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First published online on July 21, 2005.
 Copyright © 2005 by The Physiological Society
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Received May 19, 2005
Revised June 15, 2005
Accepted after revision July 20, 2005

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

Virginijus Valiunas1, Yaroslava Y Polosina1, Holly Miller1, Irina A Potapova1, Laima Valiuniene1, Sergey Doronin1, Richard T Mathias1, Richard B Robinson2, Michael R Rosen2, Ira S Cohen1, and Peter R Brink1*

1 SUNY at Stony Brook
2 Columbia University, NY

* To whom correspondence should be addressed. E-mail: peter.brink{at}sunysb.edu.

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.


Key words: Channels • Gap junction • Permeability




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