We are indebted to Pierre Mattar and Freda Miller for their assistance with the in utero electroporation and to Noel Ghanem and Bensun Fong for creating diagrams. We thank Linda Jui and Jason G. MacLaurin for excellent technical assistance. Equipment was supported by the Centre for Stroke Recovery.
This work was supported by a scholarship from HSFO and OGS to D.S.S. and a CIHR grant to R.S.S.

NOTA: Tutti gli esperimenti sono stati approvati dalla University of Animal Care comitato etico di Ottawa, aderendo alle linee guida del Consiglio canadese Animal Care. Preparazione 1. Pre-chirurgici e materiali <ol><li> Plasmidi Preparazione: <ol><li> Preparare plasmidi usano il Qiagen MegaPrep secondo il protocollo del produttore. Inoculare almeno 400 ml di brodo LB con batteri trasformati per garantire una resa elevata DNA e incubare durante la notte. </li><li> Risospendere plasmide in 100-200 microlitri TE ad una concentrazione finale di almeno 5 mg / mL. Aliquota DNA e conservare a -20 ° C. Se la concentrazione è troppo bassa, non precipitare e risospendere come questo può aggiungere impurità. Iniziare con una nuova preparazione. </li><li> Prima della chirurgia, diluire un singolo plasmide (Cre-GFP per esempio) per 2 mg / mL in acqua sterile con 1 ml di 0,1% (w / v) colorante Fast verde per 10 o 20 ml di soluzione diluita plasmide. Se GFPe il gene di interesse sono da co-elettroporate su plasmidi separati, iniettare il plasmide GFP e il plasmide che porta il gene di interesse ad un rapporto di 1: 4 per massimizzare la probabilità che tutte le celle contrassegnate con GFP sono co-elettroporate con entrambi plasmidi. </li></ol></li><li> Tirare i tubi microcapillari di vetro (diametro esterno 1 mm - di seguito denominati aghi) su un estrattore standard di ago, utilizzando un unico tiro passo a 62 ° C. </li><li> Autoclave una confezione chirurgica contenente i seguenti: porta-aghi, divaricatore ferita, pinze, forbici per la chirurgia, forbici per aghi, cucitrice, graffette, garza, le coperture della ferita, teli chirurgici (Figura 1A, B; Materials Table). </li><li> Raccogliere altri strumenti necessari: elettroporatore, FemtoJet Microinjecter, Tweezertrodes (5 o 7 mm), saline, sutura, siringa da 5 ml (Materials Table). </li></ol> 2. Preparare Mouse for Surgery <ol><li> Per la gestione del dolore, iniettare un-p temporizzatoMouse regnant con buprenorfina (0,05 mg / kg di peso corporeo) un&#39;ora prima dell&#39;intervento. Applicare questo protocollo per topi a tempo ovunque da embrionale giorno 12,5 (E12.5) a E17.5. </li><li> Utilizzare microloader puntali delle pipette per eseguire il carico tirato aghi con la soluzione plasmide. Non riempire fino in fondo la punta dell&#39;ago ma ciò causerebbe l&#39;ago ad occludersi. </li><li> Anaesthetize mouse usando 2-5% gas isoflurano con 1 L / min O 2. Una volta immobile, posto in maschera e applicare una pomata per gli occhi per evitare l&#39;essiccazione. Iniettare con 1 ml di soluzione fisiologica (0,9% NaCl) per via sottocutanea in indietro. NOTA: Quando si lavora con gas isoflurano, utilizzare un scavenger gas di riconquistare gas in eccesso ed evitare la fuoriuscita nella stanza. </li><li> Shave addome. Lavare addome con Chlorohexadine scrub, risciacquare con acqua, e applicare una soluzione di preparazione finale del chlorohexadine e il 70% di etanolo. Mouse copertura con telo sterile o garza per coprire la pelliccia, ma lasciare l&#39;addome esposto (Figura 1C, D). </li></ol>In utero elettroporazione <ol><li> Fare un&#39;incisione nella pelle sopra l&#39;addome tra i capezzoli inferiori. Tirare la pelle lontano dallo strato muscolare sottostante strappando il tessuto connettivo, poi tagliare attraverso il peritoneo nella cavità addominale lungo la linea alba. Inserire il riavvolgitore ferita per tirare i bordi della ferita parte. </li><li> Rimuovere l&#39;utero dalla cavità addominale e stenderlo come mostrato (Figura 1E). <ol><li> Se diversi cuccioli deve essere iniettato con differenti plasmidi (come GFP vs GFP con il gene di interesse), contare i cuccioli su ogni lato e decidere quali e quanti iniettare con ciascun plasmide. Sostituire un lato dell&#39;utero posteriore nell&#39;addome per mantenere gli embrioni caldo e lubrificati. Inumidire Subito dell&#39;utero esposta con soluzione salina (preferibilmente riscaldato alla temperatura corporea). </li><li> Se iniettando tutti cuccioli con lo stesso plasmide (come quando l&#39;iniezione Cre-GFP in embrioni transgenici) only rimuovere un corno dell&#39;utero alla volta. </li></ol></li><li> Mettere un ago caricata nella testa di presa della bacchetta di iniezione e con attenzione tagliare la punta con un paio di piccole forbici chirurgiche o pinza sottile. Tagliare la punta in modo che l&#39;ago rimane più a lungo possibile, pur consentendo al fluido di passare attraverso (Figura 1Ji-iv). <ol><li> Regolare la lunghezza di iniezione (in secondi) e la pressione di iniezione sul microinjector modo che una piccola goccia e facilmente visibile di 0,1-0,2 microlitri appare sulla punta dell&#39;ago quando viene premuto il pedale paddle. Tenere il volume di questa goccia più coerente possibile tra gli aghi diversi. NOTA: questo passaggio è cruciale per la sopravvivenza degli embrioni (vedi la discussione). </li><li> Utilizzare i seguenti parametri di iniezione per la FemtoJet: iniezione di pressione (pi) = 250-300 hPa; tempo di iniezione (ti) = 0,8-1,2 sec; Pressione compensazione = 10-50 hPa. Se necessario, modificare questi parametri per accogliere variazione tra gli aghi. </li></ol></li><li> Seleggere un embrione e posizionarla delicatamente sulla schiena, la testa inclinata verso l&#39;alto (Figura 1F). NOTA: Questo può richiedere ruotando o spostando l&#39;embrione; fare attenzione a non applicare troppa pressione ed evitare la rottura del sacco amniotico durante la manipolazione dell&#39;embrione. </li><li> Una volta che l&#39;embrione è a posto, individuare il ventricolo, che presenta una forma di mezzaluna ombra parallelo al seno sagittale. Premere la punta dell&#39;ago verso l&#39;utero sopra il ventricolo con una leggera angolazione e guidare l&#39;ago attraverso la parete uterina. La quantità di pressione richiesta dipende dalla nitidezza dell&#39;ago. Spingere l&#39;ago attraverso la corteccia nel ventricolo. NOTA: Anche se vi è raramente qualsiasi ulteriore resistenza rilevabile, la testa del embrione è spesso spinto via un po &#39;e poi si ritrae, come l&#39;ago passa attraverso la corteccia nel ventricolo. </li><li> Pompare il paddle piede per iniettare la soluzione plasmide. Continuare a pompare fino a una zona verde è visibile ed è conforme alfigura dell&#39;arco del ventricolo (Figura 1G). Nei giovani embrioni (E12.5 a E14.5) il colorante spesso diffondere nel ventricolo controlaterale. NOTA: Il numero di pompe dipende dal diametro della punta dell&#39;ago, l&#39;età dell&#39;embrione (e conseguente