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1 Department of Physiology and Biophysics and the Institute for Molecular Cardiology at SUNY at Stony Brook, NY, USA
2 The Laboratory for Chemical Biology and the Department of Pharmacological Science at SUNY at Stony Brook, NY, USA
3 Department of Pharmacology and the Center for Molecular Therapeutics, Columbia University, New York, NY, USA
The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase ß (pol ß) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol ß was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mß16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin-deficient N2A cells. NRK and Mß16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol ß. These two pol ß knockdown cell lines (NRK-kcdc and Mß16tsA-kcdc) were co-cultured with labelled wild type, NRK-wt or Mß16tsA-wt cells or N2A cells. The levels of pol ß mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. Co-culture of Mß16tsA-kcdc cells with Mß16tsA-wt, N2A or NRK-wt cells had no effect on pol ß levels in these cells. Similarly, co-culture of NRK-kcdc with N2A cells had no effect on pol ß levels in the N2A cells. In contrast, co-culture of NRK-kcdc with NRK-wt cells resulted in a significant reduction in pol ß in the wt cells. The inability of Mß16tsA-kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell-to-cell movement of the siRNA. These results support the novel hypothesis that non-hybridized and possible hybridized forms of siRNA can move between mammalian cells through connexin-specific gap junctions.
(Received 19 May 2005;
accepted after revision 20 July 2005;
first published online 21 July 2005)
Corresponding author P. R. Brink: Department of Physiology and Biophysics, SUNY at Stony Brook, Stony Brook, NY 11794, USA. Email: peter.brink{at}sunysb.edu
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