Establishment of transgenic fibroblasts for producing recombinant human interferon-α and erythropoietin in bovine milk

Human interferon alpha (IFN-alpha) and erythropoietin (EPO) have been used for a variety of purposes in clinical medicine. Human IFN-alpha has been used to treat several types of viral infection and cancer, as well as renal anemia, via stimulation of erythrocyte formation in the bone marrow. Transgenic cattle are excellent candidates for pharmaceutical production for humans due to their ability to produce recombinant proteins in milk. The purpose of the present study was to generate bovine transgenic fibroblasts capable of producing recombinant human IFN-alpha and EPO proteins in transgenic cattle milk. First, we analyzed the promoter activities of various bovine milk protein genes in HC11 mouse mammary epithelial cells. The bovine milk protein gene promoters were cloned into the Luc gene in a promoterless pGL3-Basic vector. Presence of the alpha S1-casein promoter (-175 to +796 nt) resulted in an up to 16-fold increase in luciferase activity compared with that of the promoter-less construct. In addition, the human IFN-alpha and EPO genes were identified as significantly overexpressed in HC11 cells compared with the promoter-less construct. Together, the present results demonstrate that the construct with the alpha S1-casein promoter may induce secretion of recombinant human IFN-alpha and EPO into bovine milk. Furthermore, we generated transgenic fibroblasts expressing human IFN-alpha and EPO cDNA controlled by the alpha S1-casein promoter and two screening markers, enhanced green fluorescent protein and neomycin resistance. These transgenic fibroblasts may be a source of somatic cells for generating transgenic cattle that produce recombinant human IFN-alpha and EPO proteins during lactation.