dimensioni del ventricolo) e il volume di iniezione desiderato. Mantenere il volume iniettato più coerente possibile tra embrioni. </li><li> Iniettare tre o quattro embrioni. Tenere tutti gli embrioni esposti umide con soluzione salina durante procedura. </li><li> Ritornare alla primi embrioni iniettati e posizionare le pale in modo che l&#39;elettrodo positivo disegnare il plasmide nella regione desiderata del cervello (Figura 1H). Angle l&#39;elettrodo negativo in modo che il percorso più breve tra gli elettrodi passa direttamente attraverso la struttura mirato per elettroporazione (Figura 2). </li><li> Una volta che le pale sono a posto, applicare lo shock. A seconda dell&#39;età e delle dimensioni degli embrioni, utilizzare 5 impulsi di 35-50 V (vedi T1 grado). Utilizzare i seguenti parametri di elettroporazione: lunghezza di impulso di 5 msec, con un intervallo di impulso di 950 msec. Ripetere con tutti gli embrioni iniettati. </li><li> Tagliare la punta di nuovo ago pre-caricato come al punto 3.3, come l&#39;ultimo ago probabile secca e ostruita durante la fase 3.9. Iniettare la prossima serie di tre o quattro embrioni sia con lo stesso plasmide o un plasmide diverso e quindi applicare la scossa come al punto 3.9. Quando tutti i cuccioli in un lato dell&#39;utero vengono iniettati, rimuovere il secondo lato dell&#39;utero dall&#39;addome della femmina e quindi sostituire la parte completata per mantenerlo caldo. </li><li> Ritorno entrambi i lati dell&#39;utero alla femmina facendo attenzione a mantenere la superficie umida con soluzione fisiologica e non applicare troppa pressione per evitare traumi alle cavità amniotiche. Suturare lo strato muscolare chiusa con un semplice punto continuo. Staple la pelle chiusa. Se i punti non sono disponibili, suturare la pelle con un semplice punto interrotto. (Figura 1I) </li><li> Mentre suturing, progressivamente abbassare il isoflurano per consentire l&#39;anestesia della femmina incinta per alleggerire. NOTA: Questo accelerare l&#39;eccitazione della diga dopo l&#39;intervento chirurgico è completo; mantenendo la femmina anestetizzato per il più breve tempo possibile migliorare la sopravvivenza dell&#39;embrione. Tempo chirurgico totale, a partire da quando la femmina viene prima anestetizzato, deve essere di circa 30 min. </li></ol> 4. Aftercare <ol><li> Posizionare la femmina suturato in un incubatore o su una piastra elettrica per recuperare da anestesia. Il suo letto in biancheria pulita e posizionare un gel ripresa standard nella gabbia mantenere l&#39;idratazione. Per la gestione del dolore dopo un intervento chirurgico, dare le donne tre dosi di buprenorfina (0,05 mg / kg di peso corporeo) a 8 intervalli hr dopo l&#39;intervento, poi quattro dosi supplementari a intervalli di 12 h. </li></ol> 5. Brains raccolta NOTA: Brains possono essere raccolte a qualsiasi età fino all&#39;età adulta. Si applica la seguenteper la raccolta di cervelli prima della nascita. Si noti che una volta che i cuccioli sono nati, l&#39;ordine all&#39;interno dell&#39;utero è perso, quindi o ogni pup nella lettiera deve essere iniettata con lo stesso plasmide, o un altro metodo di distinguere tra controllo e animali da esperimento deve essere messo a punto. <ol><li> Preparare a freddo (4 ° C), filtrato 4% paraformaldeide (PFA) prima eutanasia femmina. </li><li> Euthanize femmina con una iniezione intraperitoneale di una dose letale di pentobarbital sodio (100 mg / kg di peso corporeo) e tagliare addome aperto. Estrarre entrambi i lati dell&#39;utero e lay out come quando cuccioli stati inizialmente iniettati. <ol><li> Nel caso in cui tutti i cuccioli ricevuto lo stesso plasmide, ad esempio quando si inietta Cre in una lettiera transgenico, ordine embrione non è importante, quindi tagliato l&#39;utero fuori della femmina e posto in PBS. </li><li> Se cuccioli sono stati iniettati con diversi plasmidi, assicurarsi di individuare ogni cucciolo, rilevando eventuali cuccioli morti, e determinare quali sono stati iniettati con controllo e speriplasmidi Tal prima di rimuovere l&#39;utero dalla femmina. Fare attenzione a tenere traccia di quali pup appartiene a quale gruppo dopo l&#39;utero è in PBS. </li></ol></li><li> Rimuovere ogni cucciolo individualmente, decapitarlo con forbici affilate o una lametta e mettere la testa in PBS. In caso di topi transgenici, raccogliere campioni di tessuto per la genotipizzazione e mantenere ogni testa separata, ad esempio in una piastra 12 pozzetti. </li><li> Sotto un ambito dissezione, rimuovere il cervello dal cranio, facendo attenzione a non intaccare la corteccia o strappare la linea mediana. Utilizzando un microscopio a fluorescenza, controllare per vedere se il cervello hanno un GFP + patch, che indica che sono stati elettroporate con successo. </li><li> Fissare cervelli di immersione in PFA a 4 ° C durante la notte. NOTA: Controllare il cervello per l&#39;espressione di GFP prima della fissazione da quando PFA spesso spegne il segnale. </li><li> Cervelli Cryoprotect attraverso l&#39;immersione nel 20% di saccarosio per almeno tre giorni prima del congelamento. Utilizzare un gradiente di saccarosio del 10%, 15% e 20% (24 ore in ciascuna soluzione) to prevenire danni ai tessuti a causa della pressione osmotica. Conservare il cervello congelato a -80 ° C e tagliare su un criostato a 10-14 micron sezioni spesse. </li></ol> 6. Co-colorazione con anti-GFP NOTA: Se il recupero dell&#39;antigene è necessario per co-macchia con anti-GFP, è importante usare un antigene protocollo di recupero delicata che non compromette il segnale GFP. Alcuni protocolli di recupero antigene sono troppo dure da utilizzare in combinazione con GFP colorazione (anche con un anticorpo contro GFP). Un acido citrico base pre-trattamento prima dell&#39;applicazione dell&#39;anticorpo primario è efficace per la maggior parte epitopi 24. <ol><li> Fare una soluzione 0,01 M di acido citrico in DDH 2 O. pH a 6 con un pH metro calibrato. </li><li> Calore acido citrico in camera di colorazione in forno a microonde o bagnomaria fino citrico raggiunge 75-80 ° C. Mettere i vetrini in camera di colorazione per 10-15 minuti. Mantenere la temperatura più stabile possibile. </li><li> Lavare i vetrini in PBS3x per 3 minuti a temperatura ambiente. Applicare l&#39;anticorpo primario e procedere con immunocolorazione secondo un protocollo standard. </li><li> Convalidare Cre escissione mediata da co-etichettatura per GFP e il gene di interesse per confermare abbattere (Figura 3A). </li></ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+E2F-1+mediates+the+autoregulation+of+RB+gene+expression.">The transcription factor E2F-1 mediates the autoregulation of RB gene expression.</a> Mol. Cell. Biol. 14 (1), 299-309 .</li><li>Ferland, R. J., Cherry, T. J., Preware, P. O., Morrisey, E. E., Walsh, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain. 460 (2), 266-279 (2003).</li></ol>

The authors have nothing to disclose

Due delle principali sfide in svolgimento in electroporations utero sono 1) la prevenzione embrione letalità e 2) riduzione della variabilità tra electroporations. Uno dei fattori più importanti nella prevenzione letalità è la qualità ago, come aghi smussati infliggere più danni per l&#39;embrione. Quando si taglia l&#39;ago, mantenere la punta più a lungo possibile, pur consentendo una gocciolina visibile di 0,1-0,2 ml di fluido apparire dopo una pompa del paddle piede (Figura 1J). Se l&#39;ago è troppo noioso o l&#39;albero troppo breve, il danno ai tessuti risultante potrebbe uccidere l&#39;embrione. Al contrario, se la punta dell&#39;ago è troppo fine, ci vorrà troppo tempo per iniettare il volume plasmide sufficiente; lasciando l&#39;ago nel cervello per troppo tempo i danni cause a causa di vibrazioni inevitabili di mani del ricercatore e lievi rotazioni dell&#39;embrione. Inoltre, utilizzare un microbeveler per fare gli aghi con un forte, consigli smussati per permettere iniezioni in più giovane, Gli embrioni più fragili, anche se questo non dovrebbe essere necessario per embrioni E13.5 e anziani. Il livello di stress della donna incinta è un altro fattore chiave che influenzano la sopravvivenza dell&#39;embrione. Il livello di stress più alto della diga, più è probabile che verrà interrotta la lettiera. Per questo motivo è importante ridurre al minimo il tempo di anestesia e cambiare alloggiamento il meno possibile prima e dopo l&#39;intervento chirurgico. Il ceppo di fondo può anche influenzare il livello di stress e può essere necessario per le colonie outcross inclini all&#39;ansia su un ceppo, come CD1, partendo dal presupposto che il fenotipo in esame è mantenuto tra i diversi ceppi di sfondo. Il singolo più grande limitazione per IUE è la variabilità intrinseca tra electroporations. Ogni cervello elettroporate è diverso a causa di variazioni di volume di iniezione, il posizionamento paddle, contatto paddle e l&#39;età degli embrioni. Poiché gli aghi sono variabili, non è possibile iniettare un volume fisso - piuttosto le invetigator deve approssimare la dimensione della macchia verde per fornire simili quantità di tutti gli embrioni. Analogamente, poiché l&#39;embrione fluttua in un fluido riempito sac, non è possibile misurare sistematicamente la posizione e l&#39;orientamento delle pale sopra la testa dell&#39;embrione, come avviene per preparazioni iniettabili stereotassica. Infine, leggere differenze di età embrionale possono avere effetti significativi sui risultati sperimentali. Per questo motivo, si raccomanda confronto tra cucciolata.

