Enhanced Delivery of siRNA Complexes by Sonoporation in Transgenic Rice Cell Suspension Cultures

  • Cheon, Su-Hwan (Department of Biological Engineering, Inha University) ;
  • Lee, Kyoung-Hoon (Department of Biological Engineering, Inha University) ;
  • Kwon, Jun-Young (Department of Biological Engineering, Inha University) ;
  • Choi, Sung-Hun (Department of Biological Engineering, Inha University) ;
  • Song, Mi-Na (Department of Biological Engineering, Inha University) ;
  • Kim, Dong-II (Department of Biological Engineering, Inha University)
  • Published : 2009.08.31


Small interfering synthetic double-stranded RNA (siRNA) was applied to suppress the expression of the human cytotoxic-T-Iymphocyte 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 (PEl) at a mass ratio of 1:10 (0.33 ${\mu}g$ siRNA:3.3 ${\mu}g$ PEl) 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 419W 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.


  1. Chen, T. L., Y. L. Lin, Y. L. Lee, N. S. Yang, and M. T. Chan. 2004. Expression of bioactive human interferon-gamma in transgenic rice cell suspension cultures. Transgenic Res. 13: 499-510
  2. Cheon, S.-H., K.-H. Lee, J.-Y. Kwon, H.-N. Ryu, D.-H. Yu, Y.- S. Choi, and D.-I. Kim. 2007. Effects of silkworm hemolymph on cell viability and hCTLA4Ig production in transgenic rice cell suspension cultures. J. Microbiol. Biotechnol. 17: 1944- 1948
  3. Duxbury, M. S. and E. E. Whang. 2004. RNA Interference: A practical approach. J. Surg. Res. 117: 339-344
  4. Elouahabi, A. and J. M. Ruysschaert. 2005. Formation and intracellular trafficking of lipoplexes and polyplexes. Mol. Ther. 11: 336-347
  5. Emans, N., S. Zimmermann, and R. Fischer. 2002. Uptake of a fluorescent marker in plant cells is sensitive to Brefeldin A and Wortmannin. Plant Cell 14: 71-86
  6. Florea, B. I., C. Meaney, H. E. Junginger, and G. Borchard. 2002. Transfection efficiency and toxicity of polyethylenimine in differentiated Calu-3 and nondifferentiated COS-1 cell cultures. AAPS PharmSci. 4: 1-11
  7. Godbey, W. T., K. K. Wu, and A. G. Mikos. 1999. Poly (ethylenimine) and its role in gene delivery. J. Control. Release 60: 149-160
  8. Hellwig, S., J. Drossardk, R. M. Twyman, and R. Fisher. 2004. Plant cell cultures for the production of recombinant proteins. Nat. Biotechnol. 22: 1415-1422
  9. Hong, S. Y., T. H. Kwon, Y. S. Jang, S. H. Kim, and M. S. Yang. 2006. Production of bioactive human granulocyte-colony stimulating factor in transgenic rice cell suspension cultures. Protein Expr. Purif. 47: 68-73
  10. Huang, L. F., Y. K. Liu, C. A. Lu, S. L. Hsieh, and S. M. Yu. 2005. Production of human serum albumin by sugar starvation induced promoter and rice cell culture. Transgenic Res. 14: 569-581
  11. Laborde, J. L., C. Bouyer, J. P. Caltagirone, and A. Gerard. 1998. Acoustic cavitation field prediction at low and high frequency ultrasounds. Ultrasonics 36: 581-587
  12. Lee, J. M., M. Takahashi, H. Mon, K. Koga, Y. Kawaguchi, and T. Kusakabe. 2005. Efficient gene transfer into silkworm larval tissues by a combination of sonoporation and lipofection. Cell Biol. Int. 29: 976-979
  13. Lee, S.-J., C.-I. Par, M.-Y. Park, H.-S. Jung, W.-S. Ryu, S.-M. Lim, et al. 2007. Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures. Protein Expr. Purif. 51: 293-302
  14. Lee, S.-Y. and D.-I. Kim. 2006. Perfusion cultivation of transgenic Nicotiana tabacum suspensions in bioreactor for recombinant protein production. J. Microbiol. Biotechnol. 16: 673-677
