Generation of transgenic fibroblasts producing doxycycline-inducible human interferon-alpha or erythropoietin for a bovine mammary bioreactor

摘要

Interferon (IFN-) is a cytokine, produced predominantly in immune cells in response to pathogens, which interferes with viral replication in host cells. Another cytokine hormone, erythropoietin (EPO), is synthesized in interstitial fibroblasts of the kidney and acts as a stimulator for the production of red blood cells. Importantly, the two cytokines have been used in the treatment of certain hematological malignancies, including renal anemia. In the production of recombinant proteins, a transgenic expression system in bovine species is an efficient strategy for pharmaceutical production. In the present study, recombinant constructs capable of producing recombinant human IFN- and EPO proteins were established and were generated containing the mammary gland-specific S1-casein promoter region (between -175 and +796 nt), as this promoter was revealed to have the highest level of activity in a previous promoter study. In order to minimize developmental toxicity by constitutive exogenous expression, a doxycycline (dox)-inducible system was introduced to the IFN-/EPO-expressing constructs. Therefore, a unitary tetracycline (tet)-on the IFN-/EPO vector was established, which combined a tet-on activator cassette controlled by the S1-casein promoter, with a responder cassette encoding the IFN-/EPO gene, controlled by the tetracycline response element (TRE) promoter. In these systems, the tet-controlled transactivator is affected by mammary gland-specific S1-casein promoter, and binding of the transcriptional activator to the TRE results in transcription of the downstream IFN-/EPO genes in the presence of dox. To assess this, the unitary tet-on IFN-/EPO vector was introduced into a bovine mammary gland cell line (MAC-T), and the cells were then treated with 0.1-1 g/ml dox. A marked increase was observed in the expression levels of IFN-/EPO. In addition, bovine transgenic fibroblasts containing a mammary gland-specific and dox-inducible IFN-/EPO construct were generated. These transgenic fibroblasts may provide a source for somatic cell nuclear transfer for the generation of transgenic cattle producing recombinant human IFN-/EPO protein during lactation.