Migrazione Radial è un processo fondamentale per primo sviluppo corticale. Essa dipende da una varietà di fattori cellulari autonomi, come uscita corretta mitosi e la differenziazione neuronale, polarità cellulare, regolazione della dinamica del citoscheletro e l&#39;espressione dei recettori transmembrana, così come non-cellulari fattori autonomi, come la formazione del ponteggio gliali radiali e secrezione di orientamento migratorio molecole 1-3. Poiché rottura di uno di questi meccanismi può compromettere migrazione neuronale in un modello di topo transgenico 4-8, determinare la causa di fondo di un difetto di migrazione può essere un processo complesso e difficile. In utero elettroporazione (IUE) può essere utilizzato per integrare e semplificare interpretazione del fenotipo di un modello knock-out genetico e quindi chiarire i meccanismi importanti necessari per la migrazione radiale 7,9-11. In utero elettroporazione (IUE) è il processo mediante il quale un carryi plasmide<html> ng sia un gene di interesse e un reporter viene iniettato nel ventricolo nel cervello di un topo o ratto embrionale e quindi trascinato nelle cellule che rivestono il ventricolo attraverso l&#39;uso di una corrente elettrica 12-14. Questo permette al ricercatore di analizzare l&#39;effetto di alto o in basso regolazione di un gene di interesse per lo sviluppo o la funzione dei neuroni elettroporate. Dopo IUE, il cervello possono essere trattati per immunoistochimica 7,9-11, elettrofisiologia 15 o coltura cellulare 16,17. Il principale vantaggio di IUE è che si permette la manipolazione altamente specifica dell&#39;espressione genica. Inoltre, IUE può essere utilizzato per mirare ad una regione specifica del cervello sviluppo attraverso una corrente diretta (Figura 2). IUE può anche essere utilizzato per mirare ad un tipo di cellula specifico mediante iniezione di plasmidi che trasportano geni sotto il controllo di vari promotori o sistemi di attivazione plasmide (tetraciclina indotta l&#39;espressione del gene è un esempio) 10,18-21. jove_content &quot;&gt; IUE può essere utilizzato in combinazione con il sistema di escissione gene mediato Cre-Lox 7-9,11,22. Un plasmide contenente un gene che codifica il messaggio per la proteina Cre può essere elettroporate in un embrione omozigote per l&#39;allele floxed ( in cui le regioni del gene o DNA specifico sono fiancheggiate da due siti loxP per Cre mediata ricombinazione DNA). Cre quindi indurrà ricombinazione del gene di interesse specifico dei precursori neuronali elettroporate, generando un knock-out della floxed allele.The effetto proteine ​​atterramento sulla migrazione neuronale e lo sviluppo all&#39;interno dei singoli neuroni può essere studiata. elettroporazione di Cre induce ricombinazione in solo una piccola popolazione di cellule colpite, lasciando intatto l&#39;ambiente di supporto. Al contrario, l&#39;espressione tessuto specifico di Cre sotto il controllo di un tipo di cellula specifico promotore, si verifica durante tutto il tessuto in modo che entrambi neuroblasti migrano e l&#39;ambiente circostante potrebbero essere influenzate. Pertanto, juxtaposition di questi due approcci può determinare se una data difetto di migrazione è dovuta alla cella meccanismi non autonomi autonomi o cellulari. Migrazione difettoso in entrambi i sistemi sperimentali suggeriscono che i risultati fenotipo osservati da un meccanismo autonomo di cellule; migrazione normale dopo elettroporazione di Cre migrazione difettoso in un modello specifico Cre tessuto indica che il gene di interesse agisce attraverso un meccanismo autonomo non-cellula. IUE può anche essere usato per eseguire esperimenti di soccorso da electroporating potenziali geni interagiscono in knock out animali 7,9,10. Ad esempio, un ricercatore potrebbe tentare di salvare un fenotipo migrazione in un modello transgenico da electroporating un bersaglio a valle e determinare se il difetto di migrazione viene corretto in neuroni elettroporate. Questo ha il vantaggio aggiuntivo che un salvataggio di successo indica che la migrazione normale può essere ripristinato manipolando l&#39;espressione della proteina in una specifica neurone, anche se l&#39;ambiente circostante è ancora carente per il gene bersaglio. Ancora una volta, questo approccio è più tempo e conveniente che attraversa o generare linee transgeniche, con il vantaggio di determinare se il meccanismo è difettoso cellule autonoma. IUE può essere utilizzato per monitorare la migrazione dei neuroni attraverso elettroporazione di un plasmide giornalista in knock out embrioni (Figura 4C). Poiché solo i neuroni che rivestono il ventricolo al momento dell&#39;intervento chirurgico sono elettroporate 17, IUE può essere utilizzata per seguire i neuroni nati in un momento specifico con il vantaggio di visualizzazione della loro morfologia in vivo. Infine, è possibile utilizzare per indirizzare IUE regioni cerebrali come la telencefalo mediale o dorsale, ippocampo o eminenza gangliare (Figura 2). Questo dà ricercatori il potere di indagare la funzione del gene in una determinata area indipendente complicazioni risultatoing da proteine ​​atterramento in strutture vicine. Proteina retinoblastoma (pRb), P107 e P130 comprendono la famiglia di proteine ​​tasca e sono ben regolatori del ciclo cellulare uscita stabilita. Tuttavia, vi è una crescente evidenza che queste proteine ​​regolano anche del ciclo cellulare aspetti indipendenti di sviluppo neurale. Come abbiamo già dimostrato, pRb e P107 svolgono un ruolo cruciale sia in tangenziale 4,23 e migrazione radiale 6. Qui, dimostriamo il ruolo della proteina tasca familiari pRb e P107 sulla migrazione neuronale 6 per esemplificare l&#39;uso di elettroporazione in utero di Cre in un modello transgenico. In sintesi, IUE fornisce un modo potente di analizzare cellule effetti autonome di delezione del gene (o sovraespressione). Se combinato con tessuto specifico knock out modelli, IUE può fornire ulteriori informazioni riguardanti i meccanismi che controllano la migrazione neuronale.

