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

Etica Dichiarazione: Il trattamento dei topi e le procedure sperimentali sono state condotte in conformità agli orientamenti europei, nazionali e istituzionali per la cura degli animali. 1. In Utero Elettroporazione Nota: In utero elettroporazione è stato eseguito come precedentemente pubblicato 12,14. Pertanto, il metodo è descritto brevemente nel seguito (figura 1). <ol><li> Preparativi <ol><li> Preparare veloce verde soluzione priva di endotossine color avanzato trasfezione qualità plasmide (contenente 1,5 pCAGGS) come descritto in precedenza 12. </li><li> Tirare e macinare (35 ° angolo) capillari di vetro borosilicato (0,8-0,9 diametro) per preparazioni iniettabili. </li><li> Sterilizzare gli strumenti. </li></ol></li><li> Chirurgia <ol><li> Somministrare analgesici (carprofen, 4 mg / kg di peso corporeo, sc, 24 ore) deposito di almeno 30 minuti prima di iniziare l&#39;intervento chirurgico. </li><li> Mantenere la lunghezza dellaanestesia ad un massimo di 35-45 min (Fase III - fase dell&#39;anestesia chirurgici secondo Guedel 15 - massima 25-30 min). </li><li> Anestetizzare topi cronometrati in gravidanza con isoflurano (induzione in una camera a 2,8%, la chirurgia tramite maschera: 2,5%). Confermare l&#39;anestesia da non responsività ai piedi-pizzico. </li><li> Applicare pomata occhio per prevenire la secchezza oculare durante l&#39;anestesia. </li><li> Sterilizzare l&#39;area chirurgica con 7,5% di soluzione Providone-iodio e coprire il mouse con garza sterile (umido con una soluzione allo 0,9% di alcool benzilico soluzione fisiologica), con solo l&#39;area chirurgica esposta. </li><li> Tagliare aprire la cavità addominale con un bisturi (incisione cutanea: 1,5-2 cm, incisione muscolo: 1-1,5 cm). </li><li> Preservare la cavità addominale aperta da secchi da inumidire con soluzione salina riscaldata alcool benzilico allo 0,9% o altra soluzione isotonica sterile, come indicato dal personale veterinario o del comitato. </li><li> Estrarre attentamente corna uterine (con pinze ad anello, non toccare l&#39;utero con le ditaS). </li></ol></li><li> L&#39;iniezione di DNA ed elettroporazione. Nota: I dettagli esatti per il targeting regioni specifiche sono indicati nella sezione 2. <ol><li> Iniettare lentamente la soluzione di DNA colorato nel ventricolo come indicato nella figura 1 e la sezione 2 tramite i capillari borosilicato preparati. </li><li> Applicare la tensione appropriata per il corrispondente stadio embrionale (ad es., 36-38 V per E14) tramite elettrodi specializzati forcipe tipo platino (ciclo intervallo di lunghezza 50 msec, intervallo di pausa 950 msec). Pulse le palette elettroporazione durante l&#39;applicazione della tensione per minimizzare i danni della parete uterina. </li></ol></li><li> Messaggio elettroporazione / assistenza post-operatoria <ol><li> Ricollocare accuratamente le corna uterine nella cavità addominale. </li><li> Suturare il muscolo e l&#39;incisione cutanea separatamente. </li><li> Lasciate che il resto del mouse su un dispositivo di supporto per il recupero termico. </li><li> Sorvegliare il mouse durante il recupero (comportamento, alimentazione econsumo di acqua), 4 ore, 24 ore e 48 ore dopo l&#39;intervento chirurgico. Se necessario, applicare analgesici supplementari (carprofen, 4 mg / kg di peso corporeo, 24 ore deposito) per altri 2 giorni. </li></ol></li></ol> 2. Trasfezione di aree cerebrali specifiche Nota: La posizione del polo positivo è mostrato come l&#39;angolo tra la linea centrale verticale attraverso il ventricolo riempito con la soluzione di DNA (ventricolo destro o terzo) e la posizione del polo positivo. Durante il riempimento del ventricolo sinistro le posizioni sono speculare. In Figura 4 sono mostrate le posizioni primordiale dell&#39;orecchio, che forniscono importanti punti di riferimento. <ol><li> Trasfezione di aree corticali (Figura 