade-Plasmid-Lösung (enthaltend 1,5 pCAGGS), wie zuvor beschrieben 12. </li><li> Ziehen und Schleifen (35 ° Winkel) Borosilikatglas Kapillaren (0,8-0,9 Durchmesser) für Injektionszwecke. </li><li> Sterilisieren Instrumente. </li></ol></li><li> Chirurgie <ol><li> Verwalten Analgetika (Carprofen, 4 mg / kg Körpergewicht, sc, 24-Stunden-Depot) mindestens 30 Minuten vor Beginn der Operation. </li><li> Halten Sie die Längedie Anästhesie zu einem Maximum von 35-45 min (Stufe III - Phase der chirurgischen Anästhesie nach Guedel 15 - maximal 25-30 min). </li><li> Anesthetize zeitlich trächtige Mäuse mit Isofluran (Induktion in einer Kammer: 2,8%, Chirurgie über Maske: 2,5%). Bestätigen Anästhesie durch Teilnahmslosigkeit bis zu den Zehen Prise. </li><li> Bewerben Augensalbe, um ins Auge Trockenheit während der Narkose zu verhindern. </li><li> Sterilisieren Sie den OP-Bereich mit 7,5% Providon-Jod-Lösung und decken Sie die Maus mit steriler Gaze (feucht mit 0,9% Benzylalkohol Kochsalzlösung) nur mit dem OP-Bereich ausgesetzt. </li><li> Schneiden Sie die Bauchhöhle mit einem Skalpell (Hautschnitt: 1,5-2 cm, Muskel Einschnitt: 1-1,5 cm). </li><li> Erhaltung der geöffneten Bauchhöhle vor dem Austrocknen durch Befeuchten mit erwärmtem 0,9% Benzylalkohol Kochsalzlösung oder einem anderen sterilen isotonischen Lösung wie von Tierärzten oder Ausschuss gerichtet. </li><li> Vorsichtig heraus Gebärmutterhörner (mit Ringzangen, die Gebärmutter mit dem Finger nicht berührens). </li></ol></li><li> Injektion von DNA und Elektroporation. Hinweis: Die genauen Details für die auf bestimmte Regionen in Punkt 2. <ol><li> Langsam spritzen die farbigen DNA-Lösung in die Herzkammer, wie in Abbildung 1 und Abschnitt 2 über die vorbereiteten Borosilikatglas Kapillaren angezeigt. </li><li> Tragen Sie die entsprechende Spannung für den entsprechenden embryonalen Phase (z. B. 36 bis 38 V zur E14) über den Fachzange Typ Platinelektroden (Intervall Zykluslänge 50 msec, Intervallpause 950 ms). Pulse die Elektroporation Paddel während des Anlegens der Spannung zu Schäden an der Gebärmutterwand zu minimieren. </li></ol></li><li> Beitrag Elektroporation / post-operative Betreuung <ol><li> Vorsichtig wieder die Gebärmutterhörner in der Bauchhöhle. </li><li> Naht der Muskeln und der Hautschnitt getrennt. </li><li> Lassen Sie die Maus Rest auf einer Wärmevorrichtung für die Wiederherstellung. </li><li> Beaufsichtigen Sie die Maus während der Wiederherstellung (Verhalten, Futtermittel undWasserverbrauch), 4 Stunden, 24 Stunden und 48 Stunden nach der Operation. Falls erforderlich, zusätzliche Analgetika (Carprofen, 4 mg / kg Körpergewicht, 24-Stunden-Depot) für weitere 2 Tage. </li></ol></li></ol> 2. Transfektion von spezifischen Hirnarealen Anmerkung: Die Position des positiven Pols wird als der Winkel zwischen der vertikalen Mittellinie durch die Herzkammer mit der DNA-Lösung (die rechte oder dritten Ventrikel) und der Position der mit dem positiven Pol gezeigt gefüllt. Beim Befüllen des linken Ventrikels die Positionen spiegelbildlich. In Figur 4 sind die Positionen der Gehör primordialen gezeigt, die wichtige Anhaltspunkte liefern. <ol><li> Transfektion von kortikalen Arealen (2A, Tabelle 1). <ol><li> Durchführung von Operationen, wie in Abschnitt 1.2 beschrieben. Für die in utero Elektroporation von kortikalen Arealen verwenden E14 Embryonen. </li><li> Injizieren 1,5-2 ul DNA-Lösung, die 4 &amp; mgr; g DNA in der rechten lateralen Ventrikelüber die vorbereiteten Borosilikatglas Kapillaren. <ol><li> Der Embryo sorgfältig zu positionieren, indem Ringzange, so dass es keine sichtbaren Gefäße über der