IFN is an important cytokine for immune responses against viral and bacterial infections and the mediation of anticancer activity either indirectly, through regulation of anti‑inflamma- tory and anti‑angiogenic responses, or directly, by affecting the proliferation and differentiation of cancer cells (30). IFN- α / β is known to be essential in the activation of natural killer cells and macrophages (2-4). As key cytokines, IFN- α / β links the innate and adaptive immune systems (31,32). Another cytokine involved in the maintenance of red blood cell mass is EPO, which is involved in the proliferation and differentiation of erythrocytic progenitors (8). If plasma oxygen levels are low, EPO, which is released by the interstitial cells of the kidney, stimulates bone marrow to produce more red blood cells and increases the aerobic capacity of blood (6,7).
IFN- α and EPO are glycoproteins, which are major therapeutic agents in the treatment of certain hematological malignancies, including chronic renal anemia and other diseases (1,8). The mass production of these therapeutic agents is important in the pharmaceutical industry. Recombinant DNA technology has been widely used to obtain therapeutic agents in eukaryotic cell‑based culture systems (33,34). However, the productivity of these systems is limited in large-scale production. Although the productivity of a prokaryotic system is sufficient for the generation of recombinant proteins, the functional activity of recombinant proteins is restricted due to an absence of protein post‑translational modification (11). Therefore, it was hypothesized that the generation of transgenic cattle, termed bioreactors, is a more effective method for the production of relatively large quantities of recombinant therapeutic agents. This technology using livestock has significant potential and economic merit in biomedicine, agriculture, human health industries and environmental sustainability (35,36).
In the present study, bovine fibroblast cell lines expressing human IFN- α /EPO transgenes were developed in order to generate transgenic cattle. Milk is the easiest body fluid to obtain from ruminants (35). The capacity for mass‑production of the mammary gland, coupled with the relative ease of harvesting milk, means it is the organ of choice for the produc- tion of pharmaceutical products from animals. In our previous study, a α S1‑casein promoter region spanning between ‑175 and +796 nt was assessed, which demonstrated the highest expression activity among all the assessed promoters (29). Therefore, this promoter was applied in the present study for the generation of bovine transgenic fibroblasts containing dox‑inducible human IFN‑ α and EPO genes, as a material for SCNT procedures in the production of an animal bioreactor.
IFN- α and EPO are useful therapeutic agents, As over- expression by constitutively active promoters may cause unexpected side effects, including erythrocytosis involved with the induction of EPO (25), a dox‑inducible expres- sion system was adopted in parallel with a tissue-specific promoter in the present study. The expression of mammary gland‑specific and dox‑inducible human IFN‑ α or EPO was observed in an MAC‑T bovine mammary epithelial cell line, which is an immortalized epithelial cell line isolated from bovine mammary tissue (37). Mammary epithelial cells are the functional unit of the mammary gland. MAC-T cells provide a useful tool in the evaluation of foreign gene expression and the assessment of mammary tissue‑specific expression in vitro, as they retain a number of biochemical and morphological characteristics of the mammary gland in vivo (38). The unitary tet-on IFN- α /EPO induction system has mammary gland‑specific and dox‑inducible traits. When generating animal bioreactors, these cellular traits may reduce rates of stillbirth during pregnancy and unwanted outcomes caused by uncontrollable gene expression.
For the generation of transgenic cattle, these transgenic fibroblasts may be used in SCNT, a method used to perform sequential genetic modifications, targeted DNA insertions and artificial chromosome transfer using long‑term cultured somatic cells. The human IFN- α /EPO‑transgenic fibroblasts established in the present study may be used for the generation of transgenic cattle using SCNT technology. In this process, the nucleus containing chromosomal DNA in an egg cell may be removed and replaced with a nucleus containing genetically modified chromosomal DNA obtained from the transgenic fibroblast. Fusion between the enucleated egg cell and the donor somatic nucleus creates a new cell, which gains a complete set of chromosomes derived from the donor nucleus. The SCNT method has several advantages in the genetic cloning of valuable transgenic animals. The production of live offspring by SCNT using adult skin fibroblast cells was reported in goats in 1999 (39) and in cattle in 2000 (40), demonstrating the potential for use in the agricultural and pharmaceutical industries. If the animal bioreactors in the present study are born, they may produce recombinant human IFN‑ α and EPO proteins in milk on a daily basis and serve as a promising model for the production of recombinant therapeutic agents.
Acknowledgements
The present study was supported by a grant from the Next‑Generation BioGreen 21 Program (grant no. PJ00956301), Rural Development Administration, Republic of Korea.
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