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>

<ol><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294 (5544), 1071-1074 (2001).</li>
<li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev Biol. 240 (1), 237-246 (2001).</li>
<li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation%3A+Visualization+of+neuronal+migration+in+the+developing+cortex%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Efficient in utero gene transfer system to the developing mouse brain using electroporation: Visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103 (4), 865-872 (2001).</li>
<li>LoTurco, J., Manent, J. B., Sidiqi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+and+improved+tools+for+in+utero+electroporation+studies+of+developing+cerebral+cortex%5BTitle%5D)+AND+%22Cereb+Cortex%22%5BJournal%5D)">New and improved tools for in utero electroporation studies of developing cerebral cortex.</a> Cereb Cortex. 19 Suppl 1. (July), i120-i125 (1991).</li>
<li>Nakahira, E., Yuasa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuronal+generation%2C+migration%2C+and+differentiation+in+the+mouse+hippocampal+primoridium+as+revealed+by+enhanced+green+fluorescent+protein+gene+transfer+by+means+of+in+utero+electroporation%5BTitle%5D)+AND+%22J+Comp+Neurol%22%5BJournal%5D)">Neuronal generation, migration, and differentiation in the mouse hippocampal primoridium as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation.</a> J Comp Neurol. 483 (3), 329-340 (2005).</li>
<li>Navarro-Quiroga, I., Chittajallu, R., Gallo, V., Haydar, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term%2C+selective+gene+expression+in+developing+and+adult+hippocampal+pyramidal+neurons+using+focal+in+utero+electroporation%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Long-term, selective gene expression in developing and adult hippocampal pyramidal neurons using focal in utero electroporation.</a> J Neurosci. 27 (19), 5007-5011 (2007).</li>
<li>Borrell, V., Yoshimura, Y., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+gene+delivery+to+telencephalic+inhibitory+neurons+by+directional+in+utero+electroporation%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation.</a> J Neurosci Methods. 143 (2), 151-158 (2005).</li>
<li>Kolk, S. M., de Mooij-Malsen, A. J., Martens, G. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatiotemporal+Molecular+Approach+of+in+utero+Electroporation+to+Functionally+Decipher+Endophenotypes+in+Neurodevelopmental+Disorders%5BTitle%5D)+AND+%22FNMOL%22%5BJournal%5D)">Spatiotemporal Molecular Approach of in utero Electroporation to Functionally Decipher Endophenotypes in Neurodevelopmental Disorders.</a> FNMOL. 4 (November), 37(2011).</li>
<li>Tomita, K., Kubo, K. I., Ishii, K., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disrupted-in-schizophrenia-1+%28Disc1%29+is+necessary+for+migration+of+the+pyramidal+neurons+during+mouse+hippocampal+development%5BTitle%5D)+AND+%22Hu+Mol+Gen%22%5BJournal%5D)">Disrupted-in-schizophrenia-1 (Disc1) is necessary for migration of the pyramidal neurons during mouse hippocampal development.</a> Hu Mol Gen. 20 (14), 2834-2845 (2011).</li>
<li>Niwa, M., Kamiya, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Knockdown+of+DISC1+by+In+Utero+Gene+Transfer+Disturbs+Postnatal+Dopaminergic+Maturation+in+the+Frontal+Cortex+and+Leads+to+Adult+Behavioral+Deficits%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Knockdown of DISC1 by In Utero Gene Transfer Disturbs Postnatal Dopaminergic Maturation in the Frontal Cortex and Leads to Adult Behavioral Deficits.</a> Neuron. 65 (4), 480-489 (2010).</li>
<li>Nakahira, E., Kagawa, T., Shimizu, T., Goulding, M. D., Ikenaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+evidence+that+ventral+forebrain+cells+migrate+to+the+cortex+and+contribute+to+the+generation+of+cortical+myelinating+oligodendrocytes%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Direct evidence that ventral forebrain cells migrate to the cortex and contribute to the generation of cortical myelinating oligodendrocytes.</a> Dev Biol. 291 (1), 123-131 (2006).</li>
<li>Baumgart, J., Grebe, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((C57BL%2F6-specific+conditions+for+efficient+in+utero+electroporation+of+the+central+nervous+system%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">C57BL/6-specific conditions for efficient in utero electroporation of the central nervous system.</a> J Neurosci Methods. 240, 116-124 (2015).</li>
<li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system%5BTitle%5D)+AND+%22Nat+Protc%22%5BJournal%5D)">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat Protc. 1 (3), 1552-1558 (2006).</li>
<li>Matsui, A., Yoshida, A. C., Kubota, M., Ogawa, M., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+in+utero+electroporation%3A+controlled+spatiotemporal+gene+transfection%5BTitle%5D)+AND+%22JoVE%22%5BJournal%5D)">Mouse in utero electroporation: controlled spatiotemporal gene transfection.</a> JoVE. (54), (2011).</li>
<li>Guedel, A. E. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGuedel%2C+AE+%22Inhalation+anesthesia%3A+a+fundamental+guide%22">Inhalation anesthesia: a fundamental guide</a>. , Macmillan. New York. (1937).</li>
<li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+Animal+Perfusion+Fixation+for+Rodents%5BTitle%5D)+AND+%22JoVE%22%5BJournal%5D)">Whole Animal Perfusion Fixation for Rodents.</a> JoVE. (65), e3564(2012).</li>
<li>Mizutani, K., Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progenitors+resume+generating+neurons+after+temporary+inhibition+of+neurogenesis+by+Notch+activation+in+the+mammalian+cerebral+cortex%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Progenitors resume generating neurons after temporary inhibition of neurogenesis by Notch activation in the mammalian cerebral cortex.</a> Development. 132 (6), 1295-1304 (2005).</li>
<li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Labeling+embryonic+mouse+central+nervous+system+cells+by+in+utero+electroporation%5BTitle%5D)+AND+%22Dev%2C+Growth+%26+Differ%22%5BJournal%5D)">Labeling embryonic mouse central nervous system cells by in utero electroporation.</a> Dev, Growth & Differ. 50 (6), 507-511 (2008).</li>
</ol>

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 r...

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...

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

Research

JoVE Journal

Neuroscience

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

28.0K 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

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 ...

Forschung

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 ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

In utero und ex vivo Elektroporation für die Genexpression in Maus retinalen Ganglienzellen

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

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 ...

Subtype-selective Electroporation of Cortical Interneurons

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

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

Subtyp-selektive Elektroporation kortikaler Inter

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

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 ...

Murine In Utero Electroporation

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and maturation of cells during neural development. Electroporation enables the rapid and targeted delivery of material into cells by utilizing electrical pulses to create transient pores in cell membranes. Although electroporation has traditionally been used in in vitro studies, scientific advancements have now broadened its utilization to intact organs, such as those found in mouse embryos developing in utero. This video will introduce the key principles behind in utero electroporation in addition to reviewing the basic surgical techniques required to access developing embryos within a pregnant rodent. Details of the injection and electroporation steps are provided along with important considerations for directing gene delivery to specific brain regions. Finally, neurobiological applications of in utero electroporation are presented, such as investigating how specific genes contribute to neural development and how connections form between developing neurons.

Murine In Utero Electroporation

In utero Elektroporation bei Mäusen

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and ...

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

Zusammenfassung

Weitere Videos entdecken