Questo lavoro è stato finanziato dalla RIKEN Brain Science Institute (BSI), Human Frontier Science Program (HFSP) (assegnato a TS) e RIKEN Research Program Junior Associate (JRA, assegnato a MK) RIKEN Brain Science Institute (BSI), Human Frontier Science Program (HFSP) (assegnato a TS) e RIKEN Research Program Junior Associate (JRA, assegnato a MK) finanziato questo lavoro.

1. Preparazione della soluzione capillare e del DNA per l&#39;iniezione <ol><li> Purificare DNA plasmidico con un maxi-prep o metodo equivalente, e fare una concentrazione finale di 2-3 mg / mL. Aliquota 100 mg di DNA, aggiungere 10μl di digiuno colorante verde (1% stock) e TE (10mM Tris base, 1MM Soluzione EDTA, pH 8,0) e regolare un volume di 100 l per rendere la concentrazione finale del DNA per mix iniezione per 1μg / microlitri. La concentrazione di DNA plasmidico può essere cambiato dipende dalle dimensioni del plasmide, e anche la sua efficienza di trasfezione, che deve essere testato singolarmente. </li><li> Gira la soluzione di DNA per 5 minuti a 14000 rpm a temperatura ambiente e raccogliere il supernatante per rimuovere eventuali residui. </li><li> Tirare un capillare di vetro tubi di vetro capillari tubea con un estrattore micropipetta. Pizzicare la punta con una pinza per fare la punta ha un diametro di 20-30 micron. Misura 800 micron-1 mm dalla punta e controllare il diametro esterno è inferiore a 60 micron (Fig. 1A). </li><li> Collegare il capillare alla micromanipolatore, e riempire con olio minerale. Per riempire il capillare con la soluzione DNA, immergere la punta nella soluzione e aspirare la soluzione di DNA. </li></ol> 2. Stick elettrodi di platino Qui abbiamo usato elettrodi di platino bastone (Nepe gene, CUY611P2-1) a electroporate nella regione superficiale del cervello, come la corteccia (Fig. 1F). 3. Preparazione di micro-elettrodi Usa micro-elettrodi elettroporate in parti profonde del cervello (cioè, talamo e ipotalamo) (Fig. 2B-E). <ol><li> Un&#39;estremità del tungsteno (per elettrodi negativi) e platino (per gli elettrodi positivi) i fili è avvolto in oro placcato perni e fissati con parafilm. Inserire il filo per il gambo di un batuffolo di cotone e avvolgere entrambe le estremità con parafilm (Fig. 1B). </li><li> Uniformemente affilare la punta del filo con la carta vetrata fino a raggiungere 20-30 micron di diametro esterno e 60 micron da 800 micron-1 mm di punta (Fig. 1 C e D). </li><li> Applicare uno strato sottile di smalto al filo per l&#39;isolamento. Dopo lo smalto si è asciugato, utilizzare un acetone-tampone di cotone imbevuto di rimuoverlo dalla punta (circa 200 micron dalla punta). Nota importante: Al fine di evitare danni all&#39;utero del mouse, la porzione di 800 micron-1 mm di punta dovrebbe essere non più di 70 micron di diametro (Fig. 1E). </li></ol> 4. Preparazione della chirurgia <ol><li> Anestetizzare un topo gravide (E9.5-E15.5). Dimostriamo con anestesia di grado farmaceutico sodio pentobarbital (tabella II, 50 mcg per grammo di peso corporeo, iniettano intraperitoneale e attendere 5 min). Alternative al sodio pentobarbital come anestetico sono l&#39;iniezione Ketamina / xylazina o anestetici gas (il gas è preferibile per le procedure di breve durata). L&#39;iniezione deve essere fatta con attenzione per evitare qualsiasi organi, altrimenti si farà a danneggiare l&#39;utero o uccidere animali. Quando IP di successo è fatto, il tasso di sopravvivenza è superiore al 95%. Anche la chirurgia può essere eseguita in ambiente a cielo aperto. Prima di iniziare l&#39;operazione, prova per una mancanza di risposta da parte dell&#39;animale di pizzicare i loro piedi per confermare anestetico. </li><li> Radersi i capelli con l&#39;addome con una lametta e il 50% EtOH. Preparare la pelle con alternanza di macchie di Betadine e alcool (70%). </li><li> Fare un&#39;incisione sulla linea mediana dell&#39;addome con le forbici bene e tirare fuori tutte le corna uterine attenzione su un 37 ° C preriscaldato tampone fosfato salino (PBS)-garza di cotone inumidito, che si trova intorno alla ferita. Mantenere umido il utero con PBS per tutto il tempo. </li><li> Tenere un cavo flessibile a fibre ottiche tra l&#39;indice e il medio e lo pongo sotto il corno uterino. Cavo di pulire con il 70% EtOH prima dell&#39;uso. Nessun microscopio o lente d&#39;ingrandimento è necessario per la visualizzazione. Utero posizione tra il cavo ottico luce in fibra ottica e il pollice, e premere delicatamente per far salire l&#39;embrione più vicino alla parete uterina. </li></ol> 5. L&#39;iniezione di DNA ed elettroporazione <ol><li> Quando embrione è posizionato, inserire capillare di vetro con cautela nel ventricolo target e iniettare circa 1 ml di soluzione di DNA (Fig. 2A). </li><li> Per l&#39;elettroporazione di parte superficiale del brainIf utilizzo con elettrodi di platino per electoporation bastone, gli elettrodi al di fuori dell&#39;utero e applicare suggerito impulsi di corrente ad onda quadra secondo le istruzioni di produzione (Tabella I). Se elettroporazione usingFor alla parte più profonda del cervello, elettroporazione nmicro, elettrodi tipo eedle vengono utilizzati. Iinsert un elettrodo di tungsteno multa negativi nel DNA iniettato