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. 2006 Jan 30:7:9.
doi: 10.1186/1471-2202-7-9.

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

Anne L Cahill  1 , Bruce E Herring, Aaron P Fox
Affiliations

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

Anne L Cahill et al. BMC Neurosci. .

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.

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Figures

Figure 1
Figure 1
Plasmid-based expression of shRNA-SNAP-25. A) Plasmid map of pG418-shRNA, a plasmid designed to express shRNAs from the mouse U6 promoter. B) The two 56-base deoxyoligonucleotides used in the construction of pG418-shRNA-SNAP-25 are shown as they would be paired after annealing. The labels above and below the oligonucleotides indicate the source or the function of the nucleotides in the indicated regions. C) The predicted stem-loop structure of shRNA-SNAP-25 expressed from pG418-shRNA-SNAP-25.
Figure 2
Figure 2
Specific silencing of SNAP-25 by RNA interference. A) Immunoblots were done to assess the levels of SNAP-25, SNAP-23, synaptotagmin I, syntaxin 1A, tyrosine hydroxylase and β-actin in wild type PC12 cells, SNAP-25 knockdown cells and in control transfected cells (PC12 cells stably transfected with pG418-shRNA lacking an shRNA insert). Equal amounts of protein were loaded per lane. B) The SNAP-25 phenotype is maintained for at least 10 weeks in culture in both the parent PC12 cell line and the SNAP-25 knockdown cell line. C) Human and zebrafish SNAP-25 mRNAs are resistant to RNA interference. The specificity of the SNAP-25 shRNA was demonstrated by transiently transfecting SNAP-25 knockdown cells with plasmids designed to express SNAP-25 cDNA of rat, human, or zebrafish origin. The 19 nucleotide region of rat SNAP-25 mRNA which is targeted by the SNAP-25 shRNA differs from human SNAP-25 RNA at only 2 positions and from zebrafish SNAP-25 mRNA in 4 positions. The immunoblot shows that expression of rat SNAP-25 was silenced in the SNAP-25 knockdown cells, but human and zebrafish SNAP-25 were expressed. All three SNAP-25 cDNAs appeared to be expressed in the control transfected cells in that there was more SNAP-25 in the control transfected cells than in untransfected PC12 cells. The immunoblot was stripped and reprobed for β-actin to demonstrate approximately equal amounts of protein in each sample. D) SNAP-25 mRNA is reduced in SNAP-25 knockdown cells. RT-PCR was carried out with RNA isolated from wild type PC12 cells, SNAP-25 knockdown cells and in control transfected cells. SNAP-25 mRNA was easily detected in wild type and control transfected PC12 cells, but no SNAP-25 mRNA was detected in the SNAP-25 knockdown cells in this PCR experiments. However, if more of the reverse-transcription product was used for the PCR or if more cycles were done in the PCR reaction, some SNAP-25 mRNA was detectable in the SNAP-25 knockdown cells.
Figure 3
Figure 3
Catecholamine secretion is reduced in SNAP-25 knockdown PC12 cells. Representative amperometric traces are shown from a control cell stably transfected with pG418-shRNA lacking an shRNA insert (empty vector) (A) and from a SNAP-25 knockdown cell prior to and during a 2.5 min stimulation with 60 mM KCl (B). To the right of each of the traces is an averaged amperometric event shown on an expanded time scale. C) When compared to empty vector controls (n = 18) the silencing of SNAP-25 in PC12 cells (n = 18) did not alter the number of norepinephrine molecules per release event. D) Silencing of SNAP-25 resulted in a 63% reduction in the total number of exocytotic events produced following stimulation. Control transfected cells and SNAP-25 knockdown cells produced a mean ± SEM of 79 ± 17 events and 29 ± 10 events, respectively. *p < 0.03 (Student's t-test). E) Transient transfection with a human SNAP-25 expression plasmid rescued the deficit in catecholamine secretion in the SNAP-25 knockdown cells. *p < 0.03 (Student's t-test).
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