Unknown

Dataset Information

0

Stable silencing of SNAP-25 in PC12 cells by RNA interference.


ABSTRACT: 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.

SUBMITTER: Cahill AL 

PROVIDER: S-EPMC1373637 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

altmetric image

Publications

Stable silencing of SNAP-25 in PC12 cells by RNA interference.

Cahill Anne L AL   Herring Bruce E BE   Fox Aaron P AP  

BMC neuroscience 20060130


<h4>Background</h4>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  ...[more]

Similar Datasets

| S-EPMC2230596 | biostudies-literature
| S-EPMC1224215 | biostudies-other
| S-EPMC357029 | biostudies-literature
| S-EPMC7127813 | biostudies-literature
| S-EPMC2706123 | biostudies-literature
| S-EPMC3738543 | biostudies-literature
| S-EPMC4169796 | biostudies-literature
| S-EPMC1199674 | biostudies-other
| S-EPMC484202 | biostudies-literature
| S-EPMC1937568 | biostudies-literature