Ce travail a été financé par la Fondation allemande pour la recherche (Deutsche Forschungsgemeinschaft).

1. Préparation de l&#39;ADN et verre Micropipettes pour injection <ol><li> Bonne Pipette en verre de qualité sont essentiels pour réduire le taux élevé d&#39;avortements initiale due à la perte de liquide amniotique. La procédure de tirer micropipettes de verre a été bien documenté 13,18,25. Utiliser 1,2 mm de diamètre capillaires tirée dans une Sutter classique P-97 appareil avec les paramètres P = 500; chaleur = 300; Tirer = 40; Velocity = 50; Temps = 50. Monter l&#39;extracteur avec 3 mm de filaments de «creux» (Sutter Instrument FT330B). Les filaments de taille de 2 mm ont donné pour nous des résultats moins satisfaisants. D&#39;autre part, le biseautage des pointes de micropipette ne semble pas améliorer les résultats pour nous. </li><li> Dissoudre purifié, l&#39;ADN plasmidique sans endotoxine dans PBS (Cell Culture Grade) contenant 0,1% de Fast Green à une concentration finale de 1 à 2 pg / pl. The Green rapide fera la solution injectée visible dans le ventricule cérébral embryonnaire. </li><li> Charge de 10 pi de la solution d&#39;ADN dans la micropipette de verre. </li&gt; <li> Raccorder la micropipette de verre pour le système d&#39;injection (pompe à Pico) ou d&#39;une pipette de bouche. </li></ol> 2. Anesthésie Préparer la table d&#39;opération avec un coussin chauffant et les instruments chirurgicaux. Activer les sources de lumière froide pour faciliter la visualisation des embryons. Désinfecter les outils chirurgicaux en utilisant par exemple une bille de verre stérilisateur. Trois procédures d&#39;anesthésie sont possibles pour ce protocole (voir la discussion). Ici, nous allons décrire celui que nous considérons le meilleur, en utilisant inhalation isoflurane pour l&#39;induction de l&#39;anesthésie (débit de 0,5 L / min) ainsi que la maintenance (débit de 1 l / min). <ol><li> Introduire la souris enceinte dans un petit transparent (si la souris reste visible) récipient relié à la Komesaroff Mark 5 anesthésique périphérique par une courte longueur de tube. </li><li> Remplir le vaporisateur avec de l&#39;isoflurane, puis ouvrez la bouteille d&#39;oxygène relié à l&#39;appareil. Soufflez un ou deux impulsions de isoflurane / oxygène (0,5 L / min) dans le récipient tenant la souris et montre pour la narcose à mettre po </li><li> Prenez la souris sur, couvrir les yeux avec de la pommade pour les empêcher de sécher et monter le masque d&#39;anesthésie de l&#39;écoulement sur la tête immédiatement. </li></ol> 3. Laparotomie <ol><li> Placez la souris ventre en l&#39;air sur le coussin chauffant (sinon sa température corporelle va baisser très rapidement, ce qui diminue les chances de reprise) et sécuriser son corps en place en fixant ses quatre pattes sur les côtés avec du ruban adhésif. Évaluer la profondeur de l&#39;anesthésie en vérifiant la perte de la réponse à la stimulation réflexe (par exemple orteil ou pincement de la queue avec une pression ferme). </li><li> Raser la surface abdominale et désinfecter avec le iodophorpovidone-iode (Braunoderm). </li><li> Faire une incision longitudinale (1 à 1,5 cm de longueur) sur la peau de l&#39;abdomen. Ensuite, couper le péritoine. Placez la gaze de coton autour de l&#39;incision. Faire une corne utérine visible et retirez-le délicatement avec une pince émoussée sur le PBS rincé gauze. Rincer l&#39;utérus avec PBS très souvent pour le garder toujours humecté (Figures 2A et 2B). Pendant toute la procédure éviter de tirer le mésomètre ou de l&#39;utérus serré, puisque haute pression à l&#39;intérieur de l&#39;utérus transmettra à l&#39;embryon augmente les chances de perte de fluide lors de l&#39;injection entraîne l&#39;avortement. </li></ol> 4. Injection d&#39;ADN dans le ventricule cérébral <ol><li> Maintenir l&#39;utérus de manière à ce que les ventricules cérébraux peuvent être visualisées. Ne pas largement repositionner un embryon qui se trouve dans une position défavorable - cela ne fera qu&#39;accroître les chances de l&#39;avortement. </li><li> En regardant la tête de l&#39;embryon, de haut, de localiser visuellement l&#39;écart ou fissure entre les deux hémisphères du cortex gauche et droite. Les hémisphères