Einstichstelle. </li><li> Und injizieren die DNA-Lösung von etwa 0,75 mm koronal vom Lambdanaht und 0,5 mm lateral von der Pfeilnaht (Abbildung 3). Sicherzustellen, dass die Eintauchtiefe beträgt 1,2 mm (E14, von der Gebärmutterwand gemessen). Überprüfen Sie für eine blau-grüne Färbung mit einer scharfen Abgrenzung. </li></ol></li><li> Bewerben Spannung über den Fachzange Typ Platinelektroden (Intervall Zykluslänge 50 msec, Intervallpause 950 ms). <ol><li> Verwenden Sie ein 3 oder 5 mm-Elektrode Paddel. </li><li> Verwenden Sie eine Spannung im Bereich von 36 bis 38 V. </li><li> Legen Sie die Mitte der Elektroden nur vordere der Ohr primordia. </li><li> Um die retrosplenialen Cortex Verwendung Winkel 330-0 ° transfizieren, um die motorischen Kortex 0-40 ° transfizieren, um den somatosensorischen Kortex 40-95 ° Transfektion, zur TransfektionCortex piriformis (2A) 95-110 ° beträgt. </li></ol></li><li> Führen Sie die nach der Elektroporation / postoperative Versorgung, wie im Abschnitt 1.4 beschrieben. Opfern, die Mäuse und 4 Tage Analyse der Embryonen nach der Elektroporation (E18), wie in Kapitel 3 beschrieben. </li></ol></li><li> Transfektion des Hippocampus (2B, Tabelle 1). <ol><li> Durchführung von Operationen, wie in Abschnitt 1.2 beschrieben. Für die in utero Elektroporation von Hippocampus verwenden E15 Embryonen. </li><li> Injizieren 2 ul DNA-Lösung, die 4 &amp; mgr; g DNA in der rechten lateralen Ventrikel über den hergestellten Borosilikat Kapillaren. </li><li> Bewerben Spannung über den Fachzange Typ Platinelektroden (Intervall Zykluslänge 50 msec, Intervallpause 950 ms). <ol><li> Verwenden Sie ein 3 oder 5 mm-Elektrode Paddel. </li><li> Verwenden Sie eine Spannung im Bereich von 38 bis 40. </li><li> Legen Sie die Mitte der Minuspol nur vordere des Ohres Primordium und dem Zentrum Of den Pluspol an der Mitte des Ohr primordium. </li><li> Zur Transfektion von Gyrus dentatus verwenden Winkel von 190 bis 210 °, um die Ammonshorn (CA) 3 210 bis 230 ° zu transfizieren, um die CA2 230-250 ° transfizieren, um die CA1 250-275 ° (2B) zu transfizieren. </li></ol></li><li> Führen Sie die nach der Elektroporation / postoperative Versorgung, wie im Abschnitt 1.4 beschrieben. Führen Analyse nach vollständiger Hippocampus (nach der Geburt, p21), wie in Kapitel 3 beschrieben. </li></ol></li><li> Transfektion des lateralen Septums Kern und Striatum (2C, Tabelle 1) Hinweis: Die Winkel überschneiden sich mit denen, für die Transfektion der Hippocampus. Effektiv transfizieren die laterale Septum Kern und das Striatum ohne unerwünschte Hippocampus Transfektion früher Embryonalstadien (E12) verwendet werden müssen. <ol><li> Durchführung von Operationen, wie in Abschnitt 1.2 beschrieben. Für das in utero Elektroporation des lateralen Septums Kern und striatum zu verwenden E12 Embryonen. </li><li> Injiziert 1 ul DNA-Lösung, die 4 &amp; mgr; g DNA in der rechten lateralen Ventrikel über den hergestellten Borosilikat Kapillaren. </li><li> Bewerben Spannung über den Fachzange Typ Platinelektroden (Intervall Zykluslänge 50 msec, Intervallpause 950 ms). <ol><li> Verwenden Sie ein 0,5 mm-Elektrode. </li><li> Verwenden Sie 33 V. </li><li> Legen Sie die Mitte der Elektroden auf der Ebene der Ohr primordia. </li><li> Die seitlichen septalen Kern Verwendung Winkeln von 100-170 ° zu transfizieren, um die Striatum 170-240 ° (2C) zu transfizieren. </li></ol></li><li> Führen Sie die nach der Elektroporation / postoperative Versorgung, wie im Abschnitt 1.4 beschrieben. Opfern, die Mäuse und zu analysieren, die Embryonen 6 Tage nach der Elektroporation (E18), wie in Kapitel 3 