ventricolo e un elettrodo di platino in utero e la destinazione regione posto tra due elettrodi, quindi applicare suggerito impulsi di corrente ad onda quadra (Tabella I) (Fig. 2B). </li></ol> 6. Messaggio elettroporazione <ol><li> Posizionare la parte posteriore dell&#39;utero corno nella sua origposizione inale con e aggiungere 500 ml di 37 ° C preriscaldato PBS. </li><li> Sutura lo strato interno con sutura chirurgica e chiudere strato esterno con una clip 9 millimetri autoclipauto. </li><li> Animali luogo una piastra elettrica per 2 ore per consentire il recupero dall&#39;anestesia. Gli analgesici come il carprofen o buprenorfina può essere somministrato per dolore post-operatorio. </li></ol> 7. Rappresentante dei risultati: Alcuni esempi di elettroporazione corticale pCAG-EYFP costruire utilizzando elettrodi di platino bastone in diversi momenti sono mostrate nella figura Figura 32. Cervelli sono elettroporate a E13.5, E14.5 e E15.5, e raccolte al giorno postnatale (P) 6. AIl colorazione ntibody di RORC rivela posizione di livello 4 e la posizione delle cellule trasfettate EYFP sono rivelate dalla colorazione anticorpale GFP, e entrambe le immagini sono super imposto (Fig. 3A e B). Etichettati cellule corticali sono strato di cellule corticali elettroporate è chiaramente diversa in ogni esperimento (Fig. 32), che suggerisce dipendenti dal tempo elettroporazione rende strato corticale specifica GOF / LOF possibile. La vitalità degli embrioni e l&#39;efficienza di trasfezione delle cellule varia a seconda dell&#39;età del dell&#39;embrione e la posizione di elettroporazione. I campioni sono elencati nella Tabella 1, tuttavia, le condizioni dovrebbero essere ottimizzate per ogni elettroporazione a seconda dello stadio e la parte del cervello. Elettroporazione in un tessuto profondo come il talamo e ipotalamo con l&#39;utilizzo di elettrodi di platino bastone spesso danno bassa efficienza (Tabella 1). Questo problema può essere risolto utilizzando micro-elettrodi whichelectrodes, che raggiungerà a parti profonde del cervello. Figura 3 4 esempio mostra di pCAG-EYFP elettroporazione nel diencefalo di sviluppo. Soluzione di DNA plasmidico viene iniettato nel 3 ° ventricolo a E11.5 e seguiti da micro elettroporazione (Fig. 3C4C). L&#39;area elettroporate è limitato nel talamo dove la maggior parte del progetto assoni alla corteccia (Fig. 3B4B). Proiezione degli assoni a livello 4 della corteccia può anche essere visualizzati da EYFP, che fillesfills il neurone intero. (Fig. 3A4A). <img src="/files/ftp_upload/3024/3024fig1.jpg" alt="Figura 1"/> Figura 1. Schematico Cartoon dimostrare la forma corretta del capillare. (A) Un tubo capillare di vetro viene tirato con un estrattore micropipetta per rendere certa forma. Un consiglio è spento pizzicato da pinze per renderlo 20-30 micron. Misura 800 micron-1mm dalla punta (fascia rossa) e assicurarsi che il diametro esterno è inferiore a 60 micron (fascia verde). (B) Un capo del tungsteno o fili di platino è avvolto in oro placcato perni e fissati con parafilm. Poi usate la carta vetrata per rendere la loro punta a diventare 20-30 micron diametro esterno e 60 micron da 1 mm della punta. (C) Immagine del filo di tungsteno prima formazione. (D) Immagine del filo di tungsteno dopo la modellatura. (E) Immagine del filo di tungsteno dopo l&#39;applicazione sottile strato di smalto. Barra di scala in E è di 10 micron (più piccola scala) per il CE. (F) Stick elettrodi di platino (gene Nepa. CUY611P2-1). Barra di scala in F è di 2 mm. <img src="/files/ftp_upload/3024/3024fig2.jpg" alt="Figura 2"/> Figura 2. Cartoon schema di procedura chirurgica. (A) Schema di iniezione in ventricolo laterale o embrioni di grandi dimensioni (lato sinistro) e in 3 ° ventricolo o embrioni di piccole dimensioni (lato destro). (B) Schema di electrporation da elettrodo di platino bastone (lato sinistro) e con ago elettrodo tipo (lato destro). <img src="/files/ftp_upload/3024/3024fig3.jpg" alt="Figura 3"/> Figura 3. Sviluppo del mouse telencefalo era elettroporate con pCAG-EYFP a E13.5 (A), E14.5 (B) e E15.5 (C) e raccolte a P6. Cervelli sono stati sezionati coronale e colorate con anticorpi GFP o anticorpi RORC separatamente e immagini si fondono. (A) Elettroporazione di pCAG-EYFP plasmide a E13.5 molti strato trasfezione 4 neuroni. che si sovrappongono con livello 4 marcatori RORC. (B) Elettroporazione di pCAG-EYFP plasmide a E14.5 trasfezione delle cellule, che sono più superficiali di livello 4 neuroni. (C) E15.5 elettroporazione etichetta molti neuroni superficiali come layer 2 / 3. Scala bar 200 micron. <img src="/files/ftp_upload/3024/3024fig4.jpg" alt="Figura 4"/> Figura 4. Transfection di EYFP nel talamo-corticale assoni (TCA). (A) capolinea TCA in strato corticale 4 è rivelata dalla colorazione anticorpale GFP. Colorazione anticorpale RORC viene usato per rivelare una posizione di livello 4. Entrambe le immagini sono prese dalla sezione stessa e uniti. (B) Posizione elettroporazione nel talamo è dimostrato anche dalla colorazione anticorpale GFP e colorazione RORC, limitato espressione nel talamo 11. (C) Schema Cartoon per l&#39;elettroporazione talamo e la proiezione TCA sono mostrati. Scala bar 200 micron.

