Name: neonatal subventricular zone electroporation
Uploaded: 2013-02-11T13:00:00
Description: Watch this Scientific Journal Video about Neonatal Subventricular Zone Electroporation at JoVE.com

Dette arbejde blev støttet af tilskud fra Department of Defense (Idéudvikling award, W81XWH-10-1-0041, AB), CT Stamceller tilskud (AB), og en National Institute of Health NRSA 10.668.225 (DMF). Det foreliggende materiale er baseret på arbejde delvis støttet af staten Connecticut under Connecticut Stem Cell Research Grants Program. Dens indhold er udelukkende forfatternes ansvar og repræsenterer ikke nødvendigvis de officielle synspunkter i staten Connecticut, Institut for Folkesundhed i staten Connecticut eller CT Innovations, havde Inc. finansieringskilder ingen rolle i studie design, dataindsamling og analyse, beslutning om at offentliggøre, eller forberedelse af manuskriptet.

Denne procedure er i overensstemmelse med Yale IACUC krav. Forskerne bør sikre, at IACUC retningslinjerne godkendes og følges i henhold til deres institutionelle krav. 1. Afsnit 1. Fremstillinq af DNA, Solutions, og glaspipetter <ol><li> Genererer høj renhed (OD 260/280&gt; 1.80) høj koncentration (pg / pl&gt; 2.5) endotoxin-frit DNA. </li><li> Forbered 0,9% saltvandsopløsning og steril filtreres gennem et 22 um filter. </li><li> Place 10 cm brand poleret borosilikatglas kapillarrør ...

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Her har vi detaljeret teknikken med neonatal SVZ elektroporering, en teknik til hurtigt og robust mærke og manipulere SVZ stamceller og deres afkom. Der er flere fordele, at elektroporering har i sammenligning med andre teknikker. For det første på grund af den fokale mærkning af celler, er man i stand til at skelne celle autonome og ikke-autonome celle virkninger. Sekund, genetisk manipulation ved hjælp af inducerbare systemer gør det muligt at sammenligne virkninger før eller efter synaptisk integration. Endvid...

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neonatal subventricular zone electroporation

Neurale stamceller (NSC) line den postnatale laterale ventrikler og giver anledning til flere celletyper, der indbefatter neuroner, astrocytter, og ependymal celler 1.. Forståelse af de molekylære veje, der er ansvarlige for NSC selvfornyelse, engagement og differentiering er kritisk for at udnytte deres unikke potentiale til at reparere hjernen og bedre forstå sygdomme i centralnervesystemet. Tidligere metoder til manipulering af mammale systemer krævede tidskrævende og dyrt, for genteknologien på hele dyret niveau 2. Således har hovedparten af undersøgelser undersøgt funktioner NSC-molekyler in vitro eller in hvirvelløse dyr. Her vil vi demonstrere den enkle og hurtige teknik til at manipulere neonatale NPCs der omtales som neonatal subventricular zone (SVZ) elektroporation. Lignende teknikker blev udviklet et årti siden for at studere embryonale NSC og har hjulpet undersøgelser af cortical udvikling 3,4. For nylig dette blev anvendt til at studere den postnatale gnaver forhjerne 5-7. Denne teknik resulterer i robust mærkning af SVZ NSC og deres afkom. Således postnatal SVZ elektroporering giver en omkostningseffektiv og tid effektivt alternativ til pattedyr NSC genteknologi.

Neonatal subventricular zone elektroporation resulterer i mærkningen af næsten alle radial glia sammenhængende med ryg-, bryst-lateral, og lateral subventricular zone efter &quot;fejer&quot; bevægelse af pincetten elektrode (fig. 1E). Dog kan elektroporation være skræddersyet til kravet om den pågældende eksperiment, men ikke fejer elektroden og under anvendelse af specifikke placering og orientering som beskrevet i videoen. For eksempel, eftersom dorsalt lokaliseret radial glia afviger fra fori...

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

Neonatal Subventricular Zone Electroporation

Vi viser en minimalt invasiv teknik kaldet neonatal subventricular zone elektroporering. Teknikken består i at injicere plasmid-DNA i de laterale ventrikler af neonatale hvalpe og anvendelse af elektrisk strøm til at levere og genmanipulere neurale stamceller

Neonatal Subventricular Zone Elektroporation

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

Research

JoVE Journal

Neuroscience

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 ...

In vivo Electroporation of Developing Mouse Retina

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 ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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 ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

Fremstilling af akutte 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 ...

Neonatal Pial Surface Electroporation

Neonatal pial Surface Elektroporation

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

Subtype-selektive Elektroporation af Kortikal interneuroner

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

Celle Sortering af neurale stam- og progenitorceller fra voksne mus subventrikulære Zone og Live-billeddannelse af deres cellecyklus Dynamics

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

Isolering og dyrkning af Voksen neurale stamceller fra mus 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 ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

Spinal Cord Neurons Isolation og Culture fra Neonatal Mus

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Kontinuerlig video elektroencefalogram under hypoxi-iskæmi i neonatale mus

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying ...