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

Abstract

Abstract. Interferon α (IFN-α) 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-α and EPO proteins were established and were generated containing the mammary gland-specific αS1-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-α/EPO-expressing constructs. Therefore, a unitary tetracycline (tet)-on the IFN-α/EPO vector was established, which combined a tet-on activator cassette controlled by the αS1-casein promoter, with a responder cassette encoding the IFN-α/EPO gene, controlled by the tetracycline response element (TRE) promoter. In these systems, the tet-controlled transactivator is affected by mammary gland-specific αS1-casein promoter, and binding of the transcriptional activator to the TRE results in transcription of the downstream IFN-α/EPO genes in the presence of dox. To assess this, the unitary tet-on IFN-α/EPO vector was introduced into a bovine mammary gland cell line (MAC-T), and the cells were then treated with 0.1-1 µg/ml dox. A marked increase was observed in the expression levels of IFN-α/EPO. In addition, bovine transgenic fibroblasts containing a mammary gland-specific and dox-inducible IFN-α/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-α/EPO protein during lactation. IntroductionInterferons (IFNs) are glycoproteins produced by the immune system of host cells in response to infection of pathogens (1-4). Various forms of interferons have been evaluated as potential therapeutics in a number of solid tumors and hematological malignancies, including renal anemia, renal cell carcinoma, melanoma, acquired immune deficiency syndrome-associated Kaposi's sarcoma, follicular lymphoma, hairy cell leukemia and chronic myelogenous leukemia (1). INF-α and INF-β cytokines exert their antiviral function by eliciting antiviral activity from target cells and inducing apoptosis in infected cells. They also contribute to the immune response by activation of natural killer cells and macrophages (2-4). IFN-α was approved by the Food and Drug Administration as a treatment for hairy cell leukemia in 1986 (1) and, using a gene cloning ...

Sperm-borne miR-449b influences cleavage, epigenetic reprogramming and apoptosis of SCNT embryos in bovine

“…A low dose of doxycycline (Dox) regulates the expression of target gene without toxicity to animals or cells. Numerous studies have shown inducible Tet-On system is a convenient tool for reversible and very tightly controlled conditional gene expression in zebrafish, drosophila, murine, porcine and bovine models 23 – 27 . In our previous study, we successfully used the Tet-On system to induce expression of miR-34c in bovine fibroblasts 28 .…”

A Ubiquitous Chromatin Opening Element and DNA Demethylation Facilitate Doxycycline-Controlled Expression during Differentiation and in Transgenic Mice

“…Based on the mammalian genome architecture with its compartmentalization into various chromatin domains, , cellular regulatory DNA elements such as enhancers, insulators, or silencers and the epigenetic status of a particular chromatin domain can influence the synthetic expression cassettes. Since standard gene transfer methods rely on random integration of transfected DNA, cell clones with tight regulation of the transgenes have to be identified by appropriate selection and sorting strