SummaryThe rice a-amylase 3D promoter system, which is activated under sucrose-starved conditions, has emerged as a useful system for producing recombinant proteins. However, using rice as the production system for therapeutic proteins requires modifications of the N-glycosylation pattern because of the potential immunogenicity of plant-specific sugar residues. In this study, glyco-engineered rice were generated as a production host for therapeutic glycoproteins, using RNA interference (RNAi) technology to down-regulate the endogenous a-1,3-fucosyltransferase (a-1,3-FucT) and b-1,2-xylosyltransferase (b-1,2-XylT) genes. N-linked glycans from the RNAi lines were identified, and their structures were compared with those isolated from a wild-type cell suspension. The inverted-repeat chimeric RNA silencing construct of a-1,3-fucosyltransferase and b-1,2-xylosyltransferase (D3FT ⁄ XT)-9 glyco-engineered line with significantly reduced core a-1,3-fucosylated and ⁄ or b-1,2-xylosylated glycan structures was established. Moreover, levels of plant-specific a-1,3-fucose and ⁄ or b-1,2-xylose residues incorporated into recombinant human granulocyte ⁄ macrophage colony-stimulating factor (hGM-CSF) produced from the N44 + D3FT ⁄ XT-4 glyco-engineered line co-expressing ihpRNA of D3FT ⁄ XT and hGM-CSF were significantly decreased compared with those in the previously reported N44-08 transgenic line expressing hGM-CSF. None of the glyco-engineered lines differed from the wild type with respect to cell division, proliferation or ability to secrete proteins into the culture medium.

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