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 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.


CONCLUSIONS
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.
Cahill
Anne L
AL
Department of Neurobiology, Pharmacology & Physiology, The University of Chicago, Chicago, IL, USA. acahill@bsd.uchicago.edu
Herring
Bruce E
BE
Fox
Aaron P
AP
D000818:Animals; D002395:Catecholamines / Q000378:metabolism; D004558:Electric Stimulation; D005089:Exocytosis / Q000502:physiology; D015870:Gene Expression / Q000235:genetics; D020868:Gene Silencing; D016716:PC12 Cells; D010957:Plasmids; D051381:Rats; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D002395:Catecholamines; D050825:Synaptosomal-Associated Protein 25