  15. Liu, Y., H. Yang, and A. Sakanishi. 2006. Ultrasound: Mechanical gene transfer into plant cells by sonoporation. Biotechnol. Adv. 24: 1-16
  16. Lungwitz, U., M. Breunig, T. Blunk, and A. Gopferich. 2005. Polyethylenimine-based non-viral gene delivery systems. Eur. J. Pharm. Biopharm. 60: 247-266
  17. Malik, I., M. Garrido, M. Bahr, S. Kugler, and U. Michel. 2006. Comparison of test systems for RNA interference. Biochem. Biophys. Res. Commun. 341: 245-253
  18. McDonald, K. A., L. M. Hong, D. M. Trombly, Q. Xie, and A. P. Jackman. 2005. Production of human $\alpha$1-antitrypsin from transgenic rice cell culture in a membrane bioreactor. Biotechnol. Prog. 21: 728-734
  19. Mittal, V. 2004. Improving the efficiency of RNA interference in mammals. Nat. Rev. 5: 355-365
  20. Schmidt, F. R. 2005. About the nature of RNA interference. Appl. Microbiol. Biotechnol. 67: 429-435
  21. Sharp, J. M. and P. M. Doran. 2001. Characterization of monoclonal antibody fragments produced by plant cells. Biotechnol. Bioeng. 73: 338-346
  22. Shin, Y. J., S. Y. Hong, T. H. Kwon, Y. S. Jang, and M. S. Yang. 2003. High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture. Biotechnol. Bioeng. 82: 778-783
  23. Snove Jr., O. and T. Holen. 2004. Many commonly used siRNAs risk off-target activity. Biochem. Biophys. Res. Commun. 319: 256-263
  24. Snove Jr., O., M. Nedland, S.H. Fjeldstad, H. Humberset, O. R. Birkeland, T. Grunfeld, and P. Saetrom. 2004. Designing effective siRNAs with off-target control. Biochem. Biophys. Res. Commun. 325: 769-773
  25. Tang, G. and G. Galili. 2004. Using RNAi to improve plant nutritional value: From mechanism to application. Trends Biotechnol. 22: 463-469
  26. Tang, G., B. J. Reinhart, D. P. Bartel, and P. D. Zamore. 2003. A biochemical framework for RNA silencing in plants. Genes Dev. 17: 49-63
  27. Tang, W., V. Samuels, N. Whitley, N. Bloom, T. DeLaGarza, and R. J. Newton. 2004. Post-transcriptional gene silencing induced by short interfering RNAs in cultured transgenic plant cells. Geno. Prot. Bioinfo. 2: 97-108
  28. Tang, W., D. A. Weidner, B. Y. Hu, R. J. Newton, and X. H. Hu. 2006. Efficient delivery of small interfering RNA to plant cells by a nanosecond pulsed laser-induced stress wave for posttranscriptional gene silencing. Plant Sci. 171: 375-381
  29. Unnamalai, N., B. G. Kang, and W. S. Lee. 2004. Cationic oligopeptide-mediated delivery of dsRNA for post-transcriptional gene silencing in plant cells. FEBS Lett. 566: 307-310
  30. Vaucheret, H., C. Beclin, and M. Fagard. 2001. Post-transcriptional gene silencing in plants. J. Cell Sci. 114: 3083-3091
  31. Wang, M. B. and P. M. Waterhouse. 2001. Application of gene silencing in plants. Curr. Opin. Plant Biol. 5: 146-150
  32. Waterhouse, P. M. and C. A. Helliwell. 2003. Exploring plant genomes by RNA-induced gene silencing. Nat. Rev. 4: 29-38
  33. Werth, S., B. Urban-Klein, L. Dai, S. Hobel, M. Grzelinski, U. Bakowsky, F. Czubayko, and A. Aigner. 2006. A low molecular weight fraction of polyethylenimine (PEI) displays increased transfection efficiency of DNA and siRNA in fresh or lyophilized complexes. J. Control. Release 112: 257-270
  34. Wesley, S. V., C. Helliwell, M. B. Wang, and P. Waterhouse. 2004. Posttranscriptional gene silencing in plants. Methods Mol. Biol. 265: 117-129