<table><tbody><tr><td>ECM 830 Square Wave Electroporator</td><td>BTX Harvard Apparatus</td><td>45-0052</td><td>Electroporator only &amp;ndash; buy cables separately (45-0204)</td></tr><tr><td>1250FS Footswitch for ECM 830</td><td>BTX Harvard Apparatus</td><td>45-0211</td><td>Foot paddle is necessary for hands-free electroporation</td></tr><tr><td>Tweezertrodes</td><td>BTX Harvard Apparatus</td><td>45-0489 (5mm)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 45-0488 (7mm)</td><td>Platinum forcep style electrodes</td></tr><tr><td>Femtojet</td><td>Eppendorf</td><td>920010504</td><td>Microinjector</td></tr><tr><td>Griphead 0</td><td>Eppendorf</td><td>920007414</td><td>Holds the pulled needle</td></tr><tr><td>Universal Capillary Holder</td><td>Eppendorf</td><td>920007392</td><td>Connects griphead to tube</td></tr><tr><td>Injection Tube</td><td>Eppendorf</td><td>920007431</td><td>Connects capillary holder to Femtojet</td></tr><tr><td>Foot Control</td><td>Eppendorf</td><td>920005098</td><td>Foot paddle for hands-free injection</td></tr><tr><td>Microloader Tips</td><td>Eppendorf</td><td>930001007</td><td>Tips for loading the pulled capillary tubes</td></tr><tr><td>Plasmid Mega Kit</td><td>Qiagen</td><td>12181</td><td>5 QIAGEN-tip 2500, Reagents, Buffers</td></tr><tr><td>Fast Green FCF</td><td>Sigma-Aldrich</td><td>F7252-5G</td><td>Non-toxic dye</td></tr><tr><td>Extra Fine Bonn Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td>For trimming the needles</td></tr><tr><td>Bonn Strabismus Scissors</td><td>Fine Science Tools</td><td>14084-09</td><td></td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools</td><td>11050-10</td><td>Straight</td></tr><tr><td>Colibri Retractors</td><td>Fine Science Tools</td><td>17000-03</td><td></td></tr><tr><td>Stapler</td><td>Fine Science Tools</td><td>12031-07</td><td>For 7mm clips</td></tr><tr><td>Castroviejo Needle Holder</td><td>Medical Tools</td><td>SNH-6737</td><td>Straight, with lock</td></tr><tr><td>Needle Puller</td><td>Narishige</td><td>PC-10</td><td>For pulling microcappillaries</td></tr><tr><td>Microcapillaries</td><td>Drummond</td><td>1-000-800</td><td>0.4 mm inner diameter glass capillary tubes</td></tr></tbody></table>

induction of protein deletion through in utero electroporation to define deficits in neuronal migration in transgenic models

Delezione genetica utilizzando il sistema Cre-Lox in linee di topi transgenici è un potente strumento utilizzato per studiare la funzione della proteina. Tuttavia, salvo in modelli molto particolari Cre, la cancellazione di una proteina in tutta una popolazione di tessuti o cellule, spesso conduce a fenotipi complessi derivanti da molteplici meccanismi che interagiscono. Determinare se un fenotipo risultati di interruzione di un meccanismo autonomo cella, che è intrinseco alla cella in questione, o da un meccanismo autonomo non cellule, che risulterebbe dal deterioramento dell&#39;ambiente di quella cella, possono essere difficili da distinguere. Per ottenere una visione in funzione delle proteine ​​in un contesto in vivo, in utero elettroporazione (IUE) consente di eliminare gene in un piccolo sottogruppo di cellule all&#39;interno della corteccia in via di sviluppo o di qualche altra regione del cervello selezionata. IUE può essere utilizzato per indirizzare specifiche aree cerebrali, tra cui il telencefalo dorsale, telencefalo mediale, ippocampo, o eminenza gangliare. Questo facilita l&#39;osservazione of conseguenze della cella gene delezione autonoma nel contesto di un ambiente sano. Lo scopo di questo protocollo è quello di mostrare come IUE può essere utilizzato per analizzare un difetto nella migrazione radiale in una linea di topi transgenici floxed, con un&#39;enfasi sulla distinzione tra le cellule effetti autonomi autonomi e non cellulari di delezione della proteina. Confrontando il fenotipo risultante dall&#39;eliminazione gene nell&#39;intera corteccia contro gene delezione IUE mediata in una popolazione di cellule limitata, una maggiore comprensione della funzione delle proteine ​​nello sviluppo cerebrale può essere ottenuto che utilizzando due tecniche in isolamento.

Different regions of the brain can be targeted for electroporation by varying the orientation of the paddle electrodes over the embryo&#8217;s head (Figure 2). For all parts of the forebrain, the plasmid is injected into the lateral ventricle (the hind brain can be targeted by injecting into the fourth ventricle). In general, place the electrodes so that the straight line between them passes through the region of interest, with DNA being drawn toward the positive electrode. To target the lateral cortex (Figure 2A), place the positive electrode on top of the green crescent, with a slight rostral angle. For the medial cortex (Figure 2B), switch the orientation so that the positive electrode is over the contralateral non-injected ventricle, this time with an even larger forward angle. To target the hippocampus (Figure 2C), place the positive electrode over the contralateral side, but instead angle the positive electrode caudally (electroporations must be done at an age when the neural precursors are still part of the cortical hem, prior to migration to the final location of the hippocampus). To target the ganglionic eminence (Figure 2D) place the paddles on the embryo in line with the ears, with the positive electrode slightly lower than the negative electrode. Representative images of these electroporated regions are shown in Figure 2.
It is important to validate Cre-mediated excision in electroporated brains. The ideal approach is to perform in situ hybridization or immunohistochemistry against the transcript or protein in question. Unfortunately, as is the case for pRb, reliable probes or antibodies are not always available against the deleted gene. In these cases, alternative approaches, such as staining for a downstream target regulated by the deleted gene (Figure 3A) or confirming excision of the targeted allele in electroporated sections by PCR (Figure 3B), can be used. For example, Figure 3A confirms Cre mediated excision by reproducing a well-documented effect of pRb/p107/p130 gene deletion in electroporated neurons. Following electroporation of pRb f/f; p107 -/-; p130 f/f neurons with the pCig2-Cre plasmid, these triple knock out GFP+ cells continue to express the cell cycle progression marker Ki67, which is a known effect following excision of pocket proteins25. Control GFP+ neurons electroporated with the empty pCig2 plasmid do not express Ki67. The Cre plasmid should also be tested in vitro through co-transfection with a plasmid carrying a reporter gene behind a floxed stop codon, such as the NL-dsRed plasmid (Figure 3C) in a cell line, but this should only be used in combination with other in vivo techniques.
Due to the variation between electroporated brains, analysis of electroporated neurons requires calculating the percentage of total GFP+ neurons that portray a specific characteristic, such as expression of a protein marker or location within a given cortical layer. It is crucial to compare electroporated sections from similar anatomical locations within the brain relative to known landmarks. For example, images for Figure 4 were taken at the level of the anterior commissure. Furthermore, due to horizontal migration, images taken near the edge of the electroporated regions have a higher proportion of mature neurons that have reached the cortex, while images taken near the middle of the electroporated region have both mature and immature GFP+ cells. To insure consistency, it is important to quantify images selected from similar places within the electroporated patch.
Following Emx1-Cre mediated deletion of pRb and p107 in pyramidal neurons in the dorsal telencephalon, a population of neurons expressing the upper cortical layer marker FoxP1 were mislocalized to the intermediate zone (IZ) (Figure 4A). There are significantly more FoxP1+ cells in the IZ of double knock out (DKO) brains compared to control brains with a corresponding decrease in the number of FoxP1+ cells in the upper cortical layers (above layer IV) of DKO brains than found in the upper layers of control brains (Figure 4B). There was no corresponding defect in the radial glial scaffold (Figure S2 of Svoboda et al., 2012) 6. Here we compare expression of cortical layer markers Tbr1, FoxP1 and Cux1 in pRb f/+; p017 +/-; Emx1-Cre + (wild type (WT)) brains to expression of these same markers pRb f/f; p107 -/-; Emx1-Cre + (DKO) brains. Heterozygote embryos can be used as controls since pocket proteins exhibit autoregulation at the protein level and heterozygote animals are aphenotypic 26.
It was unclear whether these FoxP1+ cells were found in the IZ because these cells were immature neurons inappropriately expressing FoxP1, or whether they were earlier born neurons that had failed to migrate. To distinguish between these possibilities, we electroporated GFP into the wild type (WT) and double knock out (DKO) embryos at E14.5 and tracked the progress of these neurons over 4 days. Although this technique does not conclusively identify the final cell division, as with BrdU birthdating, it does allow the tracking of newborn neurons since it marks the discrete set of neurons derived from cells lining the ventricle at the time of electroporation 17. As shown in Figure 4C, all of the GFP+ neurons reach the uppermost cortical layer in the WT brains, but only a subset of these neurons reach the top of the cortex in DKO brains, indicating that there is in fact a defect in the migration of upper layer neurons, rather than improper expression of neuronal markers (similar results were obtained through traditional BrdU birthdating 6).
Neuronal migration is a highly complex process that is regulated by a multitude of interconnected mechanisms 1-3. As it is very difficult to identify the mechanism underlying a migration defect in an uncharacterized transgenic model, distinguishing cell autonomous versus non-cell autonomous defects can facilitate phenotype characterization. If following electroporation of Cre-GFP, GFP+ neurons fail to migrate in an otherwise wild type brain, this indicates that the mechanism disrupted by gene deletion is specific to the electroporated cell. Alternatively, if the electroporated neurons migrate normally in the wild type brain, this suggests that the cause of aberrant migration in the transgenic embryo is due to an environmental defect. Following electroporation of Cre-GFP in control pRb f/+; p107 +/- and double knock out pRb f/f; p107 -/- embryos, GFP+ neurons were found to migrate into the cortex in pRb f/f; p107 -/- brains, but many of these neurons failed reach the upper cortical layers as they did in control brains (Figure 4D) 6. There were significantly lower percentage of GFP+ neurons within the layer III/IV, marked by expression of FoxP127, in pRbf/f; p107 -/- electroporated brains than in control brains (Figure 4E). A Cre-GFP fusion protein was used to limit GFP fluorescence to the cell body and facilitate localization of the neuron to a specific cortical layer. Since the phenotype following electroporation of Cre is less severe than the phenotype following gene deletion throughout the cortex, this suggests that the original phenotype shown in Figure 4A is a combination of multiple mechanisms, both cell autonomous and not cell autonomous. Alternatively, it is possible that the time delay following electroporation, during which Cre is expressed, DNA recombination is performed, pRb is downregulated and the effects are manifested, is long enough to allow the electroporated neurons to reach the cortex before a migration defect becomes apparent.
