Nous remercions Ekaterina Karelina de l&#39;aide pour l&#39;enregistrement bande son pour la vidéo, Ivan Molotkov d&#39;animation 3D et le Dr Peter Blaesse d&#39;ACG-EGFP préparation de plasmide. Le travail a été soutenu par des subventions du Centre de mobilité internationale de la Finlande, le finnois Fondation culturelle et l&#39;Académie de Finlande.

1. Présentation Création d&#39;animaux transgéniques est une méthode puissante d&#39;investigation des fonctions des gènes chez les animaux vivants et pour une maladie de mécanisme de démêler 2,3 ainsi que pour la manipulation des propriétés des cellules 4. Toutefois, la procédure est assez laborieuse, extrêmement longue et coûteuse, ce qui justifie l&#39;utilisation de méthodes alternatives de livraison de gènes viraux tels que l&#39;injection 5, injection ventriculaire néonatale avec électroporation in utero 6 et 7,8 électroporation. La méthode de non-ventriculaire postnatale injection et électroporation a un ensemble unique d&#39;avantages: l&#39;utilisation des non-viraux constructions génétiques; possibilité de créer un profil d&#39;expression locale dans la zone d&#39;intérêt, dans la transfection in situ de types de cellules non-migrateurs, par exemple corticale astrocytes. Dans cette vidéo, nous démontrons la procédure détaillée de la livraison de gènes dans le cerveau post-natal chez le rat nouveau-né en utilisant un plasmide codant pour la protéine fluorescente verte améliorée par un promoteur de l&#39;actine de poulet bêta avec CMV Enhancer (pCAG-EGFP) 9. Nous illustrons la préparation de la solution injectable contenant le plasmide, la fabrication de pipettes en verre mince et de montage de l&#39;microinjecteur sur un instrument stéréotaxique. Puis on parle de anesthésiant ratons avec l&#39;isoflurane, sur l&#39;exécution de la chirurgie, au sujet de la procédure d&#39;injection et sur ​​les électroporation en utilisant des électrodes et les pinces de porator vivo. Enfin, nous examinons brièvement les résultats anticipés, des perspectives et des difficultés qui peuvent surgir lors de ces expériences. 2. Préparation de plasmide pour l&#39;électroporation <ol><li> Nous préparent généralement 10 pl de la solution injectable dans un plasmide de 200 ul paroi du tube mince. Cette solution est suffisante pour plusieurs expériences. </li><li> Nous mélangeons 8 ul de la solution de plasmide (voir Matériel et équipement) avec 1 pl de la PBS 10X (voir Matériel et équipement) et 1 ul de 0,1% de solution d&#39;eau vert rapide (voir Matériel et équipement). </li><li> La concentration finale du plasmide dans la solution injectable doit être comprise entre 1 et 3 pg / pl. Des concentrations plus faibles d&#39;ADN plasmidique permettra de réduire l&#39;efficacité de transfection, tandis que la concentration élevée d&#39;ADN seront trop visqueux pour être injecté à travers la pointe fine du verre capillaire. </li></ol> 3. Préparation aiguille de verre <ol><li> Pour la préparation pipette en verre, nous utilisons verre borosilicate capillaire avec un filament (voir Matériel et équipement) et un extracteur électrode verticale en verre (voir Matériel et équipement) sur un maximum de traction et de chauffage des paramètres. </li><li> Nous avons ensuite briser la pointe de la pipette à l&#39;aide d&#39;un morceau de papier pour atteindre une pointe de 10 à 20 um de diamètre. </li><li> Nous vérifions la diamètre de la pointe et la forme sous l&#39;objectif du microscope. </li></ol> 4. Microinjecteur Assemblage <ol><li> L&#39;utilisation d&#39;un mince de polymère capillaire (voir Matériel et équipement), nous remplissons environ 1 / 3 du volume d&#39;une seringue de 10 ul de Hamilton (voir Matériels et équipements) avec de l&#39;huile minérale (voir Matériel et équipement). Eviter les bulles d&#39;air! </li><li> Ensuite, nous remplir la pipette en verre avec de l&#39;huile minérale et d&#39;insérer la pipette dans la seringue Hamilton. Eviter les bulles d&#39;air! </li><li> La seringue en verre pipette est ensuite fixé sur le microinjecteur connecté à un contrôleur (voir Matériel et équipement). </li><li> La configuration microinjection fixé sur un instrument stéréotaxique (voir Matériel et équipement) nous permet en toute sécurité Immerger l&#39;embout de la pipette en verre dans le tube avec la solution injectable de plasmide. </li><li> Lorsque la pointe touche la surface de la solution, Dip It mm plus de 1 ou 2 et de remplir la pipette avec environ 1 ul du mélange par injection en utilisant les micro-injecteur contrôleur. </li></ol> 5. Animaux anesthésiant Toutes les procédures présentées ici ont été effectués selon les règlements de l&#39;Université de Helsinki pour les expérimentations animales. <ol><li> Pour chaque expérience, nous remplissons une seringue étanche au gaz (voir Matériels et équipements) avec environ 2 ml de l&#39;isoflurane (voir Matériel et équipement). </li><li> Que la seringue est fixée sur l&#39;appareil d&#39;anesthésie (voir Matériel et équipement) connecté à la source de flux d&#39;air, la cage et le masque fixé sur l&#39;installation stéréotaxique. </li><li> Sur l&#39;unité d&#39;anesthésie, nous ajustons le flux d&#39;air à environ 250 ml / min et le niveau de