Expression Of Pharmaceutical Proteins Using Transgenic Rice With A Double Built-in Cont-Ainment Strategies

Transgenic technology has been widely utilized for engineering crops for trait improvements and as bioreactors for the production of pharmaceutical proteins and industrial enzymes. However, a major concern over such pharmaceutical transgenic crops is the risk of its unintended spreading into environment and our food supplies. The existing strategies can only supervise parts of the development and application steps of the genetically modified (GM) crops. And the existing detection methods for transgenic seeds were tedious and time-consuming. Reliable containment technologies and simple detection methods for transgenic crops and seeds are badly in need. In this study, we created a double check system that combines a built-in selectively termination strategy for transgenic plants with the visual detection of transgenic rice seeds, which were used to express human proinsulin and etanercept. The results we obtained are as follows:(1) In this study, we reported a double check system, which was pre-set into pharmaceutical transgenic rice. The suppression of the expression of rice bentazon detoxification enzyme CYP81A6by RNAi technology made the rice lose bentazon resistance. The far-red fluorescent protein mKates158A gene was used as marker gene, which was visually detectable using bare eyes. We created transgenic rice via a standard Agrobacterium tumefaciens-mediated method with an inserted T-DNA that harbors a human proinsulin gene fused with the far-red fluorescent protein gene, an RNAi cassette suppressing the expression of the rice bentazon detoxification enzyme CYP81A6, and an EPSPS gene used as the selection marker for rice transformation. We totally obtained272trangenic lines. The transgenic lines were identified by PCR, western blot, southern blot, and herbicides spray test. The results suggested that transgenic events R-6, R-11and R-42not only have red seeds, high expression of human proinsulin, but also sensitive to bentazon. Our study demonstrated that the unintended transgenic pharmaceutical rice plants reported here could be killed selectively by bentazon during their growth stage, and also could be visually detected in the manufacturing process during and after harvest. This double check system will greatly reduce the chance for the transgenic pharmaceutical rice to get into environment and our food supplies. Moreover, any other genes of pharmaceutical and industrial proteins of our interest could be inserted into the T-DNA of the binary vector reported in this study.(2) Etanercept (Trade name, Enbrel) is one of the most effective drugs for the treatment of a series of disorders that TNF caused, and is now badly in needs. In this study, we try to explore rice seeds as a general bioreactor platform for the production of Enbrel. The gene fragment of Enbrel was synthesized according to the published sequence using optimal codons, and ligated with the rice storage protein Gtl promoter and then cloned into the binary plasmid, which contained an RNAi cassette suppressing the expression of bentazon detoxifying enzyme gene CYP81A6and an EPSPS gene conferring glyphosate tolerance. Transgenic rice was obtained via Agrobacterium tumefaciens-mediated method, and total of113independent transgenic lines were obtained. These transgenic lines were identified by southern blot, western blot, and bentazon spray test. The results showed that transgenic rice lines Enb-7and Enb-19were single copy lines. They expressed Enbrel successfully, and were sensitive to bentazon. Such pharmaceutical transgenic rice contained a built-in selectively termination strategy, and could be selected and deselected as you wish by two commonly used herbicides, glyphosate and bentazon.