It should be noted that the distinction between cell autonomous and non-cell autonomous mechanisms should be interpreted with caution in brains with high electroporation efficiencies, where 100-200 or more neurons are electroporated in a single 14 &#181;m thick section.If a high enough proportion of neurons are electroporated, GFP+ neurons may influence each other and create a small microenvironment. To be truly cell autonomous, migration defects must be detected even when electroporation efficiency is very low (10-30 GFP+ neurons per section). If necessary, the number of transfected cells can be lowered by decreasing voltage, pulse number or DNA concentration. All of these factors must be taken into consideration when analyzing the results following electroporation of Cre in a transgenic model.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51983/51983fig1highres.jpg" width="475" /> 
Figure 1: Performing In utero electroporation. (A) Sterile surgical set up. (B) Surgical tools from left: fine scissors for cutting needle tips; stapler; forceps; wound retractor; surgical scissors; needle holder. (C) Prepped pregnant mouse with shaved and disinfected abdomen. (D) Mouse covered with sterile drape prior to incision. (E) Uterus with E13.5 embryos removed from abdominal cavity. (F) Injecting pups in the ventricle. (G) Close up of injected pup; note the blue crescent on the embryo&#8217;s head indicating that the ventricle was successfully injected with the plasmid solution (arrow). (H) Paddles are placed to electroporate the lateral cortex. (I) Mouse post-surgery. (J) Preparing sharp needles is a crucial part of in utero electroporation. Examples of uncut needles (i, iv), good needles (ii, v) and poor needles (iii, vi) are shown at low (i-iii) and high (iv-vi) magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51983/51983fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51983/51983fig2highres.jpg" width="600" /> 
Figure 2: Targeting different brain regions. Diagrams A-D show paddle placement for targeting different regions of the forebrains. The dark blue circle represents the paddle in front of the embryo while the light blue circle represents the paddle behind the embryo. Positive and negative electrodes are indicated with the appropriate sign. (A) Electrodes angled to target the lateral cortex. (A&#8217;, A&#8221;) Example of electroporation of the lateral cortex, three days after surgery. (B) Electrodes angled to target the medial cortex. (B&#8217;, B&#8221;) Example of electroporation of the medial cortex. (C) Electrodes angled to target the hippocampus. (C&#8217;, C&#8221;) Example of electroporation of the hippocampus. (D) Electrodes angled to target the ganglionic eminence (GE). (D&#8217;, D&#8221;) Example of electroporation of the GE. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51983/51983fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/51983/51983fig3highres.jpg" width="600" /> 
Figure 3: Confirming Cre-mediated gene excision in electroporated neurons. (A) Cre mediated excision can be confirmed by co-staining for a downstream target of the deleted gene. pRb f/f; p107 -/-; p130 f/f embryos were electroporated with either the empty pCig2 plasmid or the pCig2-Cre plasmid and stained for GFP and Ki67, a marker of cell cycle entry. Double stained cells were seen in neurons electroporated with Cre (arrows in inset) but not in neurons electroporated with pCig2 alone (arrowheads). (B) Excision of pRb was also confirmed by performing PCR with genotyping primers on DNA extracted from the tissue sections removed from the slides. Sections were divided down the midline and the electroporated side of the section was pooled. The upper band represents the floxed allele and the lower band represents the wild type allele. The arrow indicates the excised allele in the electroporated side of the brain only. Because pRb f/+; p107 +/- heterozygotes were used as controls, an excised allele can be seen in the electroporated side of the brain in both the control and knockout brains. (C) Cre-mediated excision can also be confirmed in vitro using plasmids with floxed stop codons upstream of RFP, such as NL-dsRed. RFP is only expressed in HEK cells when co-transfected with Cre-GFP in Hek293 cells (bottom row), indicating that excision was successful. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51983/51983fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/51983/51983fig4highres.jpg" width="600" /> 
Figure 4: Using IUE to study migration in transgenic mouse models. (A) Expression of FoxP1, a marker for layer III/IV cortical neurons, was examined in pRb f/+; p107 +/-; Emx1-Cre + (WT), and pRb f/f; p107 -/-; Emx1-Cre + (DKO) brains at E17.5. Overlay includes Cux1 (blue - marker for layers II-IV) and Tbr1 (green - marker for layers V/VI). A population of cells expressing FoxP1 was observed in the intermediate zone of DKO embryos (arrow), but not WT embryos, with a concurrent decrease in the number of FoxP1+ neurons in layers II-IV of the cortex in DKO brains relative to WT brains (B). The white line indicates the delineation between the cortical plate and intermediate zone, which was determined using DAPI staining (not shown). (C) To trace the migration of a discrete population of late born neurons, WT and DKO embryos were electroporated with GFP at E14.5 and collected the brains at E18.5. Brains were co-stained with FoxP1 (red) and Tbr1 (blue) to show that GFP+ neurons are found almost exclusively in layer II in the WT brain but are delayed in the intermediate zone (IZ) and lower cortical layers in the DKO. (D) To determine if pRb and p107 regulate radial migration cell autonomously, pRb f/f; p107-/- and pRb f/+; p107-/+ embryos were electroporated with Cre-GFP at E13.5 and brains were collected at E17.5. The location of GFP+ neurons was quantified according to cortical layer. Layer II was defined as being above FoxP1 staining (red), layer III/IV was defined as within FoxP1 staining, above Tbr1 (blue) and layer V/VI was defined as within the Tbr1 positive layer (E). We show that a portion pRb and p107 deficient neurons fail to reach layer II but instead remain in layer III/IV. The figure has been modified from Svoboda et al., 2012. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51983/51983fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Embryo Age</td>
			<td>Voltage</td>
		</tr>
		<tr>
			<td>E12.5 &#8211; E13.5</td>
			<td>35V</td>
		</tr>
		<tr>
			<td>E14.5 &#8211; E15.5</td>
			<td>40V</td>
		</tr>
		<tr>
			<td>E16.5</td>
			<td>45V</td>
		</tr>
		<tr>
			<td>E17.5</td>
			<td>50V</td>
		</tr>
	</tbody>
</table>
Table 1: Electroporation voltage by embryo age.

