Questo lavoro è stato finanziato dalla German Research Foundation (Deutsche Forschungsgemeinschaft).

1. Preparazione del DNA e vetro Micropipette per Iniezione <ol><li> Buone micropipette di vetro di qualità sono essenziali per ridurre il tasso iniziale aborto elevato a causa della perdita di liquido amniotico. La procedura di tirare micropipette di vetro è stato ben documentato 13,18,25. Uso 1.2 mm di diametro capillari tirato in un convenzionale Sutter P-97 dispositivo con le impostazioni P = 500; calore = 300; Pull = 40; Velocità = 50; Tempo = 50. Montare l&#39;estrattore con 3 millimetri filamenti &quot;di valle&quot; (Sutter Instrument FT330B). Le due millimetri filamenti dimensioni hanno prodotto per noi i risultati meno soddisfacenti. D&#39;altra parte, smussatura della micropipetta punte non sembra migliorare i risultati per noi. </li><li> Sciogliere purificato, DNA plasmidico privo di endotossine in PBS (Cell Culture Grade) contenente 0,1% Verde fast ad una concentrazione finale di 1 a 2 mg / mL. Il oVerde renderà la soluzione iniettata visibile nel ventricolo cerebrale embrionale. </li><li> Carico 10 microlitri della soluzione di DNA nel micropipetta di vetro. </li&gt; <li> Collegare la micropipetta di vetro al sistema di iniezione (pompa Pico) o bocca pipetta. </li></ol> 2. Anestesia Preparare il tavolo chirurgico con una piastra elettrica e gli strumenti chirurgici. Accendere le sorgenti di luce fredda per facilitare la visualizzazione degli embrioni. Disinfettare gli strumenti chirurgici utilizzando per esempio una perlina di vetro sterilizzatore. Sono possibili tre diverse procedure di anestesia per questo protocollo (vedi discussione). Qui descriveremo quello che consideriamo il meglio, utilizzando inalazione isoflurano per l&#39;induzione dell&#39;anestesia (portata 0,5 l / min) e di manutenzione (portata 1 l / min). <ol><li> Introdurre il mouse incinta in un piccolo trasparente (così il mouse rimane visibile) contenitore collegato al Komesaroff Mark 5 anestetico dispositivo da un breve tratto di tubo. </li><li> Riempire il vaporizzatore con isoflurano, quindi aprire la bombola di ossigeno collegato al dispositivo. Saltare uno o due impulsi di isoflUrane / ossigeno (0,5 l / min) nel contenitore tenendo premuto il mouse e guardare per narcosi a prendere piede </li><li> Prendete il mouse fuori, coprire gli occhi con unguento per evitare che si secchi e si adattano la maschera anestesia flusso sulla sua testa immediatamente. </li></ol> 3. Laparotomia <ol><li> Posizionare il mouse a pancia in su sul pad riscaldamento (altrimenti la sua temperatura corporea scende molto velocemente, diminuendo le possibilità di recupero) e fissare il suo corpo in posizione fissando le sue quattro gambe ai lati con del nastro adesivo. Valutare la profondità dell&#39;anestesia controllando per la perdita di risposta alla stimolazione riflessa (es. punta o la coda pizzico con una forte pressione). </li><li> Radersi la superficie addominale e disinfettarlo con il iodophorpovidone-iodio (Braunoderm). </li><li> Effettuare una incisione longitudinale (lungo da 1 a 1,5 cm) sulla cute addominale. Poi tagliare il peritoneo. Posizionare garza di cotone intorno alla incisione. Fai un corno uterino visibile ed estrarlo con cautela con una pinza smussato sulla PBS-risciacquato