Amylase gene silencing by RNA interference improves recombinant hGM-CSF production in rice suspension culture.
A rice cell suspension culture system with the Ramy3D promoter, which is induced by sucrose starvation, has been previously utilized to produce large quantities of recombinant proteins. Although this expression system was reported previously to generate a good yield of recombinant hGM-CSF in transgenic rice cell suspension culture, rice alpha-amylase was a dominant protein, with 43% of total secreted proteins and an obstacle to the production and purification of secreted recombinant proteins in a rice cell suspension culture. In this study, an intron-containing self-complementary hairpin RNA (ihpRNA)-mediated post transcriptional gene silencing (PTGS) strategy for the rice alpha-amylase gene was applied in order to overcome this problem in rice cell suspension culture systems. The reduction of the mRNA level of the rice alpha-amylase gene was verified via Northern blot analysis and siRNA, an initiator of RNA interference, was detected via an RNase protection assay. The amount of rice alpha-amylase in the culture medium was reduced to 8.2% as compared to that of the wild-type. A transgenic rice cell suspension culture expressing both the hGM-CSF and ihpRNA of the rice alpha-amylase gene demonstrated that the quantity of rice alpha-amylase was reduced to 22% and that the accumulation of hGM-CSF increased by 1.9-fold as compared to that in the transgenic cell line expressing hGM-CSF only. These results indicated that RNAi technology should be of great utility for suppressing undesirable genes, and should improve accumulation and facilitate the purification of secreted recombinant proteins in rice cell suspension cultures.
Kim
Nan-Sun
NS
Division of Biological Sciences, Chonbuk National University, Dukjindong 664-14, Jeonju, Chonbuk 561-756, South Korea.
Kim
Tae-Geum
TG
Jang
Yong-Suk
YS
Shin
Yun-Ji
YJ
Kwon
Tae-Ho
TH
Yang
Moon-Sik
MS
D000681:Amylases / Q000172:deficiency* / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods*; D003470:Culture Media; D018744:DNA, Plant / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis*; D006801:Humans; D012275:Oryza / Q000166:cytology* / Q000201:enzymology* / Q000235:genetics; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins; D014170:Transformation, Genetic
D003470:Culture Media; D018744:DNA, Plant; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000681:Amylases