Watch this Scientific Journal Video about Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models at JoVE.com

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

L&#39;induzione di proteine ​​Cancellazione Through<em&gt; In Utero</em&gt; Elettroporazione per definire deficit in migrazione neuronale in modelli transgenici

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

induction-protein-deletion-through-utero-electroporation-to-define

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

10.2K Views.  University of Ottawa. Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment. 

Video: Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

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

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

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

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

In utero Elettroporazione seguita da primaria Cultura neuronale per studiare la funzione dei geni in sottoinsieme di neuroni corticali

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

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 ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

Targeting della placca neurale murina mediante nano-iniezione in utero (NEPTUNE) al giorno embrionale 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

Biologia dello sviluppo

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 ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

Una nuova strategia che unisce Array-CGH, sequenziamento dell'intero-esoma e nell'Utero elettroporazione in roditori per identificare geni causativi per le malformazioni del cervello

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

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 ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

Ex Utero Elettroporazione e colture Organotypic fetta di cervelli embrionali del Mouse per vivere-Imaging di migrazione interneuroni GABAergici

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

L'induzione di proteine Cancellazione Through

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Capitoli in questo video

In utero Elettroporazione seguita da primaria Cultura neuronale per studiare la funzione dei geni in sottoinsieme di neuroni corticali

Gene consegna efficiente in più territori SNC utilizzo In Utero Elettroporazione

Mouse in Utero elettroporazione: controllato Transefection Gene spazio-temporale

Targeting della placca neurale murina mediante nano-iniezione in utero (NEPTUNE) al giorno embrionale 7.5

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

Una nuova strategia che unisce Array-CGH, sequenziamento dell'intero-esoma e nell'Utero elettroporazione in roditori per identificare geni causativi per le malformazioni del cervello

Elettroporazione in utero approcci per studiare l'eccitabilità neuronale sottopopolazioni e connettività single-cell

Impressione confocale dei migratori dei neuroni nella cultura di fetta organotipica del cervello embrionale del mouse usando

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

Ex Utero Elettroporazione e colture Organotypic fetta di cervelli embrionali del Mouse per vivere-Imaging di migrazione interneuroni GABAergici

L'induzione di proteine ​​Cancellazione Through

Riepilogo

Esplora Altri Video

Capitoli in questo video

In utero Elettroporazione seguita da primaria Cultura neuronale per studiare la funzione dei geni in sottoinsieme di neuroni corticali

Gene consegna efficiente in più territori SNC utilizzo In Utero Elettroporazione

Mouse in Utero elettroporazione: controllato Transefection Gene spazio-temporale

Targeting della placca neurale murina mediante nano-iniezione in utero (NEPTUNE) al giorno embrionale 7.5

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

Una nuova strategia che unisce Array-CGH, sequenziamento dell'intero-esoma e nell'Utero elettroporazione in roditori per identificare geni causativi per le malformazioni del cervello

Elettroporazione in utero approcci per studiare l'eccitabilità neuronale sottopopolazioni e connettività single-cell

Impressione confocale dei migratori dei neuroni nella cultura di fetta organotipica del cervello embrionale del mouse usando

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

Ex Utero Elettroporazione e colture Organotypic fetta di cervelli embrionali del Mouse per vivere-Imaging di migrazione interneuroni GABAergici

L'induzione di proteine Cancellazione Through