gauze. Risciacquare dell&#39;utero con PBS molto spesso per mantenerlo sempre inumidito (Figure 2A e 2B). Durante l&#39;intera procedura di evitare di tirare il mesometrio o dell&#39;utero stretto, poiché l&#39;alta pressione all&#39;interno dell&#39;utero trasmetterà all&#39;embrione aumentando le probabilità di perdita di fluido in fase di iniezione con conseguente aborto. </li></ol> 4. Iniezione DNA nel ventricolo cerebrale <ol><li> Tenere l&#39;utero in modo tale che i ventricoli cerebrali possono essere visualizzati. Non estesamente riposizionare un embrione che si trova in una posizione sfavorevole - questo aumenta solo le probabilità di aborto. </li><li> Guardando la testa dell&#39;embrione dall&#39;alto, localizzare visivamente il gap o fessura tra gli emisferi corticali destro e sinistro. Gli emisferi sono facili da distinguere e ventricoli laterali al loro interno (per non essere presi di mira) di solito possono essere percepite come forme un po &#39;più scuri. Se un embrione è risultato essere perfettamente orientato per l&#39;iniezione del DNA (Figures 2C e 2D), è possibile perforare la parete uterina e entrano nel terzo ventricolo in una volta. Tenere la micropipetta di vetro a 45 ° rispetto alla parete uterina e puntura alla fine rostrale del divario tra emisferi corticali, penetrando per circa 1 mm. In questo modo la punta della micropipetta di vetro entrerà nel terzo ventricolo del cervello (non il ventricolo laterale) (Figure 2C e 2D). In embrioni meno favorevolmente orientati è utile prima perforare la parete uterina (sempre a 45 °) in prossimità della testa embrionale, posizionare la punta micropipetta nella giusta posizione tra gli emisferi corticali e solo allora perforare il cervello. Iniettare circa 1 ml di soluzione di DNA nel terzo ventricolo (una buona iniezione riempie il ventricolo con liquido verde). Ripetere la stessa procedura con tutti gli embrioni di un corno dell&#39;utero. Questo permette un certo tempo per la soluzione di DNA per mescolare uniformemente con il fluido ventricolare e raggiungere il entire neuroepitelio. </li></ol> 5. Targeting l&#39;ipotalamo per elettroporazione <ol><li> Accendere il elettroporatore e regolare le impostazioni in base all&#39;età embrionale (per E12.5 usiamo 5 impulsi a onda quadra, 50 V, 50 msec ON/950 msec OFF). Utilizzare come polo positivo all&#39;elettrodo ago in acciaio inossidabile (CUY550-10) e come polo negativo di un elettrodo piatto rotondo (CUY700P4L). (È importante che gli elettrodi sono più piccoli l&#39;embrione, poiché altrimenti la corrente scorre dietro l&#39;embrione ma non attraverso esso.) </li><li> Selezionare per elettroporazione quegli embrioni la cui parte dorsale è alto (cioè rivolta verso lo sperimentatore) (come la maggior parte di loro sono), e scartare quelle il cui orientamento non è favorevole. Ora è possibile toccare l&#39;utero con le dita etanolo-disinfettate o con i guanti. Tenendo dell&#39;utero con una pinza, tuttavia, potrebbe interferire con il flusso di corrente durante elettroporazione. </li><li> Perforare la parete uterina per la testa dell&#39;embrione tra il amnsac iotico e la placenta dalla spinta verso il basso attraverso di essa con la punta dell&#39;elettrodo dell&#39;ago. Utilizzare il dito indice dell&#39;altra mano come blocco