beschrieben. </li></ol></li><li> Transfektion des Thalamus und Hypothalamus (2D, Tabelle 1) <ol><li> Durchführung von Operationen, wie in Abschnitt 1.2 beschrieben. Für die in uTero Elektroporation der Thalamus und Hypothalamus Einsatz E12-E13 Embryonen. </li><li> Injizieren 1-1,5 &amp; mgr; l DNA-Lösung, die 4 &amp; mgr; g DNA in den lateralen Ventrikel (links oder rechts) über den hergestellten Borosilikat Kapillaren. Warten für 2-3 min, bis die Lösung in das dritte Ventrikel diffundiert. Alternativ direkt in den dritten Ventrikel zu injizieren. Hinweis: Dies verbessert Spezifität, aber vielleicht eine verminderte Überlebensrate (hohe Beschädigungsgefahr) führen. </li><li> Bewerben Spannung über den Fachzange Typ Platinelektroden (Intervall Zykluslänge 50 msec, Intervallpause 950 ms). <ol><li> Verwenden Sie eine 0,5 oder 3 mm-Elektrode. </li><li> Verwenden Sie eine Spannung im Bereich von 33 bis 35 V. </li><li> Legen Sie die Mitte der Elektroden nur posterior die Ohr primordia. </li><li> Zum Thalamus Verwendung Winkel 25-40 ° transfizieren, um die Hypothalamus 90-180 ° (2D) zu transfizieren. </li></ol></li><li> Führen Sie die nach der Elektroporation / post-operative Betreuung als described in Abschnitt 1.4. Opfern, die Mäuse und zu analysieren, die Embryonen 6 Tage nach der Elektroporation (E18), wie in Kapitel 3 beschrieben. </li></ol></li></ol> 3. Perfusionsfixierung <ol><li> Vorbereitungen <ol><li> Warm 4% Paraformaldehyd (PFA) auf 37 ° C. </li><li> Füllen Sie einen 50-ml-Spritze mit PBS (4 ° C) und eine zweite Spritze mit aufgewärmt PFA. Vermeiden Sie Luftblasen. </li><li> Verbinden der Spritzen zu einem Dreiwegehahn. Verbinden Sie das dritte Ende mit einem venenverweilkanüle (Schmetterling). </li><li> Spülen Sie die venenverweilkanüle mit PBS. </li></ol></li><li> Perfusion <ol><li> Tief betäuben die Mäuse (Schwangerschaft oder postnatale transfiziert) mit subkutanen Anwendung Ketaminhydrochlorid (60 mg / kg) und Xylazin (7,5 mg / kg). </li><li> Bestätigen Sie die chirurgische Toleranzstufe durch Teilnahmslosigkeit bis zu den Zehen Prise. </li><li> Öffnen Sie die Bauchhöhle direkt unter dem Brustkorb. </li><li> Schneiden Sie vorsichtig die Membran. </li><li> Den Brustkorb öffnen Sie vorsichtig über einen Schnitt auf each Website bis zum Schlüsselbein. </li><li> Legen Sie die Spitze des Schmetterlings in die linke Herzkammer (ausgehend von der Spitze des Herzens). </li><li> Schneiden Sie die linke Herzohr. </li><li> Langsam durchströmen die Maus mit PBS (ca. 10 ml). </li><li> Schalten Sie den Dreiwegehahn. </li><li> Perfusion der Maus mit 5-10 ml 4% PFA. </li><li> Enthaupten die Maus mit einer Schere und sezieren das Gehirn beginnend am Foramen magnum. Führen Sie die Dissektion als bisher veröffentlichten 16. </li><li> Postfixate das Gehirn für 2 Tage in 4% PFA. Halten Sie die Gehirne bei 4 ° C im Dunkeln. </li></ol></li></ol> 4. Immunhistochemie <ol><li> Erzeugen 70 um Abschnitte (zB mit einem Vibratom). </li><li> Optional: Verbessern Fluoreszenz mit Antikörperfärbung. <ol><li> Blockieren die Abschnitte für 1 h bei RT (0,1-0,2% Triton X-100, 5% normalem Ziegenserum) </li><li> Inkubieren O / N mit dem entsprechenden Antikörper (Anti-GFP, 1: 1.000) </li><li> Wash 3-5-Mal mit 0,1 M Phosphatpuffer. </li><li> Inkubieren Sie 3-5 Stunden mit dem entsprechenden Fluoreszenz-konjugierten Sekundärantikörper (Alexa Fluor 488 konjugierten Anti-Kaninchen-IgG, 1: 1.000). </li><li> Optional: 4-6-diaminodino-2-phenylindol (DAPI, 0,2 g / ml in 0,1 M Phosphatpuffer) Gegenfärbung für 1 min. </li><li> Analyse Fluoreszenz mit einem geeigneten Fluoreszenzmikroskop. </li></ol></li></ol>

