Enhanced Delivery of siRNA Complexes by Sonoporation in Transgenic Rice Cell Suspension Cultures
2009
Cheon, S.H., Inha University, Incheon, Republic of Korea | Lee, K.H., Inha University, Incheon, Republic of Korea | Kwon, J.Y., Inha University, Incheon, Republic of Korea | Choi, S.H., Inha University, Incheon, Republic of Korea | Song, M.N., Inha University, Incheon, Republic of Korea | Kim, D.I., Inha University, Incheon, Republic of Korea
Small interfering synthetic double-stranded RNA (siRNA) was applied to suppress the expression of the human cytotoxic-T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene transformed in transgenic rice cell cultures. The sequence of the 21-nucleotide siRNA was deliberately designed and synthesized with overhangs to inactivate the expression of hCTLA4Ig. The chemically synthesized siRNA duplex was combined with polyethyleneimine (PEI) at a mass ratio of 1:10 (0.33 ㎍ siRNA:3.3 ㎍ PEI) to produce complexes. The siRNA complexes (siRNA+PEI) were labeled with Cy3 in order to subsequently confirm the delivery by fluorescent microscopy. In addition, the cells were treated with sonoporation at 40 kHz and 419 W for 90 s to improve the delivery. The siRNA complexes alone inhibited the expression of hCTLA4Ig to 45% compared with control. The siRNA complexes delivered with sonoporation downregulated the production of hCTLA4Ig to 73%. Therefore, we concluded that the delivery of siRNA complexes into plant cells could be enhanced successfully by sonoporation.
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