2A, Tabella 1). <ol><li> Eseguire un intervento chirurgico come descritto nella sezione 1.2. Per l&#39;elettroporazione in utero di aree corticali utilizzare embrioni E14. </li><li> Iniettare 1,5-2 microlitri DNA-soluzione contenente 4 mg di DNA nel ventricolo laterale destroattraverso il capillare borosilicato preparati. <ol><li> Posizionare accuratamente l&#39;embrione utilizzando una pinza ad anello in modo che non ci sono vasi visibili sopra il sito di puntura. </li><li> Iniettare lentamente il DNA-soluzione circa 0,75 mm coronale dalla sutura lambdoidal e 0,5 mm lateralmente dalla sutura sagittale (Figura 3). Assicurarsi che la profondità di inserimento è di 1,2 mm (E14, misurata dalla parete uterina). Verificare la presenza di una colorazione blu-verde con una demarcazione tagliente. </li></ol></li><li> Applicare tensione tramite gli elettrodi di platino forcipe tipo specializzati (ciclo intervallo di lunghezza 50 msec, tempo di pausa 950 msec). <ol><li> Utilizzare un elettrodo pala 3 o 5 mm. </li><li> Utilizzare una tensione compresa 36-38 V. </li><li> Posizionare il centro degli elettrodi solo anteriori dei primordi dell&#39;orecchio. </li><li> Per trasfettare gli angoli uso corteccia retrosplenial 330-0 °, per trasfettare la corteccia motoria 0-40 °, per trasfettare la corteccia somatosensoriale 40-95 °, per trasfettarela corteccia piriforme 95-110 ° (Figura 2A). </li></ol></li><li> Eseguire elettroporazione / assistenza post-operatoria dopo come descritto nel paragrafo 1.4. Sacrifica i topi e analizzare gli embrioni 4 giorni dopo l&#39;elettroporazione (E18), come descritto nella sezione 3. </li></ol></li><li> Transfection dell&#39;ippocampo (Figura 2B, Tabella 1). <ol><li> Eseguire un intervento chirurgico come descritto nella sezione 1.2. Per l&#39;elettroporazione in utero di formazione dell&#39;ippocampo utilizzare embrioni E15. </li><li> Iniettare 2 microlitri DNA-soluzione contenente 4 mg di DNA nel ventricolo laterale destro attraverso il capillare borosilicato preparati. </li><li> Applicare tensione tramite gli elettrodi di platino forcipe tipo specializzati (ciclo intervallo di lunghezza 50 msec, tempo di pausa 950 msec). <ol><li> Utilizzare un elettrodo pala 3 o 5 mm. </li><li> Utilizzare una tensione compresa 38-40. </li><li> Posizionare il centro del polo negativo appena anteriore del primordio orecchio e il centro of il polo positivo al centro della primordium orecchio. </li><li> Per trasfettare giro dentato utilizzano angoli 190-210 °, per trasfettare il Ammonis cornu (CA) 3 210-230 °, per trasfettare il CA2 230-250 °, per trasfettare il CA1 250-275 ° (Figura 2B). </li></ol></li><li> Eseguire elettroporazione / assistenza post-operatoria dopo come descritto nel paragrafo 1.4. Eseguire l&#39;analisi dopo la formazione dell&#39;ippocampo completa (dopo la nascita, p21), come descritto nella sezione 3. </li></ol></li><li> Transfection del laterali nucleo settale e striato (Figura 2C, tabella 1) Nota: Gli angoli si sovrappongono con quelli transfecting l&#39;ippocampo. Per trasfettare efficacemente il nucleo settale laterale e striato senza indesiderate ippocampo transfezione fasi precedenti embrionali (E12) devono essere utilizzati. <ol><li> Eseguire un intervento chirurgico come descritto nella sezione 1.2. Per l&#39;elettroporazione in utero del nucleo settale laterale e striatum utilizzare embrioni E12. </li><li> Iniettare 1 ml di DNA-soluzione contenente 4 mg di DNA nel ventricolo laterale destro attraverso il capillare borosilicato preparati. </li><li> Applicare tensione tramite gli elettrodi di platino forcipe tipo specializzati (ciclo intervallo di lunghezza 50 msec, tempo di pausa 950 msec). <ol><li> Utilizzare un elettrodo 0,5 mm. </li><li> Utilizzare 33 V. </li><li> Posizionare il centro degli elettrodi a livello dei primordi dell&#39;orecchio. </li><li> Per trasfettare le uso angoli del setto nucleo laterali 100-170 °, per