Reduction of plant specific N-glycans by triple RNA interference in Oryza sativa
2011
Hochschulschrift
Zugriff:
99
Recombinant proteins are important in pharmaceutical market. Plant recombinant protein systems have no risk of pathogen contamination. Also, mammalian glycoproteins produced in transgenic plants have biological activities and are low in cost. However, the glycosylation systems in plant cells is not compatible with mammalian cells, because they carry plant-specific β1, 2-xylosyl and α1, 3-fucosyl residues in N-glycosylation system and it may induce human immunity and affect its application. In addition, plant cells lack galactose residues. Therefore, we try to humanize N-glycosylation pathways for the most benefits without side effects of recombinant mammalian glycoproteins produced in transgenic rice cells To reduce plant specific xylose in rice cells, we generated RNA interference (RNAi) of β-1,2-xylosyltransferrase (XT) transgenic rice cell lines, called XTi. Previously, we got XTi-19 and XTi-50 transgenic cell lines and we analyzed their xylose contents in total glycoproteins. They only remained 61% and 66% of non-transformant cells, respectively. In addition, we selected another transgenic cell lines XTi-1 with low expression of XT mRNA. The xylose level in glycoproteins reduced to 43% of WT in the XTi-1 transgenic RNAi line. Currently, we generated transgenic rice lines which are able to express a stem loop RNA molecule containing three specific gene regions to block two fucosyltransferase genes (FT1 and FT2) and one XT gene all in once. We propose that through the knockdown strategy for N-glycan engineering, transgenic rice cells will produce glycoproteins towards mammalian-like structures. By RT-PCR analysis two transgenic line showed reduction in XT、FT1 and FT2 transcripts. Compared to wild-type control, xylosyl and focosyl residues in total glycoproteins were reduced to 68 % and 57%, respectively, in one triple RNAi lines, TRIi-15. Rice cells lack mammalian specific β-1,4-galactosyltransferase (GT), and may reduce stability or bioactivity in recombinant glycoproteins produced in rice system. We transformed human GT (hGT) to rice cells and use antibody to detect the fusion protein hGT by western blot analysis. Through these genetic modifications, our goal is to engineer a mammalian glycosylation system in transgenic rice cells for pharmaceutical recombinant glycoprotein production. A high-value glycoprotein, mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) is selected to be a model glycoprotein to test whether it produced in transgenic rice cells has similar glycan structure as mammalian system. TRIi-1 and TRIi-15 are host cells to express mGM-CSF. By RT-PCR analysis, total six cell lines having mGM-CSF expression are obtained for future glycan structure comparison.
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Reduction of plant specific N-glycans by triple RNA interference in Oryza sativa
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Autor/in / Beteiligte Person: | Ku, Jung-Ting ; 顧芮庭 |
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Veröffentlichung: | 2011 |
Medientyp: | Hochschulschrift |
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