Questo lavoro è stato supportato anche da finanziamenti del Dipartimento della Difesa (sviluppo premio Idea, W81XWH-10-1-0041, AB), CT cellule staminali di assegnazione (AB), e di un Istituto Nazionale della Salute NRSA 10668225 (DMF). Il materiale presente è basato sul lavoro in parte sostenuto da parte dello Stato del Connecticut sotto la ricerca sulle cellule staminali Connecticut programma di sovvenzioni. I suoi contenuti sono di esclusiva responsabilità degli autori e non rappresentano necessariamente le opinioni ufficiali dello Stato del Connecticut, del Dipartimento di Sanità Pubblica dello Stato del Connecticut o TC Innovations, Inc. I finanziatori avuto alcun ruolo nel disegno dello studio, la raccolta dei dati e l&#39;analisi, la decisione di pubblicare, o preparazione del manoscritto.

Questa procedura è in conformità con i requisiti IACUC Yale. Gli scienziati dovrebbero fare in modo che le linee guida IACUC sono approvati e seguiti secondo le proprie esigenze istituzionali. 1. Sezione 1. Preparazione di Pipette DNA, soluzioni, e vetro <ol><li> Generazione di elevata purezza (OD 260/280&gt; 1,80) ad alta concentrazione (mg / ul&gt; 2.5) privo di endotossine DNA. </li><li> Preparare soluzione fisiologica 0,9% e filtro sterile attraverso un filtro 22 micron. </li><li> Posto 10 centimetri tubi di fuoco lucido vetro borosilicato capillari (OD: 1,5 mm, ID: 1.10 mm) in un modello PP-830 Narishige PC-10 estrattore pipetta di vetro con un filo kanthal. Impostare estrattore ad un pull un passo ponderato a 70.5 ° C. Rompere le punte con pinza circolari e ispezionare sotto un microscopio da dissezione per i bordi frastagliati. Punte coniche e posto sotto una lampada UV per 15 minuti. Pipette tirato devono essere marcati a 2 mm dal bordo della punta. </li><li> Preparare 0,1% peso / volume soluzione molto veloce verdedalla miscelazione sterile filtrata soluzione salina allo 0,9% a secco verde veloce. </li></ol> 2. Sezione 2. Preparazione degli animali, pipetta di vetro di caricamento e iniezione Plasmid <ol><li> Rimuovere giorno postnatale 0-1 cuccioli individualmente dalle gabbie, posto su un piatto di vetro Petri pre-refrigerato a 4 ° C, e quindi posto in ghiaccio. </li><li> Dopo circa 5 minuti, determinare lo stato di anesthetization utilizzando la risposta pinch piede. Se non si verifica il movimento, cuccioli di soggetti ai iniezione intraventricolare. </li><li> È importante notare che la combinazione di verde veloce, DNA, e soluzione salina può provocare una rapida evaporazione, cristallizzazione, e il blocco di pipette di vetro. Di conseguenza, generare una soluzione di riserva ed aliquota 1-2 microlitri su parafilm immediatamente prima di iniezioni. </li><li> Aspirare l&#39;intero volume aliquotate con la pipetta di vetro tirato. </li><li> Tenere la testa del cucciolo tra il pollice e l&#39;indice della mano meno dominante. </li><li> Accendere una lampadad tenere la testa del cucciolo appena fuori del fascio di luce. Se necessario, mettere salino sulla testa per ridurre la quantità di luce riflessa da pup. I ventricoli dovrebbe essere illuminato. </li><li> Con la mano dominante, inserire l&#39;ago nel ventricolo laterale più vicino a voi (Figura 1A e 1B). Il sito di iniezione deve essere approssimativamente equidistante dalla sutura lambdoidea e occhio e 2 mm lateralmente alla sutura sagittale. Inserire tirato pipetta a volume 2 mm per un cucciolo P0, che dovrebbe garantire una maggiore penetrazione nel ventricolo laterale. Casualmente, è possibile visualizzare il riempimento del liquido cerebrospinale nella pipetta dal rilascio della pressione intracranica. Per ridurre la pressione intracranica, che aiuta a iniezione di plasmide, allentare la presa sulla testa del pup.Step 2.8. Iniettare circa 0,5 l di plasmide nel ventricolo laterale che utilizza l&#39;aria compressa-iniettore (Picospritzer, Parker). </li></ol> 3. Sezione 3. Elettroporazione e recupero <ol><li> Set la tensione del generatore di elettroporazione per 5 impulsi quadrati, 50 msec / impulso a 100 volt, con 950 msec. La specificità spaziale di elettroporazione plasmide è dettata dalla direzionalità del trasferimento di carica. Particolare attenzione dovrebbe essere data al collocamento data l&#39;eterogeneità delle popolazioni di cellule staminali lungo la SVZ 8. Le differenze nei tassi di proliferazione e il destino della cellula sono stati individuati per le posizioni-dorsale e ventrale rostrale-caudale 9,10. </li><li> Una volta che il plasmide viene iniettato, dip elettrodi pinzetta in PBS. Questo passo è quello di massimizzare il trasferimento di carica e per evitare ustioni causate da resistività. </li><li> Posizionare l&#39;elettrodo positivo sulla parete dorsale laterale del cucciolo vicino all&#39;orecchio omolaterale al sito di iniezione plasmide (Figura 1C e 1D). Posizionare l&#39;elettrodo negativo sul laterale dell&#39;emisfero controlaterale ventrale al cucciolo muso. </li><li> Avviare il trasferimento corrente premendo i piedini di impulsistrega pedale e spazzare gli elettrodi da dorsale a laterale che utilizza ~ 25 ° intervalli angolari. </li><li> Posto elettroporata cuccioli su un rilievo di riscaldamento per 5 min. Ruotare delicatamente cuccioli ogni 30 secondi con una lieve stimolazione. </li></ol>

