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Neuroscience

Régiosélective Biolistic ciblage dans les tranches de cerveau organotypique l'aide d'un pistolet à gènes modifiés

doi: 10.3791/52148 Published: October 24, 2014

Abstract

Transfection d'ADN a été inestimable pour les sciences biologiques et les progrès récents à organotypiques préparations de tranches de cerveau, l'effet de divers gènes hétérologues pourrait donc être étudiée facilement tout en conservant de nombreux aspects de la biologie in vivo. Il a un intérêt croissant pour la transfection de neurones différenciées pour lesquels les méthodes classiques de transfection ont été nombreuses difficultés telles que de faibles rendements et des pertes importantes de viabilité. Transfection biolistique peut contourner la plupart de ces difficultés encore que récemment a été modifié cette technique de sorte qu'il se prête à une utilisation dans des tissus de mammifères.

Les nouvelles modifications apportées à la chambre de l'accélérateur ont amélioré la précision de tir du canon à gènes et l'augmentation de ses profondeurs de pénétration tout en permettant l'utilisation de basse pression de gaz (50 psi) sans perte d'efficacité de transfection ainsi que de permettre une propagation régiosélective ciblée de la paARTICLES à moins de 3 mm. En outre, cette technique est simple et rapide à réaliser que microinjections fastidieuses. Les deux transitoire et l'expression stable est possible avec des nanoparticules bombardement où l'expression épisomique peut être détecté dans les 24 heures et la survie des cellules a été montré pour être supérieur ou au moins égal à des méthodes classiques. Cette technique présente cependant un avantage essentiel: il permet la transfection d'être localisées dans un seul rayon de retenue permettant ainsi à l'utilisateur d'isoler les effets de l'anatomie du gène hétérologue. Nous présentons ici un protocole en profondeur pour préparer adultes viable tranches organotypiques et les soumettre à RÉGIOSÉLECTIVE transfection en utilisant un canon à gènes améliorée.

Materials

Name Company Catalog Number Comments
Helios gene gun system Bio Rad 165-2431 Gene gun and tube prep station
HEPES  Sigma 83264-100ML-F
NaCl Sigma S7653-250G
KCl Sigma P9333-500G
Na2HPO4 Sigma S7907-100G
KH2PO4 Sigma P9791-100G
0.2 µm sterile filter unit Millipore SCGPU05RE to filter media
DMEM Gibco 21885-025
Fetal calf serum Gibco 10270-098
D-Glucose Sigma G8270-100G
N2 Life Technologies 17502-048
Penicillin/Streptomycin Sigma p4333
Polyvinylpyrrolidone Sigma 856568-100G
Ethanol  Sigma 277649-100ML
Spermidine Sigma S2626
Agarose Bio Rad 161-3100EDU
Vibroslicer Laica VT1200 We used a custom design
Gene gun mount Built in house at U of T
Humidified cell culture incubator
Gold nanoparticles cytodiagnostics G-40-20
pEYFP-N1 Clontech
Tubing cutter Bio Rad 165-2422
Tefzel tubing Bio Rad 165-2441
Barrel Custom made by the LMB
Cell culture insert Millipore PICM0RG50
Paraformaldehyde Sigma 76240 Fluka
Dissecting microscope for convenience
Confocal microscope user defined for usage
Glass slide VWR 2588-CA48323-185
Microcover glass VWR 2448-CA48366-067-1
Vectashield mounting media Vector laboratories H-1400  

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Régiosélective Biolistic ciblage dans les tranches de cerveau organotypique l'aide d'un pistolet à gènes modifiés
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Cite this Article

Arsenault, J., Nagy, A., Henderson, J. T., O'Brien, J. A. Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun. J. Vis. Exp. (92), e52148, doi:10.3791/52148 (2014).More

Arsenault, J., Nagy, A., Henderson, J. T., O'Brien, J. A. Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun. J. Vis. Exp. (92), e52148, doi:10.3791/52148 (2014).

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