Ce travail a été financé par l&#39;Institut RIKEN Brain Science (BSI), Human Frontier Science Program (HFSP) (attribué à TS) et le Programme RIKEN junior Research Associate (JRA, attribué à MK) RIKEN Brain Science Institute (BSI), Human Frontier Science Program (HFSP) (attribué à TS) et RIKEN junior Programme associé de recherche (JRA, attribué à MK) a financé ce travail.

1. Préparation de la solution capillaire et d&#39;ADN pour l&#39;injection <ol><li> Purifier l&#39;ADN plasmidique avec une Maxi-prep ou méthode équivalente, et de faire une concentration finale de 2-3 pg / pl. Aliquot de 100 pg d&#39;ADN, ajouter 10 ul de jeûne colorant vert (1% des stocks) et TE (10mM Tris Base, 1 mM EDTA, pH 8,0) et régler le volume à 100 pi de rendre la concentration d&#39;ADN finale pour le mélange d&#39;injection à 1 pg / ul. La concentration d&#39;ADN plasmidique peut être changé dépend de la taille du plasmide, et aussi son efficacité de la transfection, qui doit être testé individuellement. </li><li> Faites tourner la solution d&#39;ADN pendant 5 minutes à 14000 RPM à température ambiante et ramasser le surnageant pour enlever tout résidu. </li><li> Tirez un capillaire en verre des tubes de verre capillaires tubea aide d&#39;un extracteur micropipette. Pincez la pointe avec une pince pour faire de la pointe a un diamètre de 20 à 30 um. Mesure 800 um-1 mm de la pointe et de vérifier le diamètre extérieur est inférieur à 60 um (figure 1A). </li><li> Connectez le capillaire pour le micromanipulateur, et de remplir avec de l&#39;huile minérale. Pour remplir le capillaire avec la solution d&#39;ADN, immerger la pointe dans la solution et aspirer la solution d&#39;ADN. </li></ol> 2. Bâton électrodes de platine Ici nous avons utilisé des électrodes de platine bâton (Nepe gène, CUY611P2-1) dans la région pour électroporer superficielle du cerveau, telles que le cortex (Fig. 1F). 3. Préparation de la micro-électrodes Utiliser des micro-électrodes pour une électroporation dans les parties profondes du cerveau (c&#39;est à dire, le thalamus et l&#39;hypothalamus) (Fig. 2B-E). <ol><li> Une extrémité du tungstène (pour les électrodes négatives) et le platine (pour les électrodes positives) fils est enroulé sur plaqué or broches et fixés avec du parafilm. Insérez le fil à la tige d&#39;un coton-tige et enveloppez-les deux extrémités avec du parafilm (Fig. 1B). </li><li> Uniformément affûter la pointe du fil avec du papier de verre jusqu&#39;à ce qu&#39;il atteigne 20 à 30 pm de diamètre externe et 60 um de 800 um-1 mm de la pointe (Fig. 1C et D). </li><li> Appliquer une fine couche de vernis à ongles sur le fil pour l&#39;isolation. Après le vernis à ongles a séché, utiliser un coton-tige imbibé d&#39;acétone pour le retirer de la pointe (environ 200 um de la pointe). Important: Afin d&#39;éviter d&#39;endommager l&#39;utérus de la souris, la partie à moins de 800 um-1 mm de la pointe ne devrait pas être plus de 70 m de diamètre (figure 1E). </li></ol> 4. Préparation de la chirurgie <ol><li> Anesthetize une souris enceintes (E9.5-E15.5). Nous démontrons l&#39;anesthésie avec de qualité pharmaceutique du pentobarbital de sodium (Tableau II; 50 ug par gramme de poids corporel, injecter par voie intrapéritonéale et attendez 5 min). Alternatives à pentobarbital sodique comme anesthésique incluent l&#39;injection de kétamine / xylazine ou des anesthésiques gazeux (gaz est préférable pour les procédures de courte durée). L&#39;injection doit être fait avec attention pour éviter toute organes, sinon il fera des dommages de l&#39;utérus ou de tuer des animaux. Lorsque IP réussie est fait, le taux de survie est supérieur à 95%. Aussi l&#39;intervention chirurgicale