Ringraziamo Ekaterina Karelina per un aiuto con la registrazione colonna sonora per il video, Ivan Molotkov di animazione 3D e il Dr. Peter Blaesse per CAG-EGFP preparazione plasmide. Il lavoro è stato supportato anche da finanziamenti del Centro di Mobilità Internazionale della Finlandia, il finlandese della Fondazione Culturale e l&#39;Accademia di Finlandia.

1. Introduzione Creazione di animali transgenici è un potente metodo di indagine delle funzioni dei geni in animali vivi 1 e per il meccanismo di svelare la malattia 2,3 così come per manipolare le proprietà delle cellule 4. Tuttavia, la procedura è piuttosto laboriosa, estremamente lunga e costosa, così da giustificare l&#39;uso di metodi alternativi consegna del gene virale come l&#39;iniezione 5, iniezione ventricolare neonatale con elettroporazione 6 e in utero elettroporazione 7,8. Il metodo di postnatale non-ventricolare di iniezione ed elettroporazione è un insieme unico di vantaggi: uso di costrutti genetici non virali; possibilità di creare un modello di espressione locale della zona di interesse; in situ di transfezione non migratorie tipi di cellule, ad esempio corticale astrociti. In questo video, dimostriamo la procedura dettagliata di consegna del gene postnatale al cervello di ratto neonatale utilizzando un plasmide codifica per la proteina fluorescente verde potenziato da un promotore di pollo beta actina con CMV enhancer (pCAG-EGFP) 9. Illustriamo la preparazione del plasmide contenente soluzione iniettabile, la produzione di pipette di vetro sottile e montaggio delle microinjector su uno strumento stereotassico. Poi si parla di anestetizzante ratti con isoflurano, circa l&#39;effettuazione dell&#39;intervento, in merito alla procedura di iniezione e sulla elettroporazione utilizzando elettrodi forcipe e in porator vivo. Infine, abbiamo brevemente i risultati attesi, le prospettive e le difficoltà che possono sorgere durante questi esperimenti. 2. Preparazione plasmide per elettroporazione <ol><li> Noi di solito preparare 10 ml di soluzione per l&#39;iniezione plasmide in un tubo di 200 l parete sottile. Questa soluzione è sufficiente per alcuni esperimenti. </li><li> Noi mix 8 microlitri della soluzione del plasmide (vedi Materiali e attrezzature) con 1 microlitri della PBS 10X (vedi Materiali e attrezzature) e 1 ml di soluzione allo 0,1% Veloce acqua verde (vedi Materiali e attrezzature). </li><li> La concentrazione finale del plasmide nella soluzione iniettabile deve essere compreso tra 1 e 3 mg / mL. Concentrazioni più basse di DNA plasmidico riduce l&#39;efficienza di transfezione, mentre la più alta concentrazione di DNA sarà troppo viscoso per l&#39;iniezione attraverso la punta sottile del vetro capillare. </li></ol> 3. Vetro Ago Preparazione <ol><li> Per la preparazione pipetta di vetro, si usa vetro borosilicato capillare con un filamento (vedi Materiali e attrezzature) e una verticale estrattore elettrodo di vetro (vedi Materiali e attrezzature) per la massima trazione e riscaldamento parametri. </li><li> Abbiamo poi rompere la punta della pipetta con un pezzo di carta per ottenere una punta di 10-20 micron di diametro. </li><li> Controlliamo il diametro punta e forma sotto l&#39;obiettivo microscopio. </li></ol> 4. Microinjector Assemblaggio <ol><li> Usando un sottile