Connexin-Specific Cell to Cell Transfer of Short Interfering RNA by Gap Junctions

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 can move from one cell to another via gap junctions, thus allowing one cell to directly inhibit gene expression in another cell. To test this hypothesis, fluorescently labeled 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 Cx43. Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test if siRNA for DNA Polymerase ss (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 DNA polymerase ss (polss) These two pol ss knockdown cell lines (NRK-kcdc and Mss16tsA-kcdc) were co-cultured with labeled wild type, NRK-wt or Mss16tsA-wt cells or N2A cells. The levels of pol ss mRNA and protein were determined by semi-quantitative 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.