peut être réalisée en plein air environnement. Avant de commencer l&#39;opération, le test d&#39;une absence de réponse par l&#39;animal au pincement de leurs pieds pour confirmer l&#39;anesthésie. </li><li> Raser les poils de l&#39;abdomen à l&#39;aide d&#39;une lame de rasoir et EtOH 50%. Préparer la peau avec une alternance des gommages bétadine et de l&#39;alcool (70%). </li><li> Faire une incision sur la ligne médiane abdominale avec des ciseaux fins et retirez toutes les cornes utérines attentivement sur un 37 ° C préchauffé tampon phosphate salin (PBS)-humidifié gaze de coton, qui est placé autour de la plaie. Gardez l&#39;humidité utérus avec PBS tout le temps. </li><li> Tenir un câble à fibres optiques flexibles entre l&#39;index et le majeur et le placer sous la corne utérine. Nettoyer avec câble de EtOH à 70% avant l&#39;utilisation. Aucun microscope ou loupe est nécessaire pour la visualisation. L&#39;utérus de position entre le câble de fibre optique de lumière optique et le pouce, et pressez doucement à pousser jusqu&#39;à l&#39;embryon plus proche de la paroi utérine. </li></ol> 5. L&#39;injection de l&#39;ADN et l&#39;électroporation <ol><li> Lorsque l&#39;embryon est positionné, insérez capillaire en verre soigneusement dans le ventricule cible et d&#39;injecter environ 1 microlitre de solution d&#39;ADN (figure 2A). </li><li> Pour électroporation à la partie superficielle de la brainIf utiliser avec des électrodes de platine bâton pour electoporation, placer les électrodes en dehors de l&#39;utérus et d&#39;appliquer suggéré impulsions rectangulaires de courant selon l&#39;instruction de la fabrication (tableau I). Si l&#39;électroporation usingFor à partie profonde du cerveau, l&#39;électroporation nmicro, les électrodes de type Eedle sont utilisés. Iinsert une électrode de tungstène fines négatifs dans l&#39;ADN injecté ventricule et une électrode de platine dans l&#39;utérus et de la région cible de place entre deux électrodes, puis appliquer suggéré impulsions rectangulaires de courant (tableau I) (figure 2B). </li></ol> 6. Message électroporation <ol><li> Placez le dos corne utérine dans son origLieu inal avec et ajouter 500 uL de 37 ° C préchauffé PBS. </li><li> Suture de la couche intérieure avec suture chirurgicale et fermer la couche externe avec un clip de 9 mm autoclipauto. </li><li> Placez les animaux sur un coussin chauffant pendant 2 heures pour permettre la récupération de l&#39;anesthésie. Des analgésiques tels que la buprénorphine ou de carprofène peut être administré pour les douleurs post-opératoires. </li></ol> 7. Les résultats représentatifs: Quelques exemples de l&#39;électroporation corticale du pCAG-EYFP construire en utilisant des électrodes de platine bâton à des moments différents sont représentés dans la figure Figure 32. Les cerveaux sont électroporation à E13.5, E14.5 E15.5 et, et récolté au jour postnatal (P) 6. ALa ntibody coloration du RORC révèle la position de la couche 4 et la position des cellules transfectées EYFP sont révélées par coloration d&#39;anticorps GFP, et les deux images sont superposés (Fig. 3A et B). Étiqueté cellules corticales sont couche de cellules corticales électroporées est clairement différent dans chaque expérience (fig. 32), ce qui suggère dépendant du temps électroporation rend corticale couche spécifique GOF / LOF possible. La viabilité des embryons et l&#39;efficacité de la transfection des cellules varie en fonction de l&#39;âge de l&#39;embryon et l&#39;emplacement de l&#39;électroporation. Les échantillons sont répertoriés sur le tableau 1, toutefois, les conditions doivent être optimisés pour chaque électroporation selon le stade et une partie