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A double built-in containment strategy for production of recombinant proteins in transgenic rice.

Zhang X, Wang D, Zhao S, Shen Z - PLoS ONE (2014)

Bottom Line: Using transgenic rice as a bioreactor for mass production of pharmaceutical proteins could potentially reduce the cost of production significantly.Thus, the transgenic rice plants reported in this study could be selectively killed by a commonly used herbicide during their growth stage, and their seeds may be detected visually during processing and consumption after harvest.This double built-in containment strategy may greatly enhance the confinement of the transgenic rice.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Rice Biology, Institute of Insect Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China.

ABSTRACT
Using transgenic rice as a bioreactor for mass production of pharmaceutical proteins could potentially reduce the cost of production significantly. However, a major concern over the bioreactor transgenic rice is the risk of its unintended spreading into environment and into food or feed supplies. Here we report a mitigating method to prevent unwanted transgenic rice spreading by a double built-in containment strategy, which sets a selectively termination method and a visual tag technology in the T-DNA for transformation. We created transgenic rice with an inserted T-DNA that harbors a human proinsulin gene fused with the far-red fluorescent protein gene mKate_S158A, an RNAi cassette suppressing the expression of the rice bentazon detoxification enzyme CYP81A6, and an EPSPS gene as the selection marker for transformation. Herbicide spray tests indicated that such transgenic rice plants can be killed selectively by a spray of bentazon at regular field application dosage for rice weed control. Moreover, the transgenic rice seeds were bright red in color due to the fused far-red fluorescent protein, and could be easily visualized under daylight by naked eyes. Thus, the transgenic rice plants reported in this study could be selectively killed by a commonly used herbicide during their growth stage, and their seeds may be detected visually during processing and consumption after harvest. This double built-in containment strategy may greatly enhance the confinement of the transgenic rice.

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Related in: MedlinePlus

Analysis of the fusion protein in transgenic rice seeds.A) Protein levels of fusion protein of mKate and human proinsulin in non-transgenic control and three independent transgenic events R-6, R-11 and R-42. Left panel, protein blot with the antibody to mKate; Right panel, protein blot with the antibody to proinsulin. In both left and right panels, using protein blot with the antibody to plant actin as loading control. Lanes 1-4, event R-6, R-11, R-42 and non-transgenic control, respectively. B) Transparency of ripened rice seeds. XS-134, non-transgenic control; R-42, a transgenic rice event. Scale bar, 2 mm.
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pone-0115459-g006: Analysis of the fusion protein in transgenic rice seeds.A) Protein levels of fusion protein of mKate and human proinsulin in non-transgenic control and three independent transgenic events R-6, R-11 and R-42. Left panel, protein blot with the antibody to mKate; Right panel, protein blot with the antibody to proinsulin. In both left and right panels, using protein blot with the antibody to plant actin as loading control. Lanes 1-4, event R-6, R-11, R-42 and non-transgenic control, respectively. B) Transparency of ripened rice seeds. XS-134, non-transgenic control; R-42, a transgenic rice event. Scale bar, 2 mm.

Mentions: In order to examine the expression of the proinsulin fusion protein, we carried out western blot analysis for the seed extracts of the transgenic rice with a monoclonal antibody specific to human proinsulin or mKate. A strong signal with an estimated molecular mass closed to the calculated size of the fusion protein (36 kDa) was detected in all of the transgenic events but not in the non-transgenic control plants with antibody against either human proinsulin or mKate (Fig. 6A). Weak bands of bigger or smaller size were also observed, and they were likely resulted from cross-link or degradation of the fusion protein. This result suggested that the human proinsulin fusion protein was highly expressed with the expected size in the transgenic rice seeds. As the far-red fluorescent protein was fused to the proinsulin, the brightness of the red color in the endosperm should be a direct indicator of the expression level.


A double built-in containment strategy for production of recombinant proteins in transgenic rice.

Zhang X, Wang D, Zhao S, Shen Z - PLoS ONE (2014)

Analysis of the fusion protein in transgenic rice seeds.A) Protein levels of fusion protein of mKate and human proinsulin in non-transgenic control and three independent transgenic events R-6, R-11 and R-42. Left panel, protein blot with the antibody to mKate; Right panel, protein blot with the antibody to proinsulin. In both left and right panels, using protein blot with the antibody to plant actin as loading control. Lanes 1-4, event R-6, R-11, R-42 and non-transgenic control, respectively. B) Transparency of ripened rice seeds. XS-134, non-transgenic control; R-42, a transgenic rice event. Scale bar, 2 mm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4274026&req=5

pone-0115459-g006: Analysis of the fusion protein in transgenic rice seeds.A) Protein levels of fusion protein of mKate and human proinsulin in non-transgenic control and three independent transgenic events R-6, R-11 and R-42. Left panel, protein blot with the antibody to mKate; Right panel, protein blot with the antibody to proinsulin. In both left and right panels, using protein blot with the antibody to plant actin as loading control. Lanes 1-4, event R-6, R-11, R-42 and non-transgenic control, respectively. B) Transparency of ripened rice seeds. XS-134, non-transgenic control; R-42, a transgenic rice event. Scale bar, 2 mm.
Mentions: In order to examine the expression of the proinsulin fusion protein, we carried out western blot analysis for the seed extracts of the transgenic rice with a monoclonal antibody specific to human proinsulin or mKate. A strong signal with an estimated molecular mass closed to the calculated size of the fusion protein (36 kDa) was detected in all of the transgenic events but not in the non-transgenic control plants with antibody against either human proinsulin or mKate (Fig. 6A). Weak bands of bigger or smaller size were also observed, and they were likely resulted from cross-link or degradation of the fusion protein. This result suggested that the human proinsulin fusion protein was highly expressed with the expected size in the transgenic rice seeds. As the far-red fluorescent protein was fused to the proinsulin, the brightness of the red color in the endosperm should be a direct indicator of the expression level.

Bottom Line: Using transgenic rice as a bioreactor for mass production of pharmaceutical proteins could potentially reduce the cost of production significantly.Thus, the transgenic rice plants reported in this study could be selectively killed by a commonly used herbicide during their growth stage, and their seeds may be detected visually during processing and consumption after harvest.This double built-in containment strategy may greatly enhance the confinement of the transgenic rice.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Rice Biology, Institute of Insect Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China.

ABSTRACT
Using transgenic rice as a bioreactor for mass production of pharmaceutical proteins could potentially reduce the cost of production significantly. However, a major concern over the bioreactor transgenic rice is the risk of its unintended spreading into environment and into food or feed supplies. Here we report a mitigating method to prevent unwanted transgenic rice spreading by a double built-in containment strategy, which sets a selectively termination method and a visual tag technology in the T-DNA for transformation. We created transgenic rice with an inserted T-DNA that harbors a human proinsulin gene fused with the far-red fluorescent protein gene mKate_S158A, an RNAi cassette suppressing the expression of the rice bentazon detoxification enzyme CYP81A6, and an EPSPS gene as the selection marker for transformation. Herbicide spray tests indicated that such transgenic rice plants can be killed selectively by a spray of bentazon at regular field application dosage for rice weed control. Moreover, the transgenic rice seeds were bright red in color due to the fused far-red fluorescent protein, and could be easily visualized under daylight by naked eyes. Thus, the transgenic rice plants reported in this study could be selectively killed by a commonly used herbicide during their growth stage, and their seeds may be detected visually during processing and consumption after harvest. This double built-in containment strategy may greatly enhance the confinement of the transgenic rice.

Show MeSH
Related in: MedlinePlus