polimero capillare (vedi Materiali e attrezzature) abbiamo riempire circa 1 / 3 del volume di un 10 microlitri Hamilton siringa (vedere Materiali e attrezzature) con olio minerale (vedi Materiali e attrezzature). Evitare bolle d&#39;aria! </li><li> Poi abbiamo riempire la pipetta di vetro con olio minerale e inserire la pipetta nella siringa Hamilton. Evitare bolle d&#39;aria! </li><li> La siringa di vetro con pipetta viene poi fissata sul microinjector collegati a un controller (vedi Materiali e attrezzature). </li><li> Il setup microiniezione fisso su uno strumento stereotassico (vedi Materiali e attrezzature) ci permette di immergere in tutta sicurezza la punta della pipetta di vetro nella provetta con la soluzione di iniezione plasmide. </li><li> Quando la punta tocca la superficie della soluzione, immergetelo millimetri ulteriormente da 1 o 2 e riempire la pipetta con circa 1 ml della miscela di iniezione iniettore con il micro controller. </li></ol> 5. Animali anestetizzante Tutte le procedure qui presentate sono state eseguite secondo l&#39;Università di Helsinki regolamenti per gli esperimenti sugli animali. <ol><li> Per ogni esperimento, riempire una siringa a tenuta di gas (vedi Materiali e attrezzature) con circa 2 ml di isoflurano (vedi Materiali e attrezzature). </li><li> Che la siringa è fissato sull&#39;unità anestesia (vedi Materiali e attrezzature) collegato alla sorgente del flusso d&#39;aria, la finestra di animali e la maschera fissa sulla configurazione stereotassica. </li><li> Sull&#39;unità anestesia, correggiamo il flusso d&#39;aria a circa 250 ml / min e il livello di isoflurano al 4%. </li><li> Abbiamo posto il topo cucciolo nella casella di animali per 2-5 minuti. </li><li> Quando il cucciolo smette di muoversi, posto sulla rampa di riscaldamento (vedi Materiali e attrezzature) collegato alla configurazione stereotassica. </li><li> Inserire una parte rostrale del cucciolo la testa nella maschera anestetizzante. </li><li> Ci vogliono 5 a 10 minuti per ilcucciolo di entrare nel anestesia profonda. </li><li> Controllare profondità dell&#39;anestesia con un pizzico di coda e diminuire l&#39;afflusso isoflurano a circa 1,5 al 2,0%. </li></ol> 6. Chirurgia, microiniezione e Elettroporazione <ol><li> Trattare la pelle del cucciolo testa con l&#39;etanolo al 70%. </li><li> Utilizzando piccole forbici (vedi Materiali e attrezzature) e pinze sottili (vedi Materiali e attrezzature), tagliare la pelle da un collottola del cucciolo alla sua fronte. </li><li> Piegare pezzi pelle lateralmente e tirare le barre orecchio leggermente in orifizi sonora del cranio per la testa e la fissazione della pelle. </li><li> Utilizzando un microscopio binoculare, abbiamo individuare il punto bregma sul cranio. </li><li> Identificare la regione di interesse utilizzando le coordinate stereotassica. </li><li> Posizionare la pipetta di iniezione di sopra di questa regione e contrassegnarlo facendo cadere 25-50 nl della soluzione iniettabile sulla superficie del cranio. </li><li> Forare il cranio con delicatezza e attenzione nel punto segnato con un trapano ad alta velocità chirurgico (vedi Materiali e attrezzature) al microscopio fino liquido appare nell&#39;area forato. </li><li> Fissa un elettrodi electropotation pinza (vedi Materiali e attrezzature) ai