Abstract
BackgroundA promoter capable of driving high-level transgene expression in oviduct cells is important for developing transgenic chickens capable of producing therapeutic proteins, including monoclonal antibodies (mAbs), in the whites of laid eggs. Ovalbumin promoters can be used as oviduct-specific regulatory sequences in transgenic chickens, but their promoter activities are not high, according to previous reports.ResultsIn this study, while using a previously characterized ovalbumin promoter, we attempted to improve the expression level of mAbs using a Cre/loxP-mediated conditional excision system. We constructed a therapeutic mAb expression vector, pBS-DS-hIgG, driven by the CMV and CAG promoters, in which the expression of the heavy and light chains of humanized immunoglobulin G (hIgG) is preceded by two floxed stuffer reporter genes. In the presence of Cre, the stuffer genes were precisely excised and hIgG expression was induced in pBS-DS-hIgG-transfected 293T cells. In chicken oviduct primary culture cells, hIgG was expressed after transfection of pBS-DS-hIgG together with the ovalbumin promoter-driven Cre expression vector. The expression level of hIgG in these cells was increased 40-fold over that induced directly by the ovalbumin promoter. On the other hand, hIgG was not induced by the ovalbumin promoter-driven Cre in chicken embryonic fibroblast cells.ConclusionsThe Cre/loxP-based system could significantly increase ovalbumin promoter-driven production of proteins of interest, specifically in oviduct cells. This expression system could be useful for producing therapeutic mAbs at high level using transgenic chickens as bioreactors.
Highlights
A promoter capable of driving high-level transgene expression in oviduct cells is important for developing transgenic chickens capable of producing therapeutic proteins, including monoclonal antibodies, in the whites of laid eggs
The induction system consists of two vectors: pBS-DS-humanized immunoglobulin G (hIgG), an immunoglobulin G (IgG) expression vector with two stuffer sequences flanked by loxP and modified loxP sites, and pBS-Ova2.8-Cre, a Cre recombinase expression vector driven by an oviduct-specific ovalbumin promoter contained in a 2.8-kb fragment at the 5’ end of the coding sequence of the chicken ovalbumin gene (Ova2.8) (Figure. 1A and 1C)
HIgG expression was not observed when pBSDS-hIgG was co-transfected with pBS-Ova2.8-Cre in chicken embryonic fibroblast cells. These results suggest that following co-transfection of pBS-DS-hIgG with pBS-Ova2.8-Cre in oviduct primary cultured cells, specific expression of Cre driven by the Ova2.8 promoter excises the stuffer genes in pBSDS-hIgG and induces strong expression of hIgG in oviduct cells
Summary
A promoter capable of driving high-level transgene expression in oviduct cells is important for developing transgenic chickens capable of producing therapeutic proteins, including monoclonal antibodies (mAbs), in the whites of laid eggs. Therapeutic mAbs have become a major class of drugs, their high production cost is a major obstacle This is mainly due to the use of cultured mammalian cells in the manufacturing of mAbs, which requires a complex industrial bioreactor system. The production of recombinant pharmaceutical proteins has been demonstrated in transgenic animals including sheep, goats, cattle, rabbits, and chickens (reviewed in [3,4]) Among these animals, the use of transgenic chickens as bioreactors is expected to have several advantages, including a shorter timescale for setup, ease of scaling up, and small space requirements (reviewed in [5,6]). Several groups reported the production of therapeutic proteins, such as cytokines, mini-antibodies, and mAbs using transgenic chickens [7,8,9,10,11]. Ubiquitous promoters were used to express the transgenic products; tissue-restricted expression of exogenous proteins was not demonstrated
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