l&#39;isoflurane à 4%. </li><li> Nous plaçons le raton dans la case des animaux pendant 2-5 minutes. </li><li> Quand le chiot cesse en mouvement, il place sur le coussin chauffant (voir Matériel et équipement) attaché à la configuration stéréotaxique. </li><li> Placer une partie rostrale de la tête du chiot dans le masque anesthésiant. </li><li> Il faut de 5 à 10 minutes pour lechiot à entrer dans le anesthésie profonde. </li><li> Vérifiez profondeur d&#39;anesthésie avec un pincement de la queue et la diminution de l&#39;afflux d&#39;isoflurane à environ 1,5 à 2,0%. </li></ol> 6. La chirurgie, la microinjection et électroporation <ol><li> Traiter la peau sur la tête de chiot avec l&#39;éthanol à 70%. </li><li> L&#39;utilisation de petits ciseaux (voir Matériel et équipement) et des pinces fines (voir Matériel et équipement), coupez la peau d&#39;une peau du chiot à son front. </li><li> Bend morceaux de peau sur le côté et tirez légèrement l&#39;oreille barres dans les orifices auditifs du crâne de la tête et la fixation de peau. </li><li> L&#39;utilisation d&#39;un microscope binoculaire, nous localiser le point bregma sur le crâne. </li><li> Identifier la région d&#39;intérêt en utilisant les coordonnées stéréotaxiques. </li><li> Position de la pipette d&#39;injection-dessus de cette région et le marquer par la chute de 25 à 50 nl de la solution injectable sur la surface du crâne. </li><li> Percer le crâne doucement et soigneusement au point marqué à l&#39;aide d&#39;une perceuse à haute vitesse chirurgicale (voir Matériel et équipement), sous le microscope jusqu&#39;à ce liquide apparaît dans la zone forée. </li><li> Fixer une pince électroporation électrodes (voir Matériel et équipement) sur les côtés du crâne avec un gel conducteur de courant (voir Matériel et équipement) pour assurer une meilleure conductivité. </li><li> Retirez la goutte de liquide par le trou percé à l&#39;aide de tampons de petite taille (voir Matériel et équipement) et trempette l&#39;aiguille de verre pour le trou selon les coordonnées du site d&#39;injection. </li><li> Faire infuser 25 à 100 nl de la solution injectable au taux de 5 à 20 nl / sec. </li><li> Enlever rapidement la pipette et électroporation immédiatement. L&#39;électroporation est effectuée en appliquant cinq impulsions rectangle avec la durée de 50 ms et d&#39;amplitude de 99 V à la fréquence de 1 Hz. </li><li> Retirer les électrodes et les barres d&#39;oreille. </li><li> Coudre la peau coupée en utilisant une combinaison d&#39;une pince-muets (voir Matériel et équipement) et tranchants et les fils chirurgicaux (voir Matériel et équipement). </li><li> Placer le chiot dans la chambre chaude pendant 15-30 minutes à l&#39;aide de sa récupération après l&#39;anesthésie. </li><li> Puis remettre le chiot à la cage de sa mère. </li></ol> 7. Les résultats représentatifs L&#39;expression du transgène dans les cellules transfectées avec succès apparaîtra environ 10 heures après l&#39;électroporation, et elle restera stable pendant plusieurs semaines. Nous microinjectés le plasmide soit dans de profondes couches corticales ou de la région du striatum jour postnatal 2 (P2) ratons Wistar et exécuté dans l&#39;électroporation in vivo comme décrit ci-dessus. Tranches de cerveau (400 um d&#39;épaisseur) ont été coupées au bout de 2 à 6 jours (P4-P8) en utilisant un vibrotome en milieu de tranchage à froid (Hepes tamponné Solution saline équilibrée de Earle) 10. L&#39;acquisition des images a été réalisée à l&#39;aide Zeiss Axioplan 2 LSM5 Pascal microscope confocal (Zeiss, Allemagne) dans le milieu de tranchage à la température ambiante. Les sites d&#39;injection dans les couches profondes du cortex et le striatum contient de nombreuses cellules transfectées exprimant l&#39;EGFP qui étaient situés dans une région compacte d&#39;environ 200 à 300 um de diamètre (figure 1A, B). Dans les cellules transfectées, le niveau d&#39;expression EGFP est suffisamment élevé pour permettre l&#39;imagerie des processus cellulaires minces (figure 1C), y compris les dendrites avec des épines de formes différentes (figure 1D) et les faisceaux des axones (figure 1E). La stabilité robuste de EGFP ainsi que la viabilité des cellules après l&#39;électroporation a permis le traçage parties distales des axones (jusqu&#39;à 1,5 à 2 mm du corps cellulaire) tels que les terminaux corticale (figure 1F). <img src="/files/ftp_upload/2244/2244fig1.jpg" alt="Figure 1" /> La figure 1 (A). Succès des cellules transfectées dans la région de striatum de cerveau de rat P4 délimitant le site d&#39;injection de plasmide. (B) Les cellules transfectées dans les couches corticales profondes (environ 1000 um dessous de la surface corticale) du cerveau de rat P5. (C) une cellule transfectée dans la région de striatum de cerveau de rat P8. (D) des processus dendritiques d&#39;une cellule transfectée dans la région de striatum de cerveau de rat P8; flèches indiquent les différents types d&#39;épines dendritiques et filopodes. (E) Les axones des cellules transfectées sein voie collatérale. (F) Les axones se terminant à la surface de la pie-mère P8 cortex de rat (flèche). Barres d&#39;échelle sont de 100 um sur A, B, E et F; 10 um sur les C et D.