lati del cranio con gel conduttore di corrente (vedi Materiali e attrezzature) per ottenere una migliore conduttività. </li><li> Rimuovere la goccia di liquido dal foro con tampone di piccole dimensioni (vedi Materiali e attrezzature) e immergere l&#39;ago di vetro al foro secondo le coordinate del sito di iniezione. </li><li> Infondere 25-100 nl della soluzione iniettabile al tasso da 5 a 20 nl / sec. </li><li> Rimuovere rapidamente la pipetta e electroporate immediatamente. Elettroporazione viene effettuata mediante l&#39;applicazione di cinque impulsi rettangolo con la durata di 50 ms e l&#39;ampiezza di 99 V alla frequenza di 1 Hz. </li><li> Rimuovere gli elettrodi e bar orecchio. </li><li> Cucire la pelle tagliata usando una combinazione di una pinza muto (vedi Materiali e attrezzature) e taglienti e fili chirurgici (vedi Materiali e attrezzature). </li><li> Collocare il cucciolo nella camera calda per 15-30 minuti per aiutare il suo recupero dopo l&#39;anestesia. </li><li> Poi mettere il cucciolo torna alla gabbia della madre. </li></ol> 7. Rappresentante Risultati L&#39;espressione del transgene nelle cellule transfettate con successo apparirà circa 10 ore dopo l&#39;elettroporazione, e rimarrà stabile per diverse settimane. Noi microiniettati il plasmide sia in profondità strati corticali o regione striato del giorno post-natale 2 (P2) e cuccioli di ratto Wistar eseguita in elettroporazione in vivo come descritto sopra. Sezioni di cervello (400 micron di spessore) sono stati tagliati dopo 2 a 6 giorni (P4-P8) usando un vibrotome a freddo affettare a pezzi medi (Hepes tamponata soluzione equilibrata Salt Earle) 10. Acquisizione delle immagini è stata effettuata utilizzando Zeiss Axioplan 2 LSM5 Pascal microscopio confocale (Zeiss, Germania) nel medio taglio a temperatura ambiente. I siti di iniezione in profondità strati corticali e nello striato contenute molte cellule transfettate che esprimono EGFP che si trovavano in una regione compatta di circa 200-300 micron di diametro (Figura 1A, B). All&#39;interno delle cellule transfettate, il livello di espressione EGFP è sufficientemente elevato per consentire l&#39;imaging di sottili processi cellulari (Figura 1C) tra cui dendriti con spine di forme diverse (Figura 1D) e fasci di assoni (Fig. 1E). La stabilità robusta di EGFP così come la vitalità cellulare dopo l&#39;elettroporazione permesso tracciamento parti distali degli assoni (fino a 1,5-2 mm dal corpo cellulare), come terminali corticali (Figura 1F). <img src="/files/ftp_upload/2244/2244fig1.jpg" alt="Figura 1" /> Figura 1. (A) transfettate con successo le cellule nella regione striato di cervello di ratto P4 delineare il plasmide sito di iniezione. (B), le cellule transfettati in profondità strati corticali (circa 1000 micron sotto la superficie corticale) di cervello di ratto P5. (C) Una cellula trasfettate nella regione striato di ratto cervello P8. (D) i processi di una cellula dendritiche transfettate nella regione striato di ratto cervello P8; frecce indicano i diversi tipi di spine dendritiche e filopodia. (E) Gli assoni delle cellule transfettate in via collaterale. (F) Gli assoni che terminano presso l&#39;superficie piale di P8 ratto corteccia (punta di freccia). Barre di scala sono 100 micron su A, B, E e F; 10 micron su C e D.