trasfettare striato 170-240 ° (Figura 2C). </li></ol></li><li> Eseguire elettroporazione / assistenza post-operatoria dopo come descritto nel paragrafo 1.4. Sacrifica i topi e analizzare gli embrioni 6 giorni dopo l&#39;elettroporazione (E18), come descritto nella sezione 3. </li></ol></li><li> Trasfezione del talamo e ipotalamo (Figura 2D, Tabella 1) <ol><li> Eseguire un intervento chirurgico come descritto nella sezione 1.2. Per l&#39;in utero elettroporazione delle talamo e ipotalamo uso E12-E13 embrioni. </li><li> Iniettare 1-1.5-DNA microlitri soluzione contenente 4 mg DNA nel ventricolo laterale (sinistro o destro) attraverso il capillare borosilicato preparati. Attendere 2-3 minuti, fino a quando la soluzione viene diffuso nel terzo ventricolo. In alternativa iniettare direttamente nel terzo ventricolo. Nota: Questo migliora la specificità, ma potrebbe causare un tasso di sopravvivenza ridotta (alto rischio di danni). </li><li> Applicare tensione tramite gli elettrodi di platino forcipe tipo specializzati (ciclo intervallo di lunghezza 50 msec, tempo di pausa 950 msec). <ol><li> Utilizzare un elettrodo 0,5 o 3 mm. </li><li> Utilizzare una tensione compresa 33-35 V. </li><li> Posizionare il centro degli elettrodi appena posteriore i primordi dell&#39;orecchio. </li><li> Per trasfettare gli angoli uso talamo 25-40 °, per trasfettare l&#39;ipotalamo 90-180 ° (Figura 2D). </li></ol></li><li> Eseguire posta elettroporazione / assistenza post-operatoria come described nella sezione 1.4. Sacrifica i topi e analizzare gli embrioni 6 giorni dopo l&#39;elettroporazione (E18), come descritto nella sezione 3. </li></ol></li></ol> 3. Fissazione perfusione <ol><li> Preparativi <ol><li> Warm 4% paraformaldeide (PFA) a 37 ° C. </li><li> Riempire una siringa da 50 ml con PBS (4 ° C) e una seconda siringa con riscaldato PFA. Evitare le bolle. </li><li> Collegare le siringhe a un rubinetto a tre vie. Collegare il terzo fine di un set di infusione alato (farfalla). </li><li> Lavare il set di infusione alato con PBS. </li></ol></li><li> Perfusione <ol><li> Profondamente anestetizzare topi (gravidanza o postnatale trasfettate) con l&#39;applicazione sottocutanea di ketamina cloridrato (60 mg / kg) e xilazina (7,5 mg / kg). </li><li> Confermare la fase chirurgica tolleranza refrattarietà ai piedi-pizzico. </li><li> Aprire la cavità addominale appena sotto la gabbia toracica. </li><li> Tagliare con cautela la membrana. </li><li> Aprire con cautela la gabbia toracica mediante un taglio sull&#39;esito ach fino alla clavicola. </li><li> Inserire la punta della farfalla nella camera del cuore sinistro (partendo dalla punta del cuore). </li><li> Tagliare l&#39;appendice atriale sinistra. </li><li> Lentamente profumato il mouse con PBS (circa 10 ml). </li><li> Accendere il rubinetto a tre vie. </li><li> Profumato il mouse con 5-10 ml 4% PFA. </li><li> Decapitare il mouse utilizzando le forbici e sezionare il cervello a partire dalla forame magno. Eseguire la dissezione come precedentemente pubblicato 16. </li><li> Postfixate il cervello per 2 giorni a 4% PFA. Mantenere il cervello a 4 ° C al buio. </li></ol></li></ol> 4. immunoistochimica <ol><li> Genera 70 sezioni micron (ad esempio, con un vibratome). </li><li> Opzionale: incrementare la fluorescenza con l&#39;anticorpo colorazione. <ol><li> Bloccare le sezioni per 1 ora a RT (0,1-0,2% Triton X-100, siero di capra normale 5%) </li><li> Incubare O / N con l&#39;anticorpo appropriata (anti-GFP, 1: 1.000) </li><li> Lavare 3-5 Volte con tampone fosfato 0,1 M. </li><li> Incubare 3-5 ore con l&#39;appropriato anticorpo secondario fluorescenza coniugato (Alexa Fluor 488 coniugato anti-coniglio IgG, 1: 1.000). </li><li> Opzionale: Controcolorare con 4-6-diaminodino-2-fenilindolo (DAPI, 0,2 g / ml in tampone fosfato 0,1 M) per 1 min. </li><li> Analizzare fluorescenza con un microscopio a fluorescenza appropriata. </li></ol></li></ol>