di spinta. Circa 5 mm della punta dell&#39;elettrodo deve essere ora tra il sacco amniotico e la parete uterina, circa al livello del mesencefalo (figure 2E e 2F). Ricordate che questo è il &quot;mira elettrodo&quot;, quindi la sua posizione determinerà quale è più probabile ottenere trasfettate parte del neuroepitelio ipotalamico. L&#39;embrione deve essere molto gentilmente &quot;spremuto&quot; tra gli elettrodi. </li><li> Con l&#39;altra mano, posizionare l&#39;elettrodo piatto tondo esterna della parete dell&#39;utero sul lato opposto della testa dell&#39;embrione (figure 2E e 2G). </li><li> Utilizzare l&#39;interruttore a pedale per applicare tensione (50 V, 50 ms ON, 950 ms OFF, 5 impulsi a onda quadra). </li><li> Tirare lentamente l&#39;elettrodo ad ago fuori dell&#39;utero, mentre trattenendo l&#39;utero con il dito indice dell&#39;altra mano - se amniotic liquido viene perso attraverso la parete forata, di solito l&#39;embrione subirà aborto. Ripetere la procedura con tutti gli embrioni. </li><li> Ritorna il corno dell&#39;utero nell&#39;addome e ripetere l&#39;iniezione ed elettroporazione nel corno residuo. </li></ol> 6. Completamento della Chirurgia <ol><li> Dopo l&#39;iniezione ed elettroporazione di tutti gli embrioni, posizionare l&#39;utero torna in addome con molta attenzione, posizionato esattamente come erano prima e umide cavità peritoneale con soluzione fisiologica prima di chiuderlo. </li><li> Sutura del peritoneo con catgut (Vicryl Polyglactin 910, 5-0, Ethicon V4914). Per la chiusura della pelle uso &quot;interruppe stitch&quot; methodwith una sutura più resistente (Supramid Nylon, 6-0, Serag Wiessner TO 07171L). </li><li> Disinfettare la superficie dell&#39;addome con povidone-iodio (Braunoderm) e iniettare un non-steroidei anti-infiammatori per via sottocutanea (ad esempio 100 ml di una soluzione 1:10 di Rimadyl in 0.9% NaCl) o, meglio ancora, un oppioide come buprenorphine (Temgesic, da 0.05 a 0.1 mg / kg di peso corporeo in 0.9% NaCl) per alleviare il dolore. </li><li> Rimuovere la madre dalla macchina anestetica e metterlo in una gabbia che è riscaldata da una piastra elettrica. Monitorare il mouse continuamente fino a quando non è completamente recuperato dal anestesia. Successivamente, controllare gli animali ogni giorno per assicurare che si stanno riprendendo dalla procedura senza alcun segno di infezione o dolore. </li></ol> 7. Analisi dei risultati <ol><li> Raccogliere gli embrioni (o postnatals) secondo il giorno desiderato di analisi. Topi postnatale (1-2 giorni di età) vengono uccisi per decapitazione. </li><li> Separare ogni embrione secondo il precedente elettroporazione annotazione. Sezionare il cervello allo stereomicroscopio e controllare al microscopio per segnale fluorescente verde (GFP dal reporter) nella regione appropriata. </li><li> Il cervello selezionato può essere analizzato &quot;fresco&quot;: fissare il tessuto per un breve periodo in paraformaldeide al 4% e incorporare nel agarosio 4% o in gelatina-albumin, poi sezione su un dispositivo vibratomo-type (Figura 3). Per l&#39;analisi immunoistochimica dettagliata (Figura 4), ​​crio-proteggere il cervello in soluzione di saccarosio al 30%, incorporare nel montaggio Ottobre medio (Tissue Tek) poi tagliare 20 sezioni micron in un criostato. </li></ol>