<ol>
	<li>Kriegstein, A., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+nature+of+embryonic+and+adult+neural+stem+cells.">The glial nature of embryonic and adult neural stem cells.</a> Annual Review of Neuroscience. 32, 149-184 (2009).</li><li>Imayoshi, I., Sakamoto, M., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+methods+to+identify+and+manipulate+newly+born+neurons+in+the+adult+brain.">Genetic methods to identify and manipulate newly born neurons in the adult brain.</a> Frontiers in Neuroscience. 5, 64 (2011).</li><li>LoTurco, J., Manent, J. B., Sidiqi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+tools+for+in+utero+electroporation+studies+of+developing+cerebral+cortex.">New and improved tools for in utero electroporation studies of developing cerebral cortex.</a> Cereb Cortex. 19, i120-i125 (2009).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li><li>Boutin, C., Diestel, S., Desoeuvre, A., Tiveron, M. C., Cremer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+vivo+electroporation+of+the+postnatal+rodent+forebrain.">Efficient in vivo electroporation of the postnatal rodent forebrain.</a> PloS ONE. 3, e1883 (2008).</li><li>Chesler, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+gene+expression+by+postnatal+electroporation+during+olfactory+interneuron+nurogenesis.">Selective gene expression by postnatal electroporation during olfactory interneuron nurogenesis.</a> PloS ONE. 3, e1517 (2008).</li><li>Platel, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptors+activated+by+subventricular+zone+astrocytic+glutamate+are+critical+for+neuroblast+survival+prior+to+entering+a+synaptic+network.">NMDA receptors activated by subventricular zone astrocytic glutamate are critical for neuroblast survival prior to entering a synaptic network.</a> Neuron. 65, 859-872 (2010).</li><li>Alvarez-Buylla, A., Kohwi, M., Nguyen, T. M., Merkle, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heterogeneity+of+adult+neural+stem+cells+and+the+emerging+complexity+of+their+niche.">The heterogeneity of adult neural stem cells and the emerging complexity of their niche.</a> Cold Spring Harbor Symposia on Quantitative Biology. 73, 357-365 (2008).</li><li>Fernandez, M. E., Croce, S., Boutin, C., Cremer, H., Raineteau, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+electroporation+of+defined+lateral+ventricular+walls:+a+novel+and+rapid+method+to+study+fate+specification+during+postnatal+forebrain+neurogenesis.">Targeted electroporation of defined lateral ventricular walls: a novel and rapid method to study fate specification during postnatal forebrain neurogenesis.</a> Neural Development. 6, 13 (2011).</li><li>de Chevigny, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-7a+regulation+of+Pax6+controls+spatial+origin+of+forebrain+dopaminergic+neurons.">miR-7a regulation of Pax6 controls spatial origin of forebrain dopaminergic neurons.</a> Nature Neuroscience. 15, 1120-1126 (2012).</li><li>Lacar, B., Young, S. Z., Platel, J. C., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+recording+subventricular+zone+progenitor+cells+in+live+tissue+of+postnatal+mice.">Imaging and recording subventricular zone progenitor cells in live tissue of postnatal mice.</a> Frontiers in Neuroscience. 4, (2010).</li><li>Feliciano, D. M., Quon, J. L., Su, T., Taylor, M. M., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neurogenesis+generates+heterotopias,+olfactory+micronodules+and+cortical+infiltration+following+single-cell+Tsc1+deletion.">Postnatal neurogenesis generates heterotopias, olfactory micronodules and cortical infiltration following single-cell Tsc1 deletion.</a> Human Molecular Genetics. 21, 799-810 (2012).</li><li>Iguchi, T., Yagi, H., Wang, C. C., Sato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tightly+controlled+conditional+knockdown+system+using+the+Tol2+transposon-mediated+technique.">A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.</a> PloS ONE. 7, e33380 (2012).</li></ol>