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The authors have nothing to disclose.

This protocol describes in detail how to transfect 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 of C75BL/6 mice. With all the provided information this is the first protocol, which supplies all necessary information to easily recreate transfections of these cerebral regions in the C57BL/6 mouse. Previous publications are mostly focused only on a few specific regions and therefore the detailed information needed for specific transfections is only scarcely standardized. 
The position and the size of the electrode as well as the applied voltage for the corresponding embryonic stage, which are the critical steps for effective and specific transfections, are precisely described. The provided angle-map helps to orientate the electrodes for specific electroporation. The locations of the electrodes are additionally shown on an embryo in the uterus, an embryo in the amniotic sac and also on a pure, dissected embryo. All of this is intended to help in identifying the correct positions of the electrodes. 
Usage of the appropriate voltage is a central aspect in the provided protocol. High voltages decrease the survival rate, whereas low voltages reduce the transfection efficiency 2,3,12. Voltage recommendations given in this protocol are specific for C57BL/6 mice and slightly differ from these for CD12,3,12. CD1 mice tolerate higher voltages than C57BL/6 mice and therefore higher voltages might be used for transfecting CD1 mice12. No data exist for other mouse strains, but the standards might need some modifications. 
A further central point is the size of the electrode. The usage of a smaller electrode, rather than a bigger one, is always recommended for experiments where specificity is important. Smaller electrodes enhance specificity4,12. If the specificity of the transfection is not the main point, especially for inexperienced experimenters, bigger electrodes enhance the chance of hitting the desired region. Thereby one has to keep in mind that electrodes covering too much of the embryo, might affect the heart rhythm and lead to reduced survival. For example a 10 mm electrode covers the whole E12 embryo and therefore is not recommended for transfecting E12 embryos, even if specificity is not the primary goal. 
The stages used in this protocol were chosen exemplary. Considering the fact that progenitors that are transfected on a certain time point give rise to a particular type of neuron the embryonic stages for transfection can be adapted to the specific aim of the study. For instance during cortical development, neurons are born at different time points, forming the six cortical layers in a sequential manner. Hence, depending of the time point of transfection the transfected progenitors give rise to neurons of a specific layer2,17.
For a successful transfection the following points also should be observed. First, the careful preparation and injection of the glass capillaries play a crucial role for the survival of the embryos and therefore for effective transfection. Second, the selection of an appropriate DNA concentration can also be very important and might have to be specifically adapted for different vectors18. Third, application of high voltages affects neighboring embryos, and therefore transfection of neighboring embryos might reduce the survival of these embryos. Further it has also been recommended to avoid the embryos closest to the vaginal duct, because the damage of these embryos might lead to abortion18.
Besides great advantages the technique of in utero electroporation has its drawbacks, which should be taken into consideration while designing experiment. First the regions, which can be efficiently transfected, are manly restricted to areas surrounding the ventricles. This is due to the fact that the plasmid-solution has to be injected into a ventricle. Further, even with all the details provided in this protocol it is not possible to transfect all cells out of a specific region. Even the number of transfected cells is hardly controllable. The analysis of the gene function is not restricted to embryonic stages. Adult mice can also be analyzed because the transfection of postmitotic neurons lasts at least up to 4 months13, but is not passed to the next generation. 
Further specifications, such as specifically electroporating thalamic and hypothalamic nuclei or temporally addressing cortical layers can additionally broaden the scope. The combination of genetically engineered mice, or inducible systems in with the in utero electroporation also additionally enhances possible applications. 
With all the given facts, this protocol facilitates specific transfections and therefore reduces the number of animal used. Standardization of the protocol additionally enhances the comparability of the obtained results.