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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>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Circa l&#39;anestesia: Poiché elettroporazione in utero nell&#39;ipotalamo può essere tecnicamente ardua e richiedono tempi più lunghi narcosi, preferiamo indurre e mantenere l&#39;anestesia mediante somministrazione di una miscela di ossigeno e isoflurano. Nella nostra esperienza, gli animali possono rimanere adeguatamente anestetizzato in questo modo, per periodi fino a un&#39;ora almeno, la ripresa della madre è molto veloce, e la sopravvivenza dell&#39;embrione migliorata. Altri approcci per l&#39;anest...

Manipolazione genetica del cervello di topo embrionale è un approccio preferito per conoscere regolazione dello sviluppo. La generazione di linee mutanti topo però è lento e costoso. Un metodo efficace per introdurre specifici cambiamenti genetici nello sviluppo di neuroni del cervello dei mammiferi è elettroporazione in utero. In sostanza, la tecnica consiste transfecting DNA nel cervello embrionale neuroepithelium mediante impulsi elettrici, consentendo quindi l&#39;embrione di sopravvivere per un certo periodo di tempo, raccogliere il cervello e li esamini per possibili romanzo, fenotipi informativi. In questo modo, l&#39;sperimentatore può verificare ipotesi quasi immediatamente senza i lunghi periodi di attesa necessari per la produzione di mutanti di topo. Trasfezione di DNA in via di sviluppo è iniziato con embrioni in elettroporazione ovo su embrioni di pollo 1. L&#39;essenziale proof-of-concept per il mouse è stata eseguita in cultura <sup&gt; 2. Questo è stato presto seguito dalle prime descrizioni della tecnica del mouse in utero 3,4. Il problema principale è quello di transfettare il cervello di embrioni in via di sviluppo in utero senza ucciderli o della madre. Apprendimento per eseguire l&#39;intervento necessario (laparotomia, iniezione, elettroporazione) richiede un lungo periodo di formazione. Una volta che l&#39;intervento chirurgico è stato masterizzato al punto in cui il rapporto di sopravvivenza degli embrioni è accettabile, la prossima domanda chiave è: quali strutture cerebrali sono accessibili? Non a caso, i primi documenti pubblicati mostrano risultati ottenuti con elettroporazione in utero focalizzati su sviluppo corticale 5-9. Questo è ancora vero per la maggior parte delle pubblicazioni utilizzando questa tecnica, poiché la regione del cervello di sviluppo del mouse più accessibile per procedure chirurgiche è la corteccia (Figura 1). La procedura per elettroporazione in utero nella corteccia è stata descrittain stampa 10 e in video 11-14. Una modifica della tecnica può essere utilizzata per indirizzare una parte ventrale del telencefalo, i gangli della base 15. Al di là del telencefalo, il diencefalo (classicamente suddiviso in talamo e ipotalamo) è una regione del prosencefalo più difficile da raggiungere. Un piccolo numero di documenti di report di targeting della sua parte dorsale e più accessibile, il talamo 16-19. L&#39;ipotalamo è la parte più ventrale del prosencefalo, quindi quello localizzato più profondamente dalla superficie dorsale (corteccia) (Figura 1). Questa regione rimane una sfida difficile per i ricercatori impegnati a manipolare geneticamente il cervello di topo in utero. A nostra conoscenza, solo pochissimi articoli riferire sui risultati di in utero trasfezione nell&#39;ipotalamo del mouse 20,21. Tuttavia, l&#39;importanza funzionale dell&#39;ipotalamo cannot essere sottovalutata, in quanto regola i comportamenti come il mangiare e il bere, l&#39;accoppiamento, l&#39;allevamento e la genitorialità 22. Inoltre, alterazioni dello sviluppo dell&#39;ipotalamo contribuiscono ad originare in seguito a condizioni di vita come l&#39;obesità, l&#39;ipertensione, il diabete e la pubertà precoce 23. Essere in grado di modificare geneticamente l&#39;ipotalamo durante lo sviluppo potrebbe fornire uno strumento molto potente per capirlo. Il protocollo chirurgico di base per la laparotomia di topo gravide che utilizziamo qui è simile a quella utilizzata in altri protocolli 11,13,14,24. Noi li descrivono brevemente qui per completezza. Chiave del nostro procedimento, invece, sono il tipo di anestesia, il luogo di iniezione, del tipo di elettrodi e l&#39;inserimento e la posizione dell&#39;elettrodo positivo rispetto alla testa del embrione. Preferiamo indurre e mantenere l&#39;anestesia per inalazione gas su semplice anestesia intraperitoneale, poiché il primo consente l&#39;un po &#39;più lunghi periodi di narcosi necessari per un intervento chirurgico difficile. Risultati inalazione isoflurano in veloce recupero dall&#39;anestesia, dal momento che di solito la madre dimostra un comportamento normale già minuti dopo l&#39;intervento chirurgico. Il più facile punto di iniezione della soluzione di DNA con la micropipetta di vetro è il ventricolo laterale, che però è completamente inadatto per ipotalamo elettroporazione. Iniezione diretta del terzo ventricolo è infatti cruciale per indirizzare le strutture diencefaliche profonde. È possibile trasfettare all&#39;ipotalamo da E12.0 E12.5 o con elettrodi standard, off-the-shelf. Abbiamo trovato alcuni degli elettrodi fabbricati da Nepa Gene (Chiba, Giappone) particolarmente adatta a questo scopo. Con la nostra procedura otteniamo trasfezione di tutta neuroepitelio ipotalamica o parziale, trasfezione regionale a seconda dell&#39;orientamento elettrodo. Qui mostriamo la tecnica transfettando corpo mammillary, discutibilmenteil più profondo e incasso di tutti i nuclei ipotalamici. Inoltre, mostriamo analisi istologica dettagliata delle cellule trasfettate giù al livello cellulare di risoluzione. Un confronto di elettroporazione in utero ad altri procedimenti di trasfezione sviluppo cerebrale topo in utero può essere trovata nella sezione di discussione.