sont faciles à distinguer et les ventricules latéraux intérieur d&#39;eux (pour ne pas être la cible) peuvent généralement être perçus comme des formes un peu plus sombres. Si l&#39;embryon se trouve être parfaitement orientée pour l&#39;injection d&#39;ADN (Figures 2C et 2D), il est possible de percer la paroi utérine et entrent dans le troisième ventricule à la fois. Maintenez la micropipette de verre à 45 ° de la paroi utérine et une ponction à la fin rostrale de l&#39;écart entre les hémisphères corticaux, pénétrant pendant environ 1 mm. De cette manière, la pointe de la micropipette de verre va entrer dans le troisième ventricule cérébral (pas le ventricule latéral) (figures 2C et 2D). En moins favorablement orientés embryons, il est utile de commencer par la paroi utérine Pierce (toujours à 45 °) dans le voisinage de la tête embryonnaire, placer la pointe micropipette dans la bonne position entre les hémisphères corticaux et seulement ensuite perforer le cerveau. Injecter environ 1 pl de solution d&#39;ADN dans le troisième ventricule (une bonne injection remplit le ventricule avec du liquide vert). Répétez la même procédure avec tous les embryons d&#39;une corne utérine. Ceci permet un certain temps pour la solution d&#39;ADN à mélanger uniformément avec le fluide du ventricule et à atteindre la entire neuroépithélium. </li></ol> 5. Cibler l&#39;hypothalamus pour l&#39;électroporation <ol><li> Mettez le électroporateur et ajuster les réglages en fonction de l&#39;âge embryonnaire (E12.5 nous utilisons pour 5 impulsions rectangulaires, 50 V, 50 msec ON/950 ms OFF). Utilisation telle que le pôle positif de l&#39;électrode d&#39;aiguille en acier inoxydable (CUY550-10) et en tant que pôle négatif d&#39;une électrode plate ronde (CUY700P4L). (Il est important que les électrodes sont plus petits que l&#39;embryon, puisque le courant contraire coule juste autour de l&#39;embryon, mais pas au travers.) </li><li> Sélectionnez pour l&#39;électroporation ces embryons dont la face dorsale est en hausse (c. tourné vers l&#39;expérimentateur) (comme la plupart d&#39;entre eux sont), et jetez ceux dont l&#39;orientation est pas favorable. Il est maintenant sûr de toucher l&#39;utérus avec les doigts éthanol ou désinfectée avec des gants. Tenir l&#39;utérus avec une pince, cependant, cela nuirait à la circulation du courant au cours de l&#39;électroporation. </li><li> Percer la paroi utérine par la tête de l&#39;embryon entre le amniotic sac et le placenta en poussant vers le bas à travers elle à la pointe de l&#39;électrode à aiguille. Utilisation de l&#39;index de l&#39;autre côté en tant que bloc de poussée. Environ 5 mm de la pointe de l&#39;électrode doit être présent entre le sac amniotique et la paroi utérine, à peu près au niveau du mésencéphale (figures 2E et 2F). N&#39;oubliez pas que c&#39;est le &quot;électrode ciblage», de sorte que sa position va déterminer quelle partie de la neuroépithélium hypothalamique est le plus susceptible de se transfectées. L&#39;embryon doit être très doucement &quot;pressé&quot; entre les électrodes. </li><li> Avec l&#39;autre main, la position de l&#39;électrode, plat et circulaire en dehors de la paroi de l&#39;utérus, du côté opposé de la tête de l&#39;embryon (figures 2E et 2G). </li><li> Utilisez la pédale pour appliquer une tension (50 V, 50 ms ON, 950 ms OFF, 5 impulsions rectangulaires). </li><li> Tirez doucement sur l&#39;électrode d&#39;aiguille de l&#39;utérus tout en retenant l&#39;utérus avec l&#39;index de l&#39;autre main - si amnioliquide tic est perdue par le mur percé, généralement l&#39;embryon va subir un avortement. Répétez la procédure avec tous les embryons. </li><li> Retour de la corne de l&#39;utérus dans l&#39;abdomen et répéter l&#39;injection et l&#39;électroporation dans la corne reste. </li></ol> 6. Finition de la chirurgie <ol><li> Après l&#39;injection et l&#39;électroporation de tous les embryons, placez l&#39;utérus dans l&#39;abdomen très soigneusement, positionné exactement comme ils étaient avant et humide de la cavité péritonéale avec une solution saline avant de le refermer. </li><li> Suturer le péritoine avec Catguts (Vicryl Polyglactin 910, 5-0, Ethicon V4914). Pour la fermeture de l&#39;utilisation de