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Les méthodes de livraison de gènes dans le cerveau de rongeurs vivants sont bien établies pour l&#39;électroporation in utero 7,8,11,12 et, plus récemment, par électroporation postnatale 6. Cependant, ces méthodes sont basées sur l&#39;injection intraventriculaire de l&#39;ADN plasmidique, qui peut être limitant pour plusieurs applications. Par exemple, ces méthodes ne permettent pas de cibler des cellules dans certaines régions du cerveau telles que l&#39;hippocampe, ni la tran...

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		<div>
		<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

Création d&#39;animaux transgéniques est une approche standard dans l&#39;étude de fonctions d&#39;un gène d&#39;intérêt in vivo. Toutefois, de nombreux KO ou des animaux transgéniques ne sont pas viables dans les cas où le gène modifié qui est exprimé ou supprimés dans l&#39;organisme entier. Par ailleurs, une variété de mécanismes compensatoires font qu&#39;il est souvent difficile d&#39;interpréter les résultats. Les effets compensatoires peuvent être atténuées par le calendrier soit l&#39;expression du gène ou de limiter la quantité de cellules transfectées. La méthode de non-ventriculaire postnatale microinjection et électroporation in vivo permet l&#39;administration ciblée de gènes, siRNA ou des molécules de colorant directement à une petite région d&#39;intérêt dans le cerveau de rongeurs nouveau-né. Contrairement à la technique du ventricule injection classique, cette méthode permet la transfection de types de cellules non-migrateurs. Animaux transfectées au moyen de la méthode décrite ici peut être utilisé, par exemple, pour deux photons en imagerie in vivo ou dans des expériences électrophysiologiques sur tranches de cerveau aigu.

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

Ce protocole décrit une méthode non virale de la livraison des constructions génétiques à une certaine zone du cerveau de rongeur vivant. La méthode consiste à la préparation de plasmides, de fabrication micropipette, la chirurgie néonatale chez le rat nouveau-nés, la microinjection de la construction, et<em&gt; In vivo</em&gt; Électroporation.

Livraison de gènes au cerveau de rat postnatal par des non-ventriculaire injection Plasmide et électroporation

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

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JoVE Journal

Neuroscience

15.0K vues.  University of Helsinki. Ce protocole décrit une méthode non virale de la livraison des constructions génétiques à une certaine zone du cerveau de rongeur vivant. La méthode consiste à la préparation de plasmides, de fabrication micropipette, la chirurgie néonatale chez le rat nouveau-nés, la microinjection de la construction, et In vivo Électroporation.

Vidéo: 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 électroporation suivie par la culture neuronale primaire pour étudier la fonction des gènes dans un sous-ensemble des neurones corticaux

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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Neurosciences

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.

Analyse mosaïque de fonction des gènes dans le développement de cerveau de souris à l&#39;aide postnatale à base de virus recombinaison 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.

Livraison efficace de gènes dans les Territoires du SNC multiples à l&#39;aide In Utero L&#39;électroporation

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.

Souris dans Utero électroporation: contrôlé Transefection Gene spatio-temporelle

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.

Transfert de gènes à l&#39;oreille de souris en développement intérieur par<em&gt; In Vivo</em&gt; Électroporation

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.

Expansion du embryonnaires et cellules souches neurales adultes par<em&gt; In Utero</em&gt; Électroporation ou virale par injection stéréotaxique

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 électroporation et imagerie time-lapse de neuroblaste migrations dans Souris aiguë tranches du cerveau

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.

Sous-sélective électroporation corticaux Interneurones

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.

Analyse des modifications d&#39;expression génique dans l&#39;hippocampe de rat après stimulation cérébrale profonde de l&#39;antérieur thalamique Nucleus

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.

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