Amylase gene silencing by RNA interference improves recombinant hGM-CSF production in rice suspension culture

Amylase gene silencing by RNA interference improves recombinant hGM-CSF production in rice... 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 α-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 α-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 α-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 α-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 α-amylase gene demonstrated that the quantity of rice α-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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

Amylase gene silencing by RNA interference improves recombinant hGM-CSF production in rice suspension culture

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Publisher
Springer Netherlands
Copyright
Copyright © 2008 by Springer Science+Business Media B.V.
Subject
Life Sciences; Plant Pathology; Biochemistry, general; Plant Sciences
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1007/s11103-008-9376-7
Publisher site
See Article on Publisher Site

Abstract

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 α-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 α-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 α-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 α-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 α-amylase gene demonstrated that the quantity of rice α-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.

Journal

Plant Molecular BiologySpringer Journals

Published: Jul 17, 2008

References

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