du cerveau. L&#39;électroporation dans un tissu profond, comme le thalamus et l&#39;hypothalamus à l&#39;utilisation des électrodes de platine bâton donnent souvent un faible rendement (tableau 1). Ce problème peut être résolu en utilisant des micro-électrodes whichelectrodes, qui atteindra à certaines parties profondes du cerveau. Figure 3 4 exemple montre des pCAG-EYFP électroporation dans le diencéphale développement. Solution de l&#39;ADN plasmidique est injecté dans le ventricule 3 ème au E11.5 et suivis par des micro électroporation (fig. 3C4C). La zone est limitée par électroporation dans le thalamus, où la plupart des axones du projet vers le cortex (fig. 3B4B). Projection Axon à la couche 4 du cortex peuvent également être visualisés par EYFP, qui fillesfills le neurone entier. (Fig. 3A4A). <img src="/files/ftp_upload/3024/3024fig1.jpg" alt="Figure 1"/> Figure 1. Schématiques Cartoon démontrant la bonne forme du capillaire. (A) un tube capillaire en verre est tiré aide d&#39;un extracteur micropipette pour faire certaine forme. Un pourboire est pincé par des pinces pour faire 20-30 um. Mesure 800 um-1mm de la pointe (crochet rouge) et assurez-vous que le diamètre externe est inférieur à 60 um (support vert). (B) Une extrémité du fil de tungstène ou le platine est enroulé sur plaqué or broches et fixés avec du parafilm. Ensuite, utilisez du papier de verre pour faire leur bout de devenir 20-30 um diamètres extérieurs et 60 um de 1 mm de la pointe. (C) image du fil de tungstène avant de le façonner. (D) Image du fil de tungstène après façonnage. (E) Image du fil de tungstène après l&#39;application fine couche de vernis à ongles. La barre d&#39;échelle en E est de 10 um (petite échelle) pour le marquage CE. (F) électrodes de platine Stick (gène Nepa. CUY611P2-1). La barre d&#39;échelle dans F est de 2 mm. <img src="/files/ftp_upload/3024/3024fig2.jpg" alt="Figure 2"/> Schéma de la figure dessinée 2. De la procédure chirurgicale. (A) Schéma d&#39;injection dans le ventricule latéral ou d&#39;embryons de grande taille (côté gauche) et en 3 ème ventricule ou de petits embryons (côté droit). (B) Schéma de electrporation par électrode enrobée platine (côté gauche) et par l&#39;aiguille électrode de type (côté droit). <img src="/files/ftp_upload/3024/3024fig3.jpg" alt="Figure 3"/> Figure 3. Développer la souris a été télencéphale électroporation avec pCAG-EYFP à E13.5 (A), E14.5 (B) et E15.5 (C) et récoltés à P6. Les cerveaux ont été sectionnés coronale et colorées avec des anticorps ou d&#39;anticorps GFP RORC séparément et les images sont fusionnées. (A) Electroporation de pCAG-EYFP plasmide au E13.5 transfecter plusieurs couche 4 neurones. qui se chevauchent avec la couche 4 marqueurs du RORC. (B) Electroporation de pCAG-EYFP plasmide au E14.5 cellules transfecter, qui sont plus superficielles que la couche 4 neurones. (C) E15.5 électroporation étiquette de nombreux neurones superficielles telles que la couche 2 / 3. La barre d&#39;échelle 200 um. <img src="/files/ftp_upload/3024/3024fig4.jpg" alt="Figure 4"/> Figure 4. Transfection de EYFP dans les axones thalamo-corticaux (TCA). (A) terminale TCA dans la couche corticale 4 est révélée par immunofluorescence la GFP. Coloration des anticorps RORC est utilisée pour révéler une position de la couche 4. Les deux images sont tirées de la même section et fusionnés. (B) Position du électroporation dans le thalamus est également démontré par immunofluorescence la GFP et la coloration du RORC, dont l&#39;expression est restreinte au niveau du thalamus 11. (C) schéma de dessin animé pour l&#39;électroporation thalamique et la projection TCA sont affichés. La barre d&#39;échelle 200 um.