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

Nessun conflitto di interessi dichiarati.

In questo protocollo, abbiamo solo descritto i benefici della tecnica del pCAG-EYFP trasfezione in area riservata di un cervello piccolo embrionale utilizzando diversi elettrodi di forma. Per il confronto di efficienza di trasfezione di promotore diverso è descritto in precedenza, che mostra il tipo di specificità delle cellule 1. Tuttavia, ci sono limitazioni per la tecnica, come la transfezione è solo transitoria e varia a seconda del plasmide, che non ha alcuna specificità delle cellule ed è difficile controllare il numero di cellule transfettate. ThereforeHowever, combinando questo protocollo con altre tecniche saranno ulteriori possibilità di fornire diversi metodi di manipolazione. In primo luogo, che incorpora promotore specifiche cellule nel DNA plasmidico permetterà transfezione delle cellule di tipo specifico, come i neuroni, astrociti e cellule gliali. In secondo luogo, dato che la transfezione è transitorio, non è adatto per gli scienziati che vogliono analizzare la funzione del gene nella vita futura. Pertanto, l&#39;associazione con una trasposasi per rendere DNA plasmidico integrarsi nel genoma verrà convertito in una trasfezione stabile 8. Prossimo, l&#39;adozione del tet-on o tet-off sistema permetterà di manipolare il tempo o la cella tipo specifico di espressione genica nelle cellule neurali 8. In alternativa, si può combinare questo protocollo con tamoxifene-inducibile Cre-ER (T) ricombinasi 9 e topi reporter di 10. Questa ampia accessibilità dei tessuti drasticamente cambiare i disegni sperimentali utilizzati nel campo delle neuroscienze.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> nudo filo di tungsteno di diametro 125 micron </td><td> AM sistemi </td><td> 797600 </td></tr><tr><td> nudo filo di platino del diametro 125 micron </td><td> AM sistemi </td><td> 767000 </td></tr><tr><td> Stick elettrodi </td><td> Nepa gene </td><td> CUY610P4-1 </td></tr><tr><td> tubo di vetro capillare </td><td> Stoelting </td><td> 50611 </td></tr><tr><td> micromanipolatore </td><td> KD scientifico </td><td> KDS310 </td></tr><tr><td> forbici </td><td> Roboz </td><td> RS-5865 </td></tr><tr><td> generatore di impulsi </td><td> Sistemi AM </td><td> Modello 2100 </td></tr><tr><td> 9 millimetri autoclip </td><td> Roboz </td><td> RS-9260 </td></tr><tr><td> placcati in oro pins </td><td> WPI </td><td> 5482 </td></tr><tr><td> RORC anticorpi </td><td> Perseo Proteomica </td><td> H3925 </td></tr></tbody></table>