<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

Modificazione genetica di specifiche regioni del cervello dei mammiferi è sviluppare un approccio sperimentale molto potente. Tuttavia, generando nuovi mutanti di topo è spesso frustrante lentezza. È stato dimostrato che l&#39;accesso al cervello di sviluppo del mouse in utero con ragionevole sopravvivenza post-operatoria è possibile. Ancora, risultati con questa procedura sono stati riportati quasi esclusivamente per la parte più superficiale e facilmente accessibile del cervello di sviluppo, cioè la corteccia. Il talamo, una regione stretto e più mediale, è dimostrato più difficile da bersaglio. Trasfezione in nuclei più profondo, specialmente quelli dell&#39;ipotalamo, è forse il risultato più impegnative e, pertanto, molto pochi sono stati segnalati. Qui mostriamo una procedura per individuare l&#39;intera neuroepitelio ipotalamica o parte di essa (regioni ipotalamiche) per trasfezione mediante elettroporazione. Le chiavi del nostro approccio sono più volte narcosi, l&#39;iniezione in tHird ventricolo, e tipo e posizionamento degli elettrodi appropriata. Inoltre, vi mostriamo i risultati di targeting e la successiva analisi istologica del nucleo ipotalamico più incassata, il corpo mammillary.

La maggior parte dei neuroni dell&#39;ipotalamo sono nati tra E11.5 a E15.2, secondo l&#39;analisi nascita-dating nel ratto 26 tradotto nella po &#39;più breve di sviluppo del mouse 27,28. Il picco della neurogenesi ipotalamica è raggiunta a E12.5 29-31. Conseguentemente, all&#39;età trasfezione scelto per questo studio (E12.5), una grande proporzione di neuroni ipotalamici può essere etichettata ad ogni dato livello rostro-caudale. Analisi alla E18.5 su ...

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

Nonostante l&#39;importanza funzionale e mediche dell&#39;ipotalamo,<em&gt; In utero</em&gt; Manipolazione genetica del suo sviluppo è stato raramente tentata. Mostriamo una procedura dettagliata per<em&gt; In utero</em&gt; Elettroporazione nell&#39;ipotalamo mouse e mostrano risultati rappresentativi (regionale) trasfezione ipotalamo totale e parziale.

La manipolazione genetica del topo sviluppo dell&#39;ipotalamo attraverso<em&gt; In utero</em&gt; Elettroporazione

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

Research

JoVE Journal

Neuroscience

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

15.7K Views.  University of Heidelberg. Nonostante l'importanza funzionale e mediche dell'ipotalamo, In utero Manipolazione genetica del suo sviluppo è stato raramente tentata. Mostriamo una procedura dettagliata per In utero Elettroporazione nell'ipotalamo mouse e mostrano risultati rappresentativi (regionale) trasfezione ipotalamo totale e parziale.

Video: 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.

In elettroporazione in vivo di Retina topo in via di sviluppo

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

Ricerca

Neuroscienze

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.

Mouse in Utero elettroporazione: controllato Transefection Gene spazio-temporale

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.

Gene Transfer per l&#39;orecchio del mouse Sviluppo Inner<em&gt; In Vivo</em&gt; Elettroporazione

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.

Visualizzazione e manipolazione genetica di dendriti e spine nella corteccia cerebrale e nell&#39;ippocampo mouse utilizzando<em&gt; In utero</em&gt; Elettroporazione

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.

Manipolazione genetica delle cerebellare granuli neuroni<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Per studiare neuronale Morfologia e migrazione

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-, laterale del setto Nucleus- e Striatum specifici<em&gt; In Utero</em&gt; Elettroporazione in C57BL / 6 del mouse

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.

Elettroporazione in utero approcci per studiare l&#39;eccitabilità neuronale sottopopolazioni e connettività single-cell

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.

Impressione confocale dei migratori dei neuroni nella cultura di fetta organotipica del cervello embrionale del mouse usando<em&gt; In Utero</em&gt; Elettroporazione

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.

Induzione della ricombinazione di Cre-lox nella corteccia cerebrale del Mouse attraverso elettroporazione In Utero

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

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.

Chirurgia stereotassica per la manipolazione genetica in cellule striatali di cervelli di topo neonatale

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