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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