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Metodi di consegna del gene nel cervello dei roditori viventi sono ben stabiliti per elettroporazione in utero 7,8,11,12 e, più recentemente, per l&#39;elettroporazione postnatale 6. Tuttavia, questi metodi si basano su iniezione intraventricolare del DNA plasmide, che può essere limitante per diverse applicazioni. Per esempio, questi metodi non consentono targeting cellule in alcune aree cerebrali quali l&#39;ippocampo, né la transfezione di questi non-migratori tipi di cellule come as...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2A-sa dumb Tweezers, 115mm</td>
<td>equipment</td>
<td>XYtronic</td>
<td>XY-2A-SA</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Biological Temperature Controller with stainless steel heating pad</td>
<td>equipment</td>
<td>Supertech</td>
<td>TMP-5b</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate tube with filament</td>
<td>material</td>
<td>Sutter Instruments</td>
<td>BF120-69-10</td>
<td>Glass needle</td>
</tr>
<tr>
<td>Disposable drills</td>
<td>material</td>
<td>Meisinger</td>
<td>HP 310 104 001 001 008</td>
<td>&nbsp;</td>
<tr>
<td>Dulbeco's PBS 10X</td>
<td>reagent</td>
<td>Sigma</td>
<td>D1408</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps, 110 mm</td>
<td>equipment</td>
<td>FST</td>
<td>91150-20</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Ealing microelectrode puller</td>
<td>equipment</td>
<td>Ealing</td>
<td>50-2013</td>
<td>Vertical electrode glass puller</td>
</tr>
<tr>
<td>Ethilon monofil polyamide 6-0 FS-3 16 mm 3/8c</td>
<td>material</td>
<td>Johnson &amp; Johnson Medical</td>
<td>EH7177H</td>
<td>Surgical threads</td>
</tr>
<tr>
<td>Exmire micro syringe 10.0 ml</td>
<td>equipment</td>
<td>Exmire</td>
<td>MS*GLLX00</td>
<td>Gas-tight syringe</td>
</tr>
<tr>
<td>Fast Green</td>
<td>reagent</td>
<td>Sigma</td>
<td>F7252</td>
<td>&nbsp;</td>
<tr>
<td>Forceps electrodes</td>
<td>equipment</td>
<td>BEX</td>
<td>LF650P3</td>
<td>Treat with 70% ethanol for disinfection prior to use</td>
</tr>
<tr>
<td>Foredom drill control</td>
<td>equipment</td>
<td>Foredom </td>
<td>FM3545</td>
<td>Surgical drill power supply and control. Currently available analogue is micromotor kit K.1070 (Foredom) </td>
</tr>
<tr>
<td>Foredom micro motor handpiece</td>
<td>equipment</td>
<td>Foredom</td>
<td>MH-145</td>
<td>Currently available analogue is micromotor kit K.1070 (Foredom)</td>
</tr>
<tr>
<td>Gas anesthesia platform for mice</td>
<td>equipment</td>
<td>Stoelting</td>
<td>50264</td>
<td>Assembled on stereotaxic instrument</td>
</tr>
<tr>
<td>Isoflurane</td>
<td>reagent</td>
<td>Baxter</td>
<td>FDG9623</td>
<td>&nbsp;</td>
<tr>
<td>Micro dressing forceps, 105 mm</td>
<td>equipment</td>
<td>Aesculap</td>
<td>BD302R</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Microfil</td>
<td>material</td>
<td>WPI</td>
<td>MF34G-5</td>
<td>Micro syringe filling capillaries</td>
</tr>
<tr>
<td>Mineral oil</td>
<td>reagent</td>
<td>Sigma</td>
<td>M8410</td>
<td>&nbsp;</td>
<tr>
<td>NanoFil Syringe 10 microliter</td>
<td>equipment</td>
<td>WPI</td>
<td>NANOFIL</td>
<td>Hamilton syringe</td>
</tr>
<tr>
<td>plasmid CAG-EGFP</td>
<td>reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Extracted and purified with EndoFree Plasmid Maxi Kit (Qiagen) and dissolved in nuclease free water to concentration 1.5 mg/ml</td>
</tr>
<tr>
<td>Pulse generator CUY21Vivo-SQ</td>
<td>equipment</td>
<td>BEX</td>
<td>CUY21Vivo-SQ</td>
<td>&nbsp;</td>
<tr>
<td>Schiller electrode gel</td>
<td>reagent</td>
<td>Schiller AG</td>
<td>2.158000</td>
<td>Conductive gel</td>
</tr>
<tr>
<td>Small animal stereotaxic instrument</td>
<td>equipment</td>
<td>David Kopf Instruments</td>
<td>900</td>
<td>&nbsp;</td>
<tr>
<td>Stoelting mouse and neonatal rat adaptor</td>
<td>equipment</td>
<td>Stoelting</td>
<td>51625</td>
<td>Assembled on stereotaxic instrument.
Treat earbars with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Student iris scissors, straight 11.5 cm</td>
<td>equipment</td>
<td>FST</td>
<td>91460-11</td>
<td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td>
</tr>
<tr>
<td>Sugi absorbent swabs 17 x 8 mm</td>
<td>material</td>
<td>Kettenbach</td>
<td>31602</td>
<td>Surgical tampons</td>
</tr>
<tr>
<td>UMP3 microsyringe pump and Micro 4 microsyringe pump controller</td>
<td>equipment</td>
<td>WPI</td>
<td>UMP3-1</td>
<td>Microinjector and controller</td>
</tr>
<tr>
<td>Univentor 400 Anesthesia Unit</td>
<td>equipment</td>
<td>Univentor</td>
<td>8323001</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