mouse in utero electroporation: controlled spatiotemporal gene transfection

Per capire la funzione dei geni espressi nella specifica regione del cervello di sviluppo, tra cui molecole di segnalazione e le molecole di guida degli assoni, locale o il trasferimento di geni knock-out è richiesto. Gene targeting knock-in o knock-out in regioni locali è possibile eseguire con combinazione con una linea specifica CRE, che è laborioso, costoso e richiede tempo. Pertanto, un metodo di trasfezione semplice, un tecnica di elettroporazione in utero, che può essere eseguita con tempi brevi, sarà utile per testare la possibile funzione di geni candidati prima della generazione di animali transgenici 1,2. Oltre a questo, in elettroporazione utero obiettivi aree del cervello in cui non specifica linea CRE esiste, e limiterà letalità embrionale 3,4. Qui, vi presentiamo un metodo di elettroporazione in utero combinazione di due diversi tipi di elettrodi di trasferimento genico semplice e conveniente in aree obiettivo di sviluppo del cervello. In primo luogo, tenendo un metodo unico di embrioni con un ottica di luce in fibra ottica farà embrioni di piccole dimensioni (da E9.5), visibile per l&#39;iniezione mirata soluzione di DNA in ventricoli e il tipo di elettrodi ad ago di inserimento nella zona del cervello mirati 5,6. Il modello del cervello come la zona corticale verificarsi in fase embrionale precoce, dunque, questi primi elettroporazione da E9.5 un importante contributo per comprendere l&#39;intero evento patterning zona. In secondo luogo, la forma precisa di un capillare, previene i danni uterina facendo fori per l&#39;inserimento del capillare. Inoltre, la forma precisa del elettrodi ad ago sono creati con il tungsteno e il filo di platino e affilata con carta vetrata e isolato con smalto 7, un metodo che è descritto in dettaglio in questo protocollo. Questa tecnica unica consente la trasfezione di DNA plasmidico in aree limitate del cervello e consentirà embrioni piccolo per essere elettroporate. Ciò contribuirà a, aprire una nuova finestra per molti scienziati che stanno lavorando sulla differenziazione delle cellule, la migrazione delle cellule, l&#39;orientamento degli assoni in fase embrionale molto precoce. Inoltre, questa tecnica permetterà agli scienziati di trasfezione di DNA plasmidico in parti profonde del cervello di sviluppo, come talamo e ipotalamo, dove non molte regioni specifiche linee CRE esistono per guadagno di funzione (GOF) o la perdita di funzione (LOF) analisi.

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Un metodo di trasferimento genico nel cervello del mouse di sviluppo è descritta utilizzando un metodo unico chirurgico e la forma speciale di elettrodi. Questa tecnica unica consente la trasfezione di DNA plasmidico temporalmente e spazialmente, che aiuterà molti neuroscienziati a studiare lo sviluppo del cervello.

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 ...

mouse-utero-electroporation-controlled-spatiotemporal-gene

Research

JoVE Journal

Neuroscience

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

42.7K Views.  RIKEN Brain Science Institute. Un metodo di trasferimento genico nel cervello del mouse di sviluppo è descritta utilizzando un metodo unico chirurgico e la forma speciale di elettrodi. Questa tecnica unica consente la trasfezione di DNA plasmidico temporalmente e spazialmente, che aiuterà molti neuroscienziati a studiare lo sviluppo del cervello.

Video: Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

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

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

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

In utero 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

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 ...

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 ...

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

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

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

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

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

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

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

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

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

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

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

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

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 ...

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 ...

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 ...