la peau &quot;interrompu point&quot; methodwith un de suture plus résistant (Supramid Nylon, 6-0, Serag Wiessner AUX 07171L). </li><li> Désinfecter la surface de l&#39;abdomen avec povidone-iode (Braunoderm) et injecter un non-stéroïdien anti-inflammatoire cutanée (par exemple 100 pi d&#39;une solution à 1:10 de Rimadyl dans NaCl à 0,9%) ou, mieux encore, un opioïde comme buprenorphine (Temgesic, de 0,05 à 0,1 mg / kg de poids corporel dans NaCl à 0,9%) pour soulager la douleur. </li><li> Retirez la mère de l&#39;appareil d&#39;anesthésie et le placer dans une cage qui est chauffé par un coussin chauffant. Surveiller la souris continuellement jusqu&#39;à ce qu&#39;il soit complètement rétabli de l&#39;anesthésie. Par la suite, vérifiez les animaux tous les jours pour s&#39;assurer qu&#39;ils se remettent de la procédure, sans aucun signe d&#39;infection ou de la douleur. </li></ol> 7. Analyse des résultats <ol><li> Récolter des embryons (ou postnatals) en fonction du jour de l&#39;analyse souhaitée. Souris postnatal (vieux de 1 à 2 jour) sont tués par décapitation. </li><li> Séparez chaque embryon en fonction de la précédente annotation d&#39;électroporation. Disséquer le cerveau sous une loupe binoculaire et regarder sous le microscope de signal fluorescent vert (de la journaliste GFP) dans la région appropriée. </li><li> Le cerveau sélectionné peut être analysée &quot;frais&quot;: fixer le tissu pendant une courte période dans du paraformaldéhyde 4% et l&#39;intégrer dans agarose 4% ou dans de la gélatine-albumin, alors l&#39;article sur un périphérique vibratome type (Figure 3). Pour une analyse immunohistochimique plus détaillée (figure 4), cryo-protéger le cerveau dans une solution de saccharose à 30%, intégrer dans un milieu de montage (OCT Tissue Tek) puis réduction de 20 sections um dans un cryostat. </li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammillary+bodies:+two+memory+systems+in+one.">The mammillary bodies: two memory systems in one.</a> Nat. Rev. Neurosci. 5, 35-44 (2004).</li><li>Gratsch, T. E., De Boer, L. S., O'Shea, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+inhibition+of+BMP-4+gene+expression+in+postimplantation+mouse+embryos.">RNA inhibition of BMP-4 gene expression in postimplantation mouse embryos.</a> Genesis. 37, 12-17 (2003).</li><li>Wu, N., Yu, A. B., Zhu, H. B., Lin, X. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+silencing+of+Sry+gene+with+RNA+interference+in+developing+mouse+embryos+resulted+in+feminization+of+XY+gonad.">Effective silencing of Sry gene with RNA interference in developing mouse embryos resulted in feminization of XY gonad.</a> J. Biomed. Biotechnol. 2012, 343891 (2012).</li><li>Kikuchi, N., Nakamura, S., Ohtsuka, M., Kimura, M., Sato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+mechanism+of+gene+transfer+into+early+to+mid-gestational+mouse+fetuses+by+tail+vein+injection.">Possible mechanism of gene transfer into early to mid-gestational mouse fetuses by tail vein injection.</a> Gene Ther. 9, 1529-1541 (2002).</li><li>Foust, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat. Biotechnol. 27, 59-65 (2009).</li><li>Xin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+targeting+mechanism+of+Angiopep-conjugated+poly(ethylene+glycol)-co-poly(epsilon-caprolactone)+nanoparticles.">The brain targeting mechanism of Angiopep-conjugated poly(ethylene glycol)-co-poly(epsilon-caprolactone) nanoparticles.</a> Biomaterials. 33, 1673-1681 (2012).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108, 193-199 (1991).</li><li>Kaufman, R. J., Davies, M. V., Wasley, L. C., Michnick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+vectors+for+stable+expression+of+foreign+genes+in+mammalian+cells+by+use+of+the+untranslated+leader+sequence+from+EMC+virus.">Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.</a> Nucleic Acids Res. 19, 4485-4490 (1991).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu. Rev. Biochem. 67, 509-544 (1998).</li><li>Londrigan, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+promoters+for+driving+efficient+transgene+expression+in+neonatal+porcine+islets.">Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets.</a> Xenotransplantation. 14, 119-125 (2007).</li></ol>

Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

A propos de l&#39;anesthésie: Depuis électroporation in utero dans l&#39;hypothalamus peut être techniquement difficile et nécessiter des temps de narcose plus, nous préférons pour induire et maintenir une anesthésie par administration d&#39;un mélange d&#39;oxygène et d&#39;isoflurane. Dans notre expérience, les animaux peuvent rester suffisamment anesthésiés de cette façon pendant des périodes allant jusqu&#39;à une heure au moins, la reprise de la mère est très rapide, et la survie de l&#39;...

La manipulation génétique du cerveau embryonnaire de souris est une approche privilégiée pour en apprendre davantage sur la régulation du développement. La génération de lignées de souris mutantes est cependant lent et coûteux. Une méthode puissante pour introduire des modifications génétiques spécifiques dans le développement des neurones du cerveau des mammifères est en électroporation in utero. Essentiellement, la technique consiste à transfecter l&#39;ADN dans le neuroépithélium cerveau embryonnaire au moyen d&#39;impulsions électriques, puis permettre à l&#39;embryon de survivre pendant un certain laps de temps, de recueillir le cerveau et les examiner une éventuelle roman, phénotypes informatives. De cette façon, l&#39;expérimentateur peut tester des hypothèses presque immédiatement, sans les longues périodes d&#39;attente nécessaires pour la production de mutants de souris. Transfection d&#39;ADN dans des embryons en développement a commencé par électroporation in ovo sur des embryons de poulet 1. La preuve de concept essentiel pour la souris a été réalisée dans la culture <sup&gt; 2. Ce fut bientôt suivi par les premières descriptions de la technique sur la souris in utero 3,4. Le principal problème consiste à transfecter le cerveau d&#39;embryons en développement in utero sans eux ou la mère qui tue. Apprendre à effectuer la chirurgie nécessaire (laparotomie, l&#39;injection, l&#39;électroporation) nécessite une longue période de formation. Une fois que l&#39;opération a été maîtrisé au point où le taux de survie des embryons est acceptable, la prochaine question clé est: quelles structures cérébrales sont-ils accessibles? Sans surprise, les premiers articles publiés montrant les résultats obtenus avec électroporation in utero axés sur le développement cortical 5-9. Cela est encore vrai pour la plupart des publications utilisant cette technique, car la région du cerveau de souris en développement plus accessible à des procédures chirurgicales est le cortex (Figure 1). La procédure d&#39;électroporation in utero dans le cortex a été décriten version imprimée et en vidéo 10 11-14. Une modification de la technique peut être utilisée pour cibler une partie ventrale du télencéphale, les noyaux gris centraux 15. Au-delà du télencéphale, diencéphale (classiquement divisé en thalamus et de l&#39;hypothalamus) est une région du cerveau antérieur plus difficiles à atteindre. Un petit nombre de documents ciblant des rapports de sa partie dorsale et le plus accessible, le thalamus 16-19. L&#39;hypothalamus est la partie la plus ventrale du cerveau antérieur, donc celle localisée plus profondément de la surface dorsale (cortex) (Figure 1). Cette région reste un défi difficile pour les chercheurs engagés à manipuler génétiquement le cerveau de souris in utero. À notre connaissance, seuls quelques articles un rapport sur ​​les résultats de transfection in utero dans l&#39;hypothalamus de souris 20,21. Cependant, l&#39;importance fonctionnelle de l&#39;hypothalamus cannot être surestimée, car elle régule les comportements comme manger et de boire, l&#39;accouplement, la reproduction et la parentalité 22. En outre, les altérations dans le développement hypothalamique contribuent à provenir tard dans des conditions de vie comme l&#39;obésité, l&#39;hypertension, le diabète et la puberté précoce 23. Être capable de modifier génétiquement l&#39;hypothalamus au cours du développement constituerait un outil très puissant pour comprendre. Le protocole chirurgical de base pour la laparotomie de souris gravides que nous utilisons ici est similaire à celle utilisée dans les autres protocoles 11,13,14,24. Nous allons décrire ici brièvement par souci d&#39;exhaustivité. La clé de notre procédure, d&#39;autre part, sont le type d&#39;anesthésie, l&#39;endroit