Name: methods for study of neuronal morphogenesis: ex vivo rnai electroporation in embryonic murine cerebral cortex
Uploaded: 2012-05-18T12:00:00
Description: Watch this Scientific Journal Video about Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex at JoVE.com

Vi takker Dr. Shirin Bonni til tilvejebringelse af pSil-GFP-konstruktion, Dr. Alper Uzun til illustration af figur 1, og Leduc Bioimaging facilitet til konfokal mikroskopi. EMM er støttet af Career Award for Medical Science fra Burroughs Wellcome Fund, en NARSAD Young Investigators Award, og NIH NCRR Cobre P20 RR018728-01. SBL er støttet af NIH NCRR Cobre P20 RR018728-01, og har modtaget støtte fra PHS NRSA 5T32MH019118-20.

1. Forberedelse Kultur Solutions og medier (ikke video) <ol><li> Fremstille en liter komplet Hanks balancerede saltopløsning (HBSS) indeholdende 1X HBSS, 2,5 mM HEPES (pH 7,4), 30 mM D-glucose, 1 mM CaCl2, 1 mM MgSO4 og 4 mM NaHCO3. Tilsættes dobbelt destilleret vand (ddH2O). Filtersteriliser med en 0,2 um filter og opbevares ved 4 ° C. </li><li> Forberede skive dyrkningsmedium under anvendelse af 35 ml Basal Medium Eagle media, 12,9 ml komplet HBSS, 20 mM D-glucose (1,35...

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Disse fremgangsmåder involverer ex vivo elektroporation af plasmider, der koder dobbeltstrenget RNA hårnåle 8 og dyrkning af organotypiske skiver 4 tilvejebringer flere forskellige fordele. Første af disse fremgangsmåder giver mulighed for en hurtig vurdering af RNAi-afledte fænotyper. Optagelse af en ekspressionskassette der koder for GFP i den samme pSilencer vektor, der indeholder U6 promotoren, som driver den dobbeltstrengede RNA-hairpin muliggør en hurtig identifikation og kara...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Hamilton sprøjte </td><td> HAMILTON </td><td> 80008 </td><td> 31 gauge, 0,5 inches lang, PT-4 (niveau punkt beveling), 10 gi volumen </td></tr><tr><td> Platinum tweezertrodes </td><td> BTX </td><td> 45-0489 </td><td> 5mm størrelse </td></tr><tr><td> ECM830 elektroporator </td><td> BTX </td><td> 45-0002 </td><td></td></tr><tr><td> BTX Footswitch </td><td> BTX </td><td> 45-0208 </td><td> Til brug med ECM830 elektroporator </td></tr><tr><td> Vibrerende klinge mikrotom </td><td> LEICA </td><td> VT1000 S </td><td></td></tr><tr><td> 6 - vel ret til at bruge med indsatse </td><td> FALCON </td><td> 353502 </td><td> Indeholder indhak til at passe indsatse </td></tr><tr><td> Vævskulturplader indsatse </td><td> FALCON </td><td> 353090 </td><td> 0,4 mikrometer </td&gt; </tr><tr><td> Fast Green </td><td> SIGMA </td><td> F7252 </td><td></td></tr><tr><td> Lavtsmeltende agarose </td><td> FISHER </td><td> BP165-25 </td><td> DNA-kvalitet </td></tr><tr><td> Laminin </td><td> Sigma-Aldrich </td><td> L2020 </td><td></td></tr><tr><td> Poly-L-lysin </td><td> Sigma-Aldrich </td><td> P5899 </td><td></td></tr><tr><td> Basal Medium Eagle </td><td> Sigma Aldrich </td><td> B-1522 </td><td></td></tr><tr><td> HBSS 10x uden Ca og Mg </td><td> GIBCO </td><td> 14180-046 </td><td></td></tr><tr><td> HEPES-fri syre </td><td> Sigma-Aldrich </td><td> H4034 </td><td></td></tr></tbody></table>

methods for study of neuronal morphogenesis: ex vivo rnai electroporation in embryonic murine cerebral cortex

Hjernebarken dirigerer højere kognitive funktioner. Denne seks lagdelt struktur er dannet i en indvendig-først ud-sidste måde, hvor de første fødes neuroner forbliver tættere til ventriklen, medens de sidste fødes neuroner migrerer forbi den første fødes neuroner mod overfladen af hjernen 1. Ud over neuronal migration 2, er en vigtig fremgangsmåde til normal cortical funktion regulering af neuronale morfogenese 3. Mens neuronal morfogenese kan studeres in vitro i primære kulturer, der er meget at lære fra, hvordan disse processer er reguleret i væv miljøer. Vi beskriver teknikker til at analysere neuronal migration og / eller morfogenese i organotypiske skiver af hjernebarken 4,6. En pSilencer modificerede vektor anvendes, som indeholder både en U6 promotor, der driver de dobbeltstrengede hairpin RNA og et separat ekspressionskassette, der koder for GFP-protein drevet bya CMV-promotoren 7-9. Vores fremgangsmåde giver mulighed for hurtig vurdering af defekter i neuritudvækst på specifik knockdown af kandidatgener og er blevet anvendt i en screening for regulering af neuritudvækst 8. Fordi kun en undergruppe af celler vil udtrykke RNAI konstruktionerne de organotypiske skiver tillader en mosaik analyse af potentielle fænotyper. Endvidere, fordi denne analyse udføres i en nær tilnærmelse til in vivo-miljø, men tilvejebringer en billig og hurtig alternativ til generering af transgene eller knockout dyr gener med ukendt cortical funktion. Endelig, i sammenligning med in vivo elektroporation teknologi, er succes ex vivo elektroporation forsøg, der ikke afhængig af dygtige kirurgi udvikling af færdigheder og kan udføres med en kortere uddannelse tid og dygtighed.

Watch this Scientific Journal Video about Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex at JoVE.com

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

At foretage en hurtig vurdering af funktionen af ​​gener i udviklingen af ​​cerebral cortex, beskriver vi fremgangsmåder, der involverer<em&gt; Ex vivo</em&gt; Elektroporering af plasmider co-udtrykkende inhiberende RNA (RNAi) og GFP i murin embryonisk cortex. Denne protokol er egnet til studiet af forskellige aspekter af nervesystemets udvikling, f.eks neurogenese, neuronal migration og neuronal morfogenese herunder dendritceller og axon udvækst.

Metoder til Undersøgelse af Neuronal morfogenese:<em&gt; Ex vivo</em&gt; RNAi Elektroporation i embryoniske murin Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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 ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

Påvisning af Microregional Hypoxia i mus hjernebarken ved to-foton Imaging af endogent NADH Fluorescens

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

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

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

Visualisering og genetisk manipulering af dendritter og udløberne i mus cerebrale cortex og hippocampus under anvendelse<em&gt; I utero</em&gt; Elektroporation

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

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Samtidig Elektroencefalografi, Real-time måling af laktat Koncentration og Optogenetic Manipulation af neuronale aktivitet i Rodent Cerebral Cortex

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Dobbelte Elektrofysiologiske Optagelser af synaptisk-evoked astrogliale og Neuronal respons i Akut hippocampusskiver

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Funktionel Imaging af Auditory Cortex i voksne katte ved hjælp af High-felt fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Live-Imaging mitosens i udviklingslandene mus embryonale Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

I Utero Elektroporation Approaches to Undersøgelse ophidselse af neuronale subpopulationer og Single-celle Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Inducerende Cre-lox rekombination i mus hjernebarken gennem In Utero elektroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...