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

Aucun conflit d&#39;intérêt déclaré.

Dans ce protocole, nous avons seulement décrit les avantages de la technique de la transfection pCAG-EYFP dans la zone réglementée d&#39;un petit cerveau embryonnaire en utilisant différentes électrodes en forme. Pour la comparaison de l&#39;efficacité de transfection par le promoteur différent est décrit précédemment, ce qui montre la spécificité de type cellule 1. Cependant, il ya des limites à la technique, telles que la transfection n&#39;est que transitoire et varie dépendent du plasmide, il n&#39;a pas de spécificité cellulaire et il est difficile de contrôler le nombre de cellules transfectées. ThereforeHowever, combinant ce protocole avec d&#39;autres techniques encore offrir des possibilités de méthodes de manipulation différentes. D&#39;abord, en intégrant promoteur cellule spécifique dans l&#39;ADN plasmidique permettra de transfection de cellules de type particulier, comme les neurones, cellules gliales et astrocytes. Deuxièmement, puisque la transfection est transitoire, il n&#39;est pas adapté pour les scientifiques qui veulent analyser la fonction du gène dans la vie ultérieure. Par conséquent, la combinaison avec une transposase de rendre l&#39;ADN plasmidique s&#39;intégrer dans le génome va le convertir en une transfection stable 8. Ensuite, l&#39;adoption de la tet-on ou tet-off, il sera possible de manipuler le calendrier ou l&#39;expression des gènes cellulaires spécifiques au type de cellules neurales 8. Alternativement, on peut combiner ce protocole avec le tamoxifène inductible Cre-ER (T) recombinases 9 et 10 souris journaliste. Cette grande accessibilité des tissus va changer radicalement des modèles expérimentaux utilisés dans les neurosciences.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td></tr><tr><td> nus diamètre du fil de tungstène 125 um </td><td> Systèmes AM </td><td> 797600 </td></tr><tr><td> nus diamètre du fil de platine 125 um </td><td> Systèmes AM </td><td> 767000 </td></tr><tr><td> Électrodes enrobées </td><td> Nepa génique </td><td> CUY610P4-1 </td></tr><tr><td> tube capillaire en verre </td><td> Stoelting </td><td> 50611 </td></tr><tr><td> micromanipulateur </td><td> KD Scientific </td><td> KDS310 </td></tr><tr><td> ciseaux </td><td> Roboz </td><td> RS-5865 </td></tr><tr><td> générateur d&#39;impulsions </td><td> Systèmes AM </td><td> Modèle 2100 </td></tr><tr><td> 9 mm autoclip </td><td> Roboz </td><td> RS-9260 </td></tr><tr><td> contacts dorés </td><td> WPI </td><td> 5482 </td></tr><tr><td> D&#39;anticorps RORC </td><td> Protéomique Persée </td><td> H3925 </td></tr></tbody></table>