de l&#39;injection, le type d&#39;électrodes et de l&#39;insertion et de la position de l&#39;électrode positive par rapport à la tête de l&#39;embryon. Nous préférons pour induire et maintenir une anesthésie par inhalation de gaz plus simples anesthésie intrapéritonéale, puisque le premier permet l&#39;un peu plus longues périodes de narcose nécessaires pour une chirurgie difficile. Résultats d&#39;inhalation isoflurane chez une récupération plus rapide de l&#39;anesthésie, car, généralement, la mère démontre un comportement normal déjà minutes après la chirurgie. Le point d&#39;injection de la solution d&#39;ADN avec la micropipette de verre la plus simple est le ventricule latéral, ce qui est cependant tout à fait impropre à l&#39;électroporation de l&#39;hypothalamus. Injection directement dans le troisième ventricule est en effet crucial de cibler les structures diencéphaliques profondes. Il est possible de transfecter l&#39;hypothalamus de E12.0 ou E12.5 à la norme électrodes, off-the-shelf. Nous avons trouvé quelques-uns des électrodes fabriquées par Nepa Gene (Chiba, Japon) particulièrement adapté à cet effet. Grâce à notre procédure nous obtenons transfection de l&#39;ensemble neuroépithélium hypothalamique ou transfection régionale partielle en fonction de l&#39;orientation de l&#39;électrode. Ici, nous démontrons la technique par transfection du corps mamillaire, sans doutele plus profond et le plus en retrait de tous les noyaux hypothalamiques. En outre, nous montrons l&#39;analyse histologique détaillée des cellules transfectées jusqu&#39;au niveau cellulaire de la résolution. Une comparaison des électroporation in utero avec d&#39;autres approches de la transfection le développement du cerveau de la souris in utero peut être trouvée dans la section Discussion.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Sanofi GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>HDG9623</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Ketamin</td>
			<td>Pharma GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Fluka</td>
			<td>44715</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rimadyl</td>
			<td>Pfizer</td>
			<td>&nbsp;</td>
			<td>non-steroidal anti-inflammatory</td>
		</tr>
		<tr>
			<td>Braunoderm</td>
			<td>Braun</td>
			<td>3887138</td>
			<td>povidone-iodine</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline PBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temgesic (buprenorphine)</td>
			<td>Essex Pharma</td>
			<td>&nbsp;</td>
			<td>opioid analgesic</td>
		</tr>
		<tr>
			<td>Eye Ointment</td>
			<td>Pan-Ophtal</td>
			<td>7136926</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Anaesthetic Device Komesaroff Mark-5</td>
			<td>Medical Developments Australia</td>
			<td>ACN 004 903 682</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Capillary puller P-97</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compresstome</td>
			<td>Precisionary Instr.</td>
			<td>VF-300</td>
			<td>Vibratome-type device</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY21EDIT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrode 1</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY550-10</td>
			<td>Stainless Steel Needle Electrode, 10 mm-Tip, 0. 5 mm diam.</td>
		</tr>
		<tr>
			<td>Electrode 2</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY700P4L</td>
			<td>Cover Round Platinum Plate 4 mm diameter</td>
		</tr>
		<tr>
			<td>Fiberoptic cold light source</td>
			<td>Leica</td>
			<td>KL2500 LCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>GC120T-15</td>
			<td>1. 2 mm O.D. x 0. 94 mm I.D.</td>
		</tr>
		<tr>
			<td>Glass bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>FST250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Harvard Apparatus</td>
			<td>py872-5272</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Injection device</td>
			<td>World Precision Instruments</td>
			<td>Pneumatic Pico Pump PV820</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Suture Thread Coated Vicryl</td>
			<td>Ethicon</td>
			<td>V4914</td>
			<td>Peritoneal Suture</td>
		</tr>
		<tr>
			<td>Suture Thr. Supramid</td>
			<td>Serag Wiessner</td>
			<td>TO07171L</td>
			<td>Skin Suture</td>
		</tr>
	</tbody>
</table>

genetic manipulation of the mouse developing hypothalamus through in utero electroporation

La modification génétique des régions spécifiques du cerveau des mammifères en développement est une approche expérimentale très puissant. Cependant, générant des nouveaux mutants de souris est souvent d&#39;une lenteur décourageante. Il a été démontré que l&#39;accès au cerveau de souris en développement in utero à la survie post-opératoire raisonnable est possible. Pourtant, les résultats de cette procédure ont été signalés presque exclusivement pour la partie la plus superficielle et facilement accessibles du cerveau en développement, à savoir le cortex. Le thalamus, une région étroite et plus médial, s&#39;est avéré plus difficile à cibler. Transfection dans des noyaux plus profond, en particulier ceux de l&#39;hypothalamus, est peut-être les résultats les plus difficiles et donc très peu ont été rapportés. Ici, nous démontrons une procédure de cibler l&#39;ensemble neuroépithélium hypothalamique ou une partie de celui-ci (régions hypothalamiques) pour la transfection par électroporation. Les clés de notre approche sont plus narcose fois, l&#39;injection dans le troisi ventricule, et le type et le positionnement des électrodes approprié. En outre, nous montrons les résultats de ciblage et d&#39;analyse histologique du noyau hypothalamique plus en retrait, le corps mamillaire.

La plupart des neurones de l&#39;hypothalamus sont nés entre E11.5 à E15.2, selon l&#39;analyse naissance-dating chez le rat 26 traduit en développement un peu plus courte de souris 27,28. Le pic de la neurogenèse hypothalamique est atteint à E12.5 29-31. En conséquence, à l&#39;âge de transfection choisi pour la présente étude (E12.5), une grande proportion des neurones hypothalamiques peut être marqué à n&#39;importe quel niveau rostro-caudale donné. <p class="jove_co...

Watch this Scientific Journal Video about Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation at JoVE.com

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Malgré l&#39;importance fonctionnelle et médicale de l&#39;hypothalamus,<em&gt; In utero</em&gt; Manipulation génétique de son développement a été tentée rarement. Nous montrons une procédure détaillée pour<em&gt; In utero</em&gt; Électroporation dans l&#39;hypothalamus de souris et affichent des résultats représentatifs de totale et partielle (régional) transfection hypothalamique.

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 ...

genetic-manipulation-mouse-developing-hypothalamus-through-utero

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JoVE Journal

Neuroscience

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

15.7K vues.  University of Heidelberg. Malgré l'importance fonctionnelle et médicale de l'hypothalamus, In utero Manipulation génétique de son développement a été tentée rarement. Nous montrons une procédure détaillée pour In utero Électroporation dans l'hypothalamus de souris et affichent des résultats représentatifs de totale et partielle (régional) transfection hypothalamique.

Vidéo: Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

En électroporation in vivo de la rétine de souris en développement

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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Neurosciences

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.

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 ...

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Manipulation génétique des neurones du cervelet granules<em&gt; In Vitro</em&gt; Et<em&gt; In Vivo</em&gt; À étudier neuronale Morphologie et migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

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 ...

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 électroporation approches pour étudier l&#39;excitabilité des neurones sous - populations et connectivité unicellulaire

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

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 ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, they may function to regulate the developmental process and/or physiological function in neonatal brains. To investigate neurobiological functions of specific genes in the brain, it is essential to inactivate genes in the brain. Here, we describe a simple stereotaxic method to inactivate gene expression in the striatum of transgenic mice at neonatal time windows. AAV-eGFP-Cre viruses were microinjected into the striatum of Ai14 reporter gene mice at postnatal day (P) 2 by stereotaxic brain surgery. The tdTomato reporter gene expression was detected in P14 striatum, suggesting a successful Cre-loxP mediated DNA recombination in AAV-transduced striatal cells. We further validated this technique by microinjecting AAV-eGFP-Cre viruses into P2Foxp2fl/fl mice. Double labeling of GFP and Foxp2 showed that GFP-positive cells lacked Foxp2 immunoreactivity in P9 striatum, suggesting the loss of Foxp2 protein in AAV-eGFP-Cre transduced striatal cells. Taken together, these results demonstrate an effective genetic deletion by stereotaxically microinjected AAV-eGFP-Cre viruses in specific neuronal populations in the neonatal brains of floxed transgenic mice. In conclusion, our stereotaxic technique provides an easy and simple platform for genetic manipulation in neonatal mouse brains. The technique can not only be used to delete genes in specific regions of neonatal brains, but it also can be used to inject pharmacological drugs, neuronal tracers, genetically modified optogenetics and chemogenetics proteins, neuronal activity indicators and other reagents into the striatum of neonatal mouse brains.

We describe a protocol of stereotaxic surgery with a homemade head-fixed device for microinjecting reagents into the striatum of neonatal mouse brains. This technique allows genetic manipulation in neuronal cells of specific regions of neonatal mouse brains.

Chirurgie stéréotaxique des manipulations génétiques dans des cellules de cerveaux de souris néonatales striataux

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...