Enhanced delivery of siRNA complexes by sonoporation in transgenic rice cell suspension cultures.
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 microg siRNA:3.3 microg 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.
Cheon
Su-Hwan
SH
Department of Biological Engineering, Inha University, Incheon 402-751, Korea.
Lee
Kyoung-Hoon
KH
Kwon
Jun-Young
JY
Choi
Sung-Hun
SH
Song
Mi-Na
MN
Kim
Dong-Il
DI
D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D060908:CTLA-4 Antigen; D018929:Cell Culture Techniques; D002470:Cell Survival; D018014:Gene Transfer Techniques; D006801:Humans; D012275:Oryza / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D015703:Antigens, CD; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D034741:RNA, Small Interfering; D011994:Recombinant Proteins