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

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.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.SNAP-25 along with syntaxin and synaptobrevin are the SNARE proteins known to be involved in exocytosis of synaptic vesicles in neurons and of large dense-core vesicles in neuroendocrine cells [1,2]. The role of SNAP-25 in exocytosis has been studied in a variety of different experimental preparations [3-7]. 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 barely detectable 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 [8-10]. In mammalian cells this double-stranded RNA must be short (<30 bp), but may be either a simple RNA duplex with two unpaired nucleotides on the 3'-ends (small interfering RNA or siRNA; [11]) or a small hairpin RNA (shRNA) [12-16]. The siRNA or shRNA may be chemically synthesized, transcribed in vitro, produced by enzymatic cleavage of long double-stranded RNA, or expressed in situ from a plasmid or viral vector. For cells, such as PC12 cells, where only a modest transfection efficiency can be achieved, the use of viral vectors or the establishment of cell lines stably transfected with a vector expressing an shRNA are the best approaches to RNA i