Seit der ersten Beschreibung im Jahr 2001 von drei unabhängigen Gruppen 1-3 i n utero Elektroporation hat sich zu einem weit verbreiteten Standard-Tool zur Analyse der Genexpression in der Nagetier zentralen Nervensystems. Im Vergleich zu der Erzeugung von Knockout-Mäusen, die, trotz kontinuierlich Verbesserung der Techniken, noch Zeit und Geld raub, die in utero Elektroporation gefällt durch ihre Einfachheit. Also, in utero Elektroporation ermöglicht eine schnelle und effiziente Verstärkungs- und loss-of-function-Studien 4. Die Hirnregionen zu transfizieren, wird die Lösung, welche das negativ geladene Plasmid in einem Ventrikel injiziert. Während des elektrischen Impulses, wandert der negativ geladenen DNA zum positiven Pol und daher kann die transfizierte Region einfach durch Verändern der Position des positiven Pols ausgewählt werden. Es ist oft gezeigt worden, dass zahlreiche Regionen des zentralen Nervensystems können targeted 3,5-8. Beispielsweise zeigen neuere Studien, spezifische Transfektionen des Hippocampus, der piriformen Cortex oder Striatum 9-11. Die Information über den richtigen Positionen sind jedoch oft nur kaum standardisiert und sind nicht immer leicht zu verschiedenen Mäusestämme zu übertragen. Transfektion bestimmter Embryonalstadien ist bei weitem nicht trivial. Viele Einflussfaktoren zu beachten bei der Auswahl des Set-up für bestimmte in utero Elektroporation genommen werden. Erstens, um optimal zu transfizieren, die entsprechenden embryonalen Stadien, ist das Wissen über die geeigneten Spannungen notwendig. Hohe Spannungen verringern die Überlebensrate, wohingegen niedrige Spannungen reduzieren die Transfektionseffizienz 2,3,12. Auch die Größe der Elektrode Paddel spielt eine entscheidende Rolle, denn die Verwendung von Elektroden Paddel, die zu groß zu einer verringerten Spezifität sind oder Tod aufgrund von Erkrankung des Herzrhythmus 4,12,13 verursachen. Die angelegteSpannung und die Größe und die Position der Elektrode Paddel sind die wichtigsten Merkmale zu berücksichtigen, es gibt aber auch weitere Faktoren, die die Ergebnisse der Elektroporation, wie die aufgebrachte Menge an DNA-Lösung. Wir haben ein detailliertes Protokoll, das eine schnelle und effiziente Transfektion von verschiedenen Hirnregionen der C57BL / 6-Maus 12 ermöglicht. In diesem Protokoll detaillierte Information über die Spannungen, die verwendet werden und die Größe der Elektrode Paddel für erhöhte Spezifität bereitgestellt. Weitere Information über die Herzkammer mit Empfehlungen für die Menge der Plasmid-Lösung und der Lage der Elektrode zugeführt wird, gefüllt werden. Die Anzeige der detaillierten Positionsinformationen in der Karte und der weiteren Darstellung von diesen Positionen ermöglicht eine einfache spezifische und effiziente in utero Elektroporation des retrosplenialen Cortex, dem motorischen Cortex, somatosensorischen Cortex piriformis cortex, ter Ammonshorn 1-3, dem Gyrus dentatus, das Striatum, die laterale Septum-Kern, der Thalamus und Hypothalamus.