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Qui dettaglio la tecnica di elettroporazione neonatale SVZ, una tecnica per etichettare in modo rapido e robusto e manipolare le cellule staminali SVZ e la loro progenie. Ci sono diversi vantaggi che elettroporazione ha rispetto ad altre tecniche. Prima, data l&#39;etichettatura focale di cellule, si è in grado di discernere cellule effetti autonomi e non autonomi cella. In secondo luogo, la manipolazione genetica utilizzando sistemi inducibili permette di confrontare gli effetti prima o dopo l&#39;integrazione sinapti...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Heavy Tubi lucido borosilicato </td><td> Sutter Instrument </td><td> BF150-110-10 </td><td></td></tr><tr><td> Doppio-Stage Glass Micropipetta Puller </td><td> Narishige </td><td> PC-10H </td><td></td></tr><tr><td> Fast Green </td><td> Fischer Scientific </td><td> 0521192205 </td><td></td></tr><tr><td> ECM 830 Onda quadra generatore di impulsi </td><td> Harvard Apparatus </td><td> 45-0052 </td><td></td></tr><tr><td> Tweezertrodes </td><td> Harvard Apparatus </td><td> 45-0488 </td><td></td></tr><tr><td> Fibra ottica Sorgente luminosa </td><td> Fisher Scientific </td><td> 12-562-36 </td><td></td></tr><tr><td> Tungsteno alogenalampada </td><td> USHIO America, Inc </td><td> 1002247 </td><td></td></tr><tr><td> Picospritzer II </td><td> Parker Instruments </td><td> 052-0312-900 </td><td></td></tr></tbody></table>

neonatal subventricular zone electroporation

Cellule staminali neurali (NSC) linea ventricoli postnatali laterali e danno luogo a diversi tipi cellulari che includono neuroni, astrociti e cellule ependimali 1. La comprensione dei meccanismi molecolari responsabili della NSC auto-rinnovamento, l&#39;impegno, e la differenziazione è fondamentale per sfruttare il loro potenziale unico per riparare il cervello e capire meglio disturbi del sistema nervoso centrale. Metodi precedenti per la manipolazione dei sistemi di mammifero richiede l&#39;impegno lungo e costoso dell&#39;ingegneria genetica a livello animale intero 2. Così, la maggior parte degli studi hanno esplorato le funzioni di molecole NSC in vitro o in invertebrati. Qui, una dimostrazione della tecnica semplice e rapida per manipolare NPC neonatali che si riferisce a come neonatale zona subventricolare (SVZ) elettroporazione. Tecniche simili sono state sviluppate una decina di anni fa per studiare cellule staminali neurali embrionali e hanno aiutato studi sulla cortica3,4 l di sviluppo. Più di recente questo è stato applicato per studiare il proencefalo roditore postnatale 5-7. Questa tecnica comporta l&#39;etichettatura robusto SVZ cellule staminali neurali e la loro progenie. Così, postnatale SVZ elettroporazione fornisce un costo e un&#39;alternativa tempo efficace per mammiferi ingegneria genetica NSC.