gene delivery to postnatal rat brain by non-ventricular plasmid injection and electroporation

Creazione di animali transgenici è un approccio standard per studiare le funzioni di un gene di interesse in vivo. Tuttavia, molti knockout o animali transgenici non sono sostenibili a quei casi in cui viene espresso il gene modificato o cancellato in tutto l&#39;organismo. Inoltre, una varietà di meccanismi di compenso spesso rendono difficile l&#39;interpretazione dei risultati. Gli effetti di compensazione può essere alleviato con tempismo sia l&#39;espressione genica o limitando la quantità di cellule transfettate. Il metodo di postnatale non-ventricolare microiniezione e elettroporazione in vivo permette somministrazione mirata di geni, molecole di colorante siRNA o direttamente a una piccola regione di interesse nel cervello dei roditori neonato. In contrasto con tecnica convenzionale iniezione ventricolare, questo metodo permette di trasfezione di non-migratori tipi di cellule. Animali trasfettate mediante il metodo descritto qui può essere utilizzato, ad esempio, per i due fotoni imaging in vivo o in esperimenti elettrofisiologici su sezioni di cervello acuto.

Watch this Scientific Journal Video about Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation at JoVE.com

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Questo protocollo descrive un metodo non-virale di consegna di costrutti genetici per una certa area del cervello dei roditori viventi. Il metodo consiste di preparazione plasmide, fabbricazione micropipetta, chirurgia neonatale dei cuccioli di ratto, microiniezione del costrutto, e<em&gt; In vivo</em&gt; Elettroporazione.

Gene consegna di cervello di ratto post-natale da parte di non-ventricolare iniezione plasmidi e Elettroporazione

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

gene-delivery-to-postnatal-rat-brain-non-ventricular-plasmid

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JoVE Journal

Neuroscience

15.0K Views.  University of Helsinki. Questo protocollo descrive un metodo non-virale di consegna di costrutti genetici per una certa area del cervello dei roditori viventi. Il metodo consiste di preparazione plasmide, fabbricazione micropipetta, chirurgia neonatale dei cuccioli di ratto, microiniezione del costrutto, e In vivo Elettroporazione.

Video: Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

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 ...

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Neuroscienze

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism 
and schizophrenia1,2. Many genes have been studied in the prenatal brain and found crucial to many developmental processes3-5. However, their 
function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice 6. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain.
Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and 
systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs)7,8 encoding Cre into the neonatal brain, 
we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent 
protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of 
many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development,
 including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully 
in our own lab (unpublished results) and others8,9, and can be extended to other viruses, such as lentivirus 9, as well as to the expression of 
shRNA or dominant active proteins 10. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical 
imaging tools 11, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice 
and rats.

An in vivo method to test gene function in postnatal brain is described. Recombinant AAVs expressing Cre and/or a fluorescent protein are injected into neonatal mouse brain. Mosaic gene inactivation and sparse neuronal labeling are achieved, allowing rapid analysis of gene function in processes critical to neural circuit development.

Analisi mosaico della funzione del gene nello sviluppo postnatale cervello di topo mediante virus basati su ricombinazione Cre

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

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 ...

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 ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Ampliamento della embrionali e cellule staminali neurali adulte per<em&gt; In Utero</em&gt; Elettroporazione o Viral iniezione stereotassica

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

In vivo postnatale elettroporazione e Time-lapse imaging di migrazione neuroblasti in mouse acuta Fette cervello

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Sottotipo-selettivo elettroporazione della corticale interneuroni

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Analisi delle variazioni di espressione genica nel Rat Hippocampus Dopo stimolazione cerebrale profonda del nucleo anteriore talamiche

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

Usando fluorescenza cellulare attivo Ordinamento per esaminare Cell-Type-specifica espressione genica nel cervello di ratto tessuto

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...