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Stable silencing of SNAP-25 in PC12 cells by RNA interference
BACKGROUND: SNAP-25 is a synaptic protein known to be involved in exocytosis of synaptic vesicles in neurons and of large dense-core vesicles in neuroendocrine cells. Its role in exocytosis has been studied in SNAP-25 knockout mice, in lysed synaptosomes lacking functional SNAP-25 and in cells after...
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2006
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1373637/ https://www.ncbi.nlm.nih.gov/pubmed/16445859 http://dx.doi.org/10.1186/1471-2202-7-9 |
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author | Cahill, Anne L Herring, Bruce E Fox, Aaron P |
author_facet | Cahill, Anne L Herring, Bruce E Fox, Aaron P |
author_sort | Cahill, Anne L |
collection | PubMed |
description | BACKGROUND: SNAP-25 is a synaptic protein known to be involved in exocytosis of synaptic vesicles in neurons and of large dense-core vesicles in neuroendocrine cells. Its role in exocytosis has been studied in SNAP-25 knockout mice, in lysed synaptosomes lacking functional SNAP-25 and in cells after treatment with botulinum toxins A or E that specifically cleave SNAP-25. These studies have shown that SNAP-25 appears to be required for most but not all evoked secretion. In order to further study the role of SNAP-25 in catecholamine secretion from PC12 cells we have used the recently developed technique of RNA interference to generate PC12 cell lines with virtually undetectable levels of SNAP-25. RNA interference is the sequence-specific silencing or knockdown of gene expression triggered by the introduction of double-stranded RNA into a cell. RNA interference can be elicited in mammalian cells in a number of ways, one of which is by the expression of small hairpin RNAs from a transfected plasmid. Selection of stably transfected cell lines expressing a small hairpin RNA allows one-time characterization of the degree and specificity of gene silencing and affords a continuing source of well-characterized knockdown cells for experimentation. RESULTS: A PC12 cell line stably transfected with a plasmid expressing an shRNA targeting SNAP-25 has been established. This SNAP-25 knockdown cell line has barely detectable levels of SNAP-25, but normal levels of other synaptic proteins. Catecholamine secretion elicited by depolarization of the SNAP-25 knockdown cells was reduced to 37% of control. CONCLUSION: Knockdown of SNAP-25 in PC12 cells reduces but does not eliminate evoked secretion of catecholamines. Transient expression of human SNAP-25 in the knockdown cells rescues the deficit in catecholamine secretion. |
format | Text |
id | pubmed-1373637 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2006 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-13736372006-02-18 Stable silencing of SNAP-25 in PC12 cells by RNA interference Cahill, Anne L Herring, Bruce E Fox, Aaron P BMC Neurosci Research Article BACKGROUND: SNAP-25 is a synaptic protein known to be involved in exocytosis of synaptic vesicles in neurons and of large dense-core vesicles in neuroendocrine cells. Its role in exocytosis has been studied in SNAP-25 knockout mice, in lysed synaptosomes lacking functional SNAP-25 and in cells after treatment with botulinum toxins A or E that specifically cleave SNAP-25. These studies have shown that SNAP-25 appears to be required for most but not all evoked secretion. In order to further study the role of SNAP-25 in catecholamine secretion from PC12 cells we have used the recently developed technique of RNA interference to generate PC12 cell lines with virtually undetectable levels of SNAP-25. RNA interference is the sequence-specific silencing or knockdown of gene expression triggered by the introduction of double-stranded RNA into a cell. RNA interference can be elicited in mammalian cells in a number of ways, one of which is by the expression of small hairpin RNAs from a transfected plasmid. Selection of stably transfected cell lines expressing a small hairpin RNA allows one-time characterization of the degree and specificity of gene silencing and affords a continuing source of well-characterized knockdown cells for experimentation. RESULTS: A PC12 cell line stably transfected with a plasmid expressing an shRNA targeting SNAP-25 has been established. This SNAP-25 knockdown cell line has barely detectable levels of SNAP-25, but normal levels of other synaptic proteins. Catecholamine secretion elicited by depolarization of the SNAP-25 knockdown cells was reduced to 37% of control. CONCLUSION: Knockdown of SNAP-25 in PC12 cells reduces but does not eliminate evoked secretion of catecholamines. Transient expression of human SNAP-25 in the knockdown cells rescues the deficit in catecholamine secretion. BioMed Central 2006-01-30 /pmc/articles/PMC1373637/ /pubmed/16445859 http://dx.doi.org/10.1186/1471-2202-7-9 Text en Copyright © 2006 Cahill et al; licensee BioMed Central Ltd. |
spellingShingle | Research Article Cahill, Anne L Herring, Bruce E Fox, Aaron P Stable silencing of SNAP-25 in PC12 cells by RNA interference |
title | Stable silencing of SNAP-25 in PC12 cells by RNA interference |
title_full | Stable silencing of SNAP-25 in PC12 cells by RNA interference |
title_fullStr | Stable silencing of SNAP-25 in PC12 cells by RNA interference |
title_full_unstemmed | Stable silencing of SNAP-25 in PC12 cells by RNA interference |
title_short | Stable silencing of SNAP-25 in PC12 cells by RNA interference |
title_sort | stable silencing of snap-25 in pc12 cells by rna interference |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1373637/ https://www.ncbi.nlm.nih.gov/pubmed/16445859 http://dx.doi.org/10.1186/1471-2202-7-9 |
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