Abstract
Simple SummaryThe genetic modification of livestock genomes showed the great potential for production of industrial biomaterials as well as improving animal production. Particularly, the transgenic hen’s eggs have been considered for a massive production system of the genetically engineered biomaterials as a bioreactor animal. Virus-mediated transgene transduction is the most powerful strategy to generate the transgenic animals. However, industrial applications were hampered by many obstacles such as relatively low germline transmission and transgene silencing effects, as well as viral safety issues. In this study, a piggyBac transposon which is a non-viral integration technical platform was introduced into chicken primordial germ cells. Finally, we developed transgenic chickens and assayed the bioactivity of human cystatin C in the transgenic chicken’s tissues.A bioreactor can be used for mass production of therapeutic proteins and other bioactive substances. Although various methods have been developed using microorganisms and animal cells, advanced strategies are needed for the efficient production of biofunctional proteins. In microorganisms, post-translational glycosylation and modification are not performed properly, while animal cell systems require more time and expense. To overcome these problems, new methods using products from transgenic animals have been considered, such as genetically modified cow’s milk and hen’s eggs. In this study, based on a non-viral piggyBac transposition system, we generated transgenic bioreactor chickens that produced human cystatin C (hCST3). There were no differences in the phenotype or histochemical structure of the wild-type and hCST3-expressing transgenic chickens. Subsequently, we analyzed the hCST3 expression in transgenic chickens, mainly in muscle and egg white, which could be major deposition warehouses for hCST3 protein. In both muscle and egg white, we detected high hCST3 expression by ELISA and Western blotting. hCST3 proteins were efficiently purified from muscle and egg white of transgenic chickens using a His-tag purification system. These data show that transgenic chickens can be efficiently used as a bioreactor for the mass production of bioactive materials.
Highlights
The hCST3 nucleotide sequences were codon-optimized for chicken cell expression and synthesized to construct a piggyBac transposon-based expression vector (Figure 1A)
A His-tag (His6) was added in front of the hCST3 stop codon for efficient purification. hCST3 expression was regulated by the cytomegalovirus (CMV) promoter and the elongation factor 1 (EF1) promoter was used to express a puromycin-resistance gene for the stable selection of hCTS3 (Figure 1A)
We found that homozygous transgenic chickens deposited 20 ng/mL human epithelial growth factor in egg white [11]
Summary
A bioreactor is a device or system that supports a biologically active environment for the mass production of biofunctional proteins. Bioreactors include prokaryotic and eukaryotic cell systems and transgenic animals [1,2]. Technology, recombinant human insulin produced in E. coli was approved by the US Food and Drug Administration (FDA) for clinical use and was the first commercial recombinant pharmaceutical protein [2]. Due to inaccurate post-translational modification in bacteria and yeast bioreactor systems, recombinant pharmaceutical proteins have reduced efficacy and limited shelf life, and cause undesired side effects [3,4,5,6]. Animal cell systems are used to produce recombinant therapeutic proteins, but the production levels are relatively low and costly compared to other systems [3,4,5,6]
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