mouse in utero electroporation: controlled spatiotemporal gene transfection

Afin de comprendre la fonction des gènes exprimés dans une région spécifique du cerveau en développement, y compris des molécules de signalisation et de molécules de guidage axonal, le transfert de gènes locales ou knock-out est nécessaire. Le ciblage de gènes knock-in ou knock-out dans les régions locales est possible d&#39;effectuer avec la combinaison avec une ligne spécifique de la CRE, qui est laborieuse, coûteuse et consommatrice de temps. Par conséquent, une méthode de transfection simple, une technique d&#39;électroporation in utero, ce qui peut être réalisé avec peu de temps, sera très pratique pour tester la fonction des gènes candidats possibles avant la génération de 1,2 animaux transgéniques. En plus de cela, électroporation in utero cibles des zones du cerveau où aucune ligne spécifique de la CRE existe, et va limiter létalité embryonnaire 3,4. Ici, nous présentons une méthode d&#39;électroporation in utero combinant deux types différents d&#39;électrodes pour le transfert de gène simple et pratique dans les zones cibles du cerveau en développement. Tout d&#39;abord, une méthode unique de la tenue des embryons en utilisant un câble de fibre optique de lumière optiques fera petits embryons (de E9.5) visible pour l&#39;injection d&#39;ADN dans les ventricules solution ciblée et le type d&#39;aiguille électrodes d&#39;insertion à la zone du cerveau ciblées 5,6. La structuration du cerveau telles que l&#39;aire corticale se produisent au stade embryonnaire précoce, par conséquent, ces électroporation début du E9.5 apporter une contribution importante pour comprendre l&#39;événement toute structuration région. Deuxièmement, la forme précise d&#39;un capillaire évite d&#39;endommager l&#39;utérus en faisant des trous par l&#39;insertion de la capillarité. Par ailleurs, la forme précise des électrodes aiguilles sont créés avec le tungstène et un fil de platine et aiguisé en utilisant du papier de verre et isolés avec du vernis à ongles 7, une méthode qui est décrite en détail dans le présent protocole. Cette technique unique permet la transfection d&#39;ADN plasmidique dans des zones restreintes du cerveau et permettra aux petits embryons à électroporé. Cela aidera à, ouvrez une nouvelle fenêtre pour beaucoup de scientifiques qui travaillent sur la différenciation cellulaire, migration cellulaire, guidage axonal dans le stade embryonnaire très précoce. Par ailleurs, cette technique permettra aux scientifiques de transfecter l&#39;ADN plasmidique dans les parties profondes du cerveau en développement comme le thalamus et l&#39;hypothalamus, où pas beaucoup de lignes spécifiques à la région de la CRE existent pour gain de fonction (GOF) ou perte de fonction (LOF) analyses.

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Une méthode de transfert de gènes dans le cerveau de souris en développement est décrit en utilisant une méthode unique de chirurgie et la forme particulière des électrodes. Cette technique unique permet la transfection d&#39;ADN plasmidique temporellement et spatialement, ce qui aidera de nombreux neuroscientifiques à étudier le développement du cerveau.

Souris dans Utero électroporation: contrôlé Transefection Gene spatio-temporelle

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

mouse-utero-electroporation-controlled-spatiotemporal-gene

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JoVE Journal

Neuroscience

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

42.7K vues.  RIKEN Brain Science Institute. Une méthode de transfert de gènes dans le cerveau de souris en développement est décrit en utilisant une méthode unique de chirurgie et la forme particulière des électrodes. Cette technique unique permet la transfection d'ADN plasmidique temporellement et spatialement, ce qui aidera de nombreux neuroscientifiques à étudier le développement du cerveau.

Vidéo: Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

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

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

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

In utero électroporation suivie par la culture neuronale primaire pour étudier la fonction des gènes dans un sous-ensemble des neurones corticaux

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

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Neurosciences

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

La transfection des cellules ganglionnaires de la rétine de la souris par In vivo L&#39;électroporation

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

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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

Livraison efficace de gènes dans les Territoires du SNC multiples à l&#39;aide In Utero L&#39;électroporation

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

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

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

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

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

Transfert de gènes à l&#39;oreille de souris en développement intérieur par<em&gt; In Vivo</em&gt; Électroporation

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

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

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

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

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

Visualisation et la manipulation génétique des dendrites et des épines dans le cortex cérébral et l&#39;hippocampe de souris à l&#39;aide<em&gt; In utero</em&gt; Électroporation

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

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

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

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

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

Génération de rats transgéniques par voie topique<em&gt; In utero</em&gt; L&#39;électroporation et<em&gt; In vivo</em&gt; Bioluminescence dépistage

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

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

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

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

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

Manipulation génétique de la souris en développement à travers l&#39;hypothalamus<em&gt; In utero</em&gt; Électroporation

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

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

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

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

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

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, latéral Septal Nucleus- et le striatum spécifique<em&gt; In Utero</em&gt; L&#39;électroporation dans la souris C57BL / 6

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

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

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

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

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

L&#39;imagerie confocale temporelle des neurones migrateurs dans la culture de tranches organotypiques du cerveau embryonnaire de souris en utilisant<em&gt; Dans Utero</em&gt; Electroporation

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

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

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

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

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

Induisant la recombinaison Cre-lox dans le Cortex cérébral par l’intermédiaire de In Utero électroporation de souris

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