Neonatali subventricolare elettroporazione risultati della zona nella etichettatura di quasi tutti glia radiale contigua alla dorsale, zona dorsale subventricolare laterale e laterale seguendo il movimento &quot;spazzare&quot; dell&#39;elettrodo pinzetta (Figura 1E). Tuttavia, elettroporazione può essere adattato al requisito dell&#39;esperimento rispettivo ma non spazzare l&#39;elettrodo e utilizzando il posizionamento e l&#39;orientamento specifico come dettagliato nel video. Ad esempio, poiché loca...

Watch this Scientific Journal Video about Neonatal Subventricular Zone Electroporation at JoVE.com

Neonatal Subventricular Zone Electroporation

Dimostriamo una tecnica minimamente invasiva denominata neonatale elettroporazione zona subventricolare. La tecnica consiste nell&#39;iniettare DNA plasmidico nei ventricoli laterali di cuccioli neonati e l&#39;applicazione di corrente elettrica per fornire e manipolare geneticamente le cellule staminali neurali

Neonatale Elettroporazione zona subventricolare

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

neonatal-subventricular-zone-electroporation

Research

JoVE Journal

Neuroscience

12.7K Views.  Yale University School of Medicine. Dimostriamo una tecnica minimamente invasiva denominata neonatale elettroporazione zona subventricolare. La tecnica consiste nell'iniettare DNA plasmidico nei ventricoli laterali di cuccioli neonati e l'applicazione di corrente elettrica per fornire e manipolare geneticamente le cellule staminali neurali

Video: Neonatal Subventricular Zone Electroporation

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

La zona subventricolare En-face: colorazione Wholemount e flusso ependimale

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

Ricerca

Neuroscienze

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In elettroporazione in vivo di Retina topo in via di sviluppo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Preparazione di acuta fette zona subventricolare for Imaging di calcio

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Neonatale Pial elettroporazione Surface

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

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 ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Ordinamento delle cellule staminali neurali e di cellule progenitrici del topo adulto subventricolare Zone e Live-imaging loro dinamiche del ciclo cellulare

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Isolamento e la coltura di adulti neurali cellule staminali dal Subcallosal Zona mouse

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone (SEZ) is a thin and distinct region exposed to the ventricles and cerebrospinal fluid. The isolation of this niche allows the analysis of a neurogenic stem cell microenvironment. However, extraction of small tissues for proteome analysis&#160;is challenging, especially for the maintenance of considerable measurement depth and the achievement of reliable robustness. A new method termed cryo-section-dissection (CSD), combining high precision with minimal tissue perturbation, was developed to address these challenges. The method is compatible with state-of-the-art mass spectrometry (MS) methods that allow the detection of low-abundant niche regulators. This study compared the CSD and its proteome data to the method and data obtained by laser-capture-microdissection (LCM) and a standard wholemount dissection. The CSD method resulted in twice the quantification depth in less than half the preparation time compared to the LCM and simultaneously clearly outperformed the dissection precision of the wholemount dissection. Hence, CSD is a superior method for collecting the SEZ for proteome analysis.

Cryo-section-dissection allows fresh, frozen preparation of the largest neurogenic niche in the murine brain for deep quantitative proteome analysis. The method is precise, efficient, and causes minimal tissue perturbation. Therefore, it is ideally suited for studying the molecular microenvironment of this niche, as well as other organs, regions, and species.

Dissezione crio-sezione della zona subependimale adulta per un'analisi quantitativa accurata e profonda del proteoma

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone ...

Delivering Intrathecal Injections to Neonatal Mice

Somministrazione di iniezioni intratecali a topi neonatali

<meta charset="utf8"></head><body>Nucleofecazione efficiente di NSC isolate dalla zona subventricolare murina

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>Autore in primo piano: Nucleofection migliorata per la consegna genica ad alta efficienza in colture di cellule staminali neurali munine derivate dalla zona subven

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolamento, espansione e nucleofezione di cellule staminali neurali dalla zona subventricolare murina adulta

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...