<table><tbody><tr><td>EndoFree Plasmid Maxi Kit</td><td>QIAGEN</td><td>12362</td><td></td></tr><tr><td>Fast Green</td><td>Roth</td><td>0301.1</td><td></td></tr><tr><td>pCAGGS</td><td>Addgene</td><td></td><td></td></tr><tr><td>borosilicate glass capillaries (0.8-0.9 mm diameter)</td><td>Wold Precision Instrument Inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Isoflurane (Forene)</td><td>Abbott</td><td>PZN 4831850</td><td></td></tr><tr><td>Carprofen (Rimadyl)</td><td>Pfizer GmbH</td><td>approval number: 400684.00.00</td><td></td></tr><tr><td>eye ointment (Bepanthen Augen und Nasensalbe)</td><td>Bayer&amp;nbsp;</td><td>PZN 01578681</td><td></td></tr><tr><td>0.9% benzyl alcohol 0.9% saline solution</td><td>Pharmacy&lt;br/&gt; of the University Medical Center Mainz</td><td></td><td></td></tr><tr><td>gauze (ES-Kompressen)</td><td>Hartmann</td><td>407835</td><td></td></tr><tr><td>sterile 5-0 Perma-Hand Silk Suture</td><td>Ethicon Johnson &amp;amp; Johnson</td><td>K890H</td><td></td></tr><tr><td>ring forceps 1/ 1.5 mm</td><td>Fine Science Tools</td><td>11101-09</td><td></td></tr><tr><td>ring forceps 4.8/ 6 mm</td><td>Fine Science Tools</td><td>11106-09</td><td></td></tr><tr><td>ring forceps 2.2/ 3 mm</td><td>Fine Science Tools</td><td>11103-09</td><td></td></tr><tr><td>Adson Forceps-Serrated Straight 12 cm</td><td>Fine Science Tools</td><td>1106-12</td><td></td></tr><tr><td>IrisScissors-Delicate Straight-Sharp/Blunt 10 cm</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>Mayo-Stille Scissors-Straight 15 cm</td><td>Fine Science Tools</td><td>14012-15</td><td></td></tr><tr><td>Dumont #5 Forceps-Inox</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Castroviejo NeedleHolder-with Lock-Tungsten Carbide 14 cm</td><td>Fine Science Tools</td><td>12565-14</td><td></td></tr><tr><td>Elektroporator CUY21 SC&amp;nbsp;</td><td>Nepa Gene Co.</td><td></td><td></td></tr><tr><td>FST 250 Hot Bead Sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Microgrinder EG-44</td><td>Narishige</td><td></td><td></td></tr><tr><td>P-97 Micropette Puller</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>Platinum electrodes 650P 0.5 mm</td><td>Nepagene</td><td>CUY650P0.5</td><td></td></tr><tr><td>Platinum electrodes 650P 3 mm</td><td>Nepagene</td><td>CUY650P3</td><td></td></tr><tr><td>Platinum electrodes 650P 5 mm</td><td>Nepagene</td><td>CUY650P5</td><td></td></tr><tr><td>Platinum electrodes 650P 10 mm</td><td>Nepagene</td><td>CUY650P10</td><td></td></tr><tr><td>Anesthesia system</td><td>Rothacher-Medical GmbH</td><td>CV-30511-3 Vapor 19.3</td><td></td></tr><tr><td>Heating plate</td><td>Rothacher-Medical GmbH</td><td>HP-1M</td><td></td></tr><tr><td>Temperature Controller 220V AC</td><td>Rothacher-Medical GmbH</td><td>TCAT-2LV</td><td></td></tr></tbody></table>

