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Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun
Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transf...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MyJove Corporation
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249736/ https://www.ncbi.nlm.nih.gov/pubmed/25407047 http://dx.doi.org/10.3791/52148 |
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author | Arsenault, Jason Nagy, Andras Henderson, Jeffrey T. O'Brien, John A. |
author_facet | Arsenault, Jason Nagy, Andras Henderson, Jeffrey T. O'Brien, John A. |
author_sort | Arsenault, Jason |
collection | PubMed |
description | Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues. New modifications to the accelerator chamber have enhanced the gene gun's firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene's effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun. |
format | Online Article Text |
id | pubmed-4249736 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | MyJove Corporation |
record_format | MEDLINE/PubMed |
spelling | pubmed-42497362014-12-04 Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun Arsenault, Jason Nagy, Andras Henderson, Jeffrey T. O'Brien, John A. J Vis Exp Neuroscience Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues. New modifications to the accelerator chamber have enhanced the gene gun's firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene's effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun. MyJove Corporation 2014-10-24 /pmc/articles/PMC4249736/ /pubmed/25407047 http://dx.doi.org/10.3791/52148 Text en Copyright © 2014, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/ |
spellingShingle | Neuroscience Arsenault, Jason Nagy, Andras Henderson, Jeffrey T. O'Brien, John A. Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun |
title | Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun |
title_full | Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun |
title_fullStr | Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun |
title_full_unstemmed | Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun |
title_short | Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun |
title_sort | regioselective biolistic targeting in organotypic brain slices using a modified gene gun |
topic | Neuroscience |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249736/ https://www.ncbi.nlm.nih.gov/pubmed/25407047 http://dx.doi.org/10.3791/52148 |
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