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The authors have nothing to disclose.

This protocol describes in detail how to transfect the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus of C75BL/6 mice. With all the provided information this is the first protocol, which supplies all necessary information to easily recreate transfections of these cerebral regions in the C57BL/6 mouse. Previous publications are mostly focused only on a few specific r...

Dalla prima descrizione nel 2001 da tre gruppi indipendenti 1-3 i n utero elettroporazione è diventato uno strumento standard ampiamente utilizzato per analizzare l&#39;espressione genica nel sistema nervoso centrale dei roditori. Rispetto alla generazione di topi knockout, che è, nonostante le tecniche di continuo miglioramento, ancora di tempo e denaro che consumano, i ricorsi in utero elettroporazione a causa della sua semplicità. Così, in utero elettroporazione consente GAIN- veloce ed efficiente e la perdita di funzione studi 4. Per trasfettare le regioni cerebrali, la soluzione contenente il plasmide carica negativa viene iniettato in un ventricolo. Durante l&#39;impulso elettrico, il DNA carico negativamente migra verso il polo positivo e quindi la regione trasfettate è selezionabile semplicemente modificando la posizione del polo positivo. E &#39;stato spesso dimostrato che numerose regioni del sistema nervoso centrale possono essere targeted 3,5-8. Ad esempio, recenti studi dimostrano trasfezioni specifiche del ippocampo, la corteccia piriforme o striato 9-11. Tuttavia, le informazioni sulle posizioni appropriate sono spesso solo poco standardizzato e non sono sempre facili da trasferire a diversi ceppi di topi. Trasfezione di alcune fasi embrionali è tutt&#39;altro che banale. Molti fattori influenzano devono essere presi in considerazione quando si sceglie il set-up per specifici elettroporazione in utero. In primo luogo, per trasfettare in modo ottimale i rispettivi stadi embrionali, è necessaria la conoscenza delle tensioni del caso. Tensioni elevate riducono il tasso di sopravvivenza, mentre basse tensioni riducono l&#39;efficienza di trasfezione 2,3,12. Anche le dimensioni del paddle dell&#39;elettrodo gioca un ruolo fondamentale, perché l&#39;uso di piastre di elettrodi che sono troppo grandi risultati a ridotta specificità o possono causare la morte per affetto del ritmo cardiaco 4,12,13. La applicatatensione e la dimensione e la posizione della pala dell&#39;elettrodo sono le caratteristiche più importanti da considerare, ma ci sono anche altri fattori che influenzano il risultato della elettroporazione, come la quantità applicata di DNA-soluzione. Abbiamo sviluppato un protocollo dettagliato che consente trasfezione veloce ed efficiente di varie regioni cerebrali del C57BL / 6 del mouse 12. In questo protocollo informazioni dettagliate sulle tensioni da utilizzare e la dimensione del paddle dell&#39;elettrodo per una maggiore specificità è disponibile. Inoltre, informazioni riguardanti il ​​ventricolo da riempire con raccomandazioni per la quantità di soluzione plasmide e la posizione dell&#39;elettrodo è fornito. L&#39;indicazione delle informazioni dettagliate sulla posizione in una cartina e l&#39;ulteriore visualizzazione di queste posizioni permette specifico semplice ed efficiente in utero elettroporazione della corteccia retrosplenial, la corteccia motoria, la corteccia somatosensoriale, la corteccia piriforme, tegli cornu Ammonis 1-3, il giro dentato, lo striato, il nucleo settale laterali, il talamo e l&#39;ipotalamo.

<table border="0" cellpadding="0" cellspacing="0" width="898">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:215px;">QIAGEN</td>
			<td style="width:188px;">12362</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Green</td>
			<td>Roth</td>
			<td>0301.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCAGGS</td>
			<td>Addgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">borosilicate glass capillaries (0.8-0.9 mm diameter)</td>
			<td>Wold Precision Instrument Inc.</td>
			<td>1B100F-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane (Forene)</td>
			<td>Abbott</td>
			<td>PZN 4831850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carprofen (Rimadyl)</td>
			<td>Pfizer GmbH</td>
			<td>approval number: 400684.00.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eye ointment (Bepanthen Augen und Nasensalbe)</td>
			<td>Bayer&#160;</td>
			<td>PZN 01578681</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">0.9% benzyl alcohol 0.9% saline solution</td>
			<td style="width:215px;">Pharmacy 
			of the University Medical Center Mainz</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gauze (ES-Kompressen)</td>
			<td>Hartmann</td>
			<td>407835</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sterile 5-0 Perma-Hand Silk Suture</td>
			<td>Ethicon Johnson &#38; Johnson</td>
			<td>K890H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ring forceps 1/ 1.5 mm</td>
			<td>Fine Science Tools</td>
			<td>11101-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ring forceps 4.8/ 6 mm</td>
			<td>Fine Science Tools</td>
			<td>11106-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ring forceps 2.2/ 3 mm</td>
			<td>Fine Science Tools</td>
			<td>11103-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adson Forceps-Serrated Straight 12 cm</td>
			<td>Fine Science Tools</td>
			<td>1106-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IrisScissors-Delicate Straight-Sharp/Blunt 10 cm</td>
			<td>Fine Science Tools</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mayo-Stille Scissors-Straight 15 cm</td>
			<td>Fine Science Tools</td>
			<td>14012-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 Forceps-Inox</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Castroviejo NeedleHolder-with Lock-Tungsten Carbide 14 cm</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elektroporator CUY21 SC&#160;</td>
			<td>Nepa Gene Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FST 250 Hot Bead Sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microgrinder EG-44</td>
			<td>Narishige</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P-97 Micropette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum electrodes 650P 0.5 mm</td>
			<td>Nepagene</td>
			<td>CUY650P0.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum electrodes 650P 3 mm</td>
			<td>Nepagene</td>
			<td>CUY650P3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum electrodes 650P 5 mm</td>
			<td>Nepagene</td>
			<td>CUY650P5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum electrodes 650P 10 mm</td>
			<td>Nepagene</td>
			<td>CUY650P10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anesthesia system</td>
			<td>Rothacher-Medical GmbH</td>
			<td>CV-30511-3 Vapor 19.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating plate</td>
			<td>Rothacher-Medical GmbH</td>
			<td>HP-1M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature Controller 220V AC</td>
			<td>Rothacher-Medical GmbH</td>
			<td>TCAT-2LV</td>
			<td></td>
		</tr>
	</tbody>
</table>

cortex-, hippocampus-, thalamus-, hypothalamus-, lateral septal nucleus- and striatum-specific in utero electroporation in the c57bl/6 mouse

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

La figura 2 mostra esempi di specifica in utero elettroporazione delle regioni in via di sviluppo nella corteccia retrosplenial, la corteccia motoria, la corteccia somatosensoriale, la corteccia piriforme, il cornu Ammonis 1-3, il giro dentato, lo striato, il nucleo settale laterali , il talamo e l&#39;ipotalamo. I risultati delle trasfezioni sono confrontate con l&#39;angolazione consigliata (Figura 2). Per una migliore visualizzazione degli angoli in vivo la posizion...

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

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

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

Cortex, Hippocampus-, Thalamus-, Hypothalamus-, 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 ...

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

Research

JoVE Journal

Neuroscience

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

27.8K Views.  University Medical Center, JGU Mainz. This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

Trasfezione delle cellule gangliari della retina di topo In vivo Elettroporazione

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

Ricerca

Neuroscienze

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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

Gene consegna efficiente in più territori SNC utilizzo In Utero Elettroporazione

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

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 ...

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 ...

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

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

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

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

Generazione di topi transgenici per uso topico<em&gt; In utero</em&gt; Elettroporazione e<em&gt; In vivo</em&gt; Screening bioluminescenza

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

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

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

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

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

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 ...

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, ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

Un approccio semplice per indurre la neurite Autoimmune Sperimentale in topi C57BL/6 per le valutazioni funzionali e Neuropathological

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

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 ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

Chirurgia di impianto cocleare e registrazioni di risposta elettricamente evocate uditive del tronco cerebrale in topi C57BL/6

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Cortex, Hippocampus-, Thalamus-, Hypothalamus-, laterale del setto Nucleus- e Striatum specifici

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