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.

Abbildung 2 zeigt Beispiele für die spezifische in utero Elektroporation der Regionen entwickelt sich der retrosplenialen Hirnrinde, dem motorischen Kortex, dem somatosensorischen Kortex, die piriformen Cortex, Ammonshorn 1-3, dem Gyrus dentatus, das Striatum, die laterale Septum-Kern , Thalamus und Hypothalamus. Die Ergebnisse der Transfektionen sind gezeigt neben dem empfohlenen Winkel (Abbildung 2). Zur besseren Veranschaulichung der Winkel in vivo die Position der Elektrode (0,5 mm) werden ebenfalls auf einen Embryo (Abbildung 5, Embryos in utero, Embryos im Fruchtblase, seziert Embryo) gezeigt. Um bieten einen Überblick Tabelle 1 zusammengefasst die relevanten Fakten für spezifische Transfektion. Ferner wurde gezeigt, daß die Größe der Elektrode spielt eine entscheidende Rolle für die Spezifität (Abbildung 6). Größere Größe der Elektrode Paddel führt zu larger transfiziert Bereichen als beispielhaft in Figur 6 gezeigt ist. Ferner sind die Elektroden, die zu groß Abdeckung zu viel des Embryos sind, wodurch das Risiko einer Beeinträchtigung des Embryos Herzrhythmus (7) verbessert wird. Zum Beispiel eine 10 mm Elektrodenpaddel umfasst die gesamte E12 Embryonen (Abbildung 7). <img alt="Abbildung 1" src="/files/ftp_upload/53303/53303fig1.jpg" /> Figur 1. In utero Elektroporation. Grundschritte der Leistung des in utero Elektroporation gezeigt. (A) zeigt den gesamten Aufbau der Chirurgie, mit der Maus auf eine Heizplatte gelegt bereits mit Isofluran betäubt. (B) Die Desinfektion des Operationsfeldes ist ein entscheidender Schritt vor dem Öffnen der Bauchhöhle (C) der Maus. (D) Die DNA-haltige Lösung wird mit Fast G farbigenrüne, das die Visualisierung der erfolgreiche Injektion ermöglicht (E, F). Erfolgreiche Injektion in einen lateralen Ventrikel führt in die halbmondförmige Fleck. (G) Nach der Injektion die entsprechende Spannung wird über Zangentyp Platin-Elektroden angelegt. (H) Nach sorgfältiger Austausch der Gebärmutter in die Bauchhöhle die chirurgische Wunde vernäht. <a href="https://www.jove.com/files/ftp_upload/53303/53303fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/53303/53303fig2.jpg" /> Figur 2. Winkel-Diagramm für die Elektroporation von bestimmten Bereichen im murinen Cortex, Hippocampus, Septum seitlichen Kern, Striatum, Thalamus und Hypothalamus. Die geeigneten Positionen für die positiven und negativen Pole sind in der schematischen Zeichnung dargestellt. Für (AC </stro<html> ng&gt;) werden die Positionen zum Injizieren der DNA-Lösung in den rechten Ventrikel angegeben. Für Injektionen in die linke Herzkammer, die Winkel sind spiegelbildlich. Die spezifischen Transfektionen wurden histologisch verifiziert. (A) zeigt die Positionen der Plus- und Minuspol zur Transfektion von kortikalen Arealen (die retrosplenialen Kortex (RSC), der motorischen Kortex (MC), die somatosensorischen Kortex (SSC) und der piriformen Cortex (PC). (B) zeigt die Positionen der positiven und negativen Pol zum Transfizieren hippocampalen Bildungen (Ammonshorn 1-3 (CA1, CA2, CA3) und dem Gyrus dentatus (DG). (C) zeigt die Positionen der positiven und negativen Pol zum Transfizieren Striatum (STR) und die laterale Septum-Kern (LS). (d) zeigt die entsprechenden Positionen der Elektroden für die Transfektion der Thalamus (TH) und des Hypothalamus (HY). von Baumgart J. &amp; Grebe N. 2015 12 angepasst.<html> ps: //www.jove.com/files/ftp_upload/53303/53303fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen. <img alt="Figur 3" src="/files/ftp_upload/53303/53303fig3.jpg" /> Abbildung 3 Punktionsort. Die optimale Position für die Injektion der DNA-Lösung ist etwa 0,75 mm koronalen von der Lambdanaht und 0,5 mm lateral von der Pfeilnaht. <a href="https://www.jove.com/files/ftp_upload/53303/53303fig3large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 4" src="/files/ftp_upload/53303/53303fig4.jpg" /> Abbildung 4. Position des Ohres Primordium. Die Position des Ohr Primordium ist klar definiert (Pfeil).nk &quot;&gt; Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen. <img alt="5A" src="/files/ftp_upload/53303/53303fig5A.jpg" /><img alt="5B" src="/files/ftp_upload/53303/53303fig5B.jpg" /><img alt="5C" src="/files/ftp_upload/53303/53303fig5C.jpg" /><img alt="5D" src="/files/ftp_upload/53303/53303fig5D.jpg" /> Abbildung 5. Visualisierung der Winkel auf einem Embryo. Zur besseren Visualisierung die entsprechenden Positionen für die positive (mit einem blauen Pluszeichen gekennzeichnet) und der negative Pol für spezifische Transfektionen der kortikalen Arealen (retrosplenialen Cortex, motorischen Kortex, somatosensorischen Kortex, piriformis Kortex), die Hippocampus-Gebieten (Ammonshorn (CA) 1-3, Gyrus dentatus (DG)),die laterale Septum-Kern, das Striatum, Thalamus und Hypothalamus sind auf eines Embryos in utero gezeigt, ein Embryo in der Fruchtblase und auf einem seziert Embryo (alle E15,5). <a href="https://www.jove.com/files/ftp_upload/53303/53303fig5large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 6" src="/files/ftp_upload/53303/53303fig6.jpg" /> Figur 6. Die Spezifität der Transfektion in Abhängigkeit von der Elektrodengröße. Vergrößerung des Elektrodenpaddel reduziert die Spezifität der Transfektion. Dies ist vor allem bei jüngeren embryonalen Stadien (E12, oben) auf der Hand, sondern auch sichtbar im späten embryonalen Stadien (E17, unten). Von Baumgart J. &amp; Grebe N. 2015 12 angepasst. <a href="https://www.jove.com/files/ftp_upload/53303/53303fig6large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a><img alt="Figur 7" src="/files/ftp_upload/53303/53303fig7.jpg" /> Abbildung 7. Größe der Elektrode Paddel im Vergleich zur Größe eines E12 Embryo. Die Elektroden, die zu groß Abdeckung zu viel des Embryos sind und damit das Risiko einer Erkrankung der Herzrhythmus erhöht. Zum Beispiel eine 10 mm-Elektrode (rechts) deckt die ganze E12 Embryonen und wird deshalb nicht empfohlen, auch wenn Spezifität ist nicht das vorrangige Ziel. Von Baumgart J. &amp; Grebe N. 2015 12 angepasst. <a href="https://www.jove.com/files/ftp_upload/53303/53303fig7large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <table fo:keep-together.within-page="1"><tbody><tr><td> Zielregion </td><td> Winkel </td><td colspan="2"> Position der Elektrode </td><td> Alter des Embryos </td><td> DNA amount / Konzentration </td><td> Stromspannung </td><td> Größe der Elektrode </td></tr><tr><td> Retrosplenialen Cortex </td><td> 330 ° - 0 ° </td><td colspan="2"> Mitte der Elektroden nur anterioren des Ohres Primor </td><td> E14 (E11-E17) </td><td> 1,5- 2 ul / 2.7- 2 ug / ul </td><td> 36- 38 </td><td> 3- 5 mm </td></tr><tr><td> Motor Cortex </td><td> 0 ° - 40 ° </td><td colspan="2"> Mitte der Elektroden nur anterioren des Ohres Primor </td><td> E14 (E11-E17) </td><td> 1,5- 2 ul / 2.7- 2 ug / ul </td><td> 36- 38 </td><td> 3- 5 mm </td></tr><tr><td> Somatosensorischen Kortex </td><td> 40 ° - 95 ° </td><td colspan="2"> Mitte der Elektroden nur anterioren des Ohres Primor </td><td> E14 (E11-E17) </td><td> 1,5- 2 ul / 2.7- 2 ug / ul </td><td> 36- 38 </td><td> 3- 5 mm </td></tr><tr><td> Piriform Cortex </td><td> 95 ° - 110 ° </td><td colspan="2"> Mitte der Elektroden nur anterioren des Ohres Primor </td><td> E14 (E11-E17) </td><td> 1,5- 2 ul / 2.7- 2 ug / ul </td><td> 36-3 8 </td><td> 3- 5 mm </td></tr><tr><td> Ammonshorn (CA) 1 </td><td> 250 ° - 275 ° </td><td colspan="2"> Mitte der positive Pol an der Mitte des Ohr primordium </td><td> (E14-) E15 </td><td> 2 ul / 2 ug / ul </td><td> 38- 40 </td><td> 3- 5 mm </td></tr><tr><td> CA2 </td><td> 230 ° - 250 ° </td><td colspan="2"> Mitte der positive Pol an der Mitte des Ohr primordium </td><td> (E14-) E15 </td><td> 2 ul / 2 ug / ul </td><td> 38- 40 </td><td> 3- 5 mm </td></tr><tr><td> CA3 </td><td> 210 ° - 230 ° </td><td colspan="2"> Mitte der positive Pol an der Mitte des Ohr primordium </td><td> (E14-) E15 </td><td> 2 ul / 2 ug / ul </td><td> 38- 40 </td><td> 3- 5 mm </td></tr><tr><td> Gyrus dentatus </td><td> 190 ° - 210 ° </td><td colspan="2"> Mitte der positive Pol an der Mitte des Ohr primordium </td><td> (E14-) E15 </td><td> 2 ul / 2 ug / ul </td><td> 38- 40 </td><td> 3- 5 mm </td></tr><tr><td> Striatum </td><td> 170 ° - 240 ° </td><td colspan="2"> Mitte der Elektroden auf der Ebene des Ohrprimor </td><td> E12 </td><td> 1 ul / 4 ug / ul </td><td> 33 </td><td> 0,5 mm </td></tr><tr><td> Lateral Septal Nucleus </td><td> 100 ° - 170 ° </td><td colspan="2"> Mitte der Elektroden auf der Ebene des Ohrprimor </td><td> E12 </td><td> 1 ul / 4 ug / ul </td><td> 33 </td><td> 0,5 mm </td></tr><tr><td> Thalamus </td><td> 25 ° - 40 ° </td><td colspan="2"> Mitte der electodes nur posterior das Ohr primordia </td><td> E12- E13 </td><td> 1-1,5 &amp; mgr; l / 4-2,7 &amp; mgr; g / &amp; mgr; </td><td> 33- 35 </td><td> 0,5- 3 mm </td></tr><tr><td> Hypothalamus </td><td> 90 ° - 180 ° </td><td colspan="2"> Mitte der electodes nur posterior das Ohr primordia </td><td> E12- E13 </td><td> 1-1,5 &amp; mgr; l / 4-2,7 &amp; mgr; g / &amp; mgr; </td><td> 33- 35 </td><td> 0,5- 3 mm </td></tr></tbody></table> Tabelle 1:. Zusammenfassung der Ergebnisse in dieser Tabelle nicht enthalten ist, alle relevanten Informationen für spezifische Transfektion der beschriebenen kortikalen Arealen. Sie enthält Informationen über den Zielbereich, wobei der Winkel, der Position der Elektrode, Embryonalstadium, der aufgebrachten Menge und Konzentration der DNA-Lösung, der Spannung und der Größe der Elektrode.

Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

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 ...

cortex-hippocampus-thalamus-hypothalamus-lateral-septal-nucleus

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

27.9K Aufrufe.  University Medical Center, JGU Mainz. 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.

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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Ex Utero Elektroporation und Organotypischen Kultur der Maus Hippocampus-Gewebe

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Forschung

Entwicklungsbiologie

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

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

In Utero intraventrikuläre Injektion und Elektroporation von E15 muriner Embryonen

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

Biologie

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 ...

Neurowissenschaften

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 ...

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 ...

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 ...

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 ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

In Utero Ansätze Elektroporation die Erregbarkeit neuronaler Subpopulationen und Einzelzell - Konnektivität zu studieren

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

Double In Utero Elektroporation, um zeitlich und räumlich getrennte Zellpopulationen anzuvisieren

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

Presented here is a protocol to perform genetic manipulation in the embryonic ferret brain using in utero electroporation. This method allows for targeting of neural progenitor cells in the neocortex in vivo.

In Vivo Targeting von neuronalen Vorläuferzellen in Ferret Neocortex durch Utero Elektroporation

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian ...

Cortex, Hippocampus-, Thalamus-, Hypothalamus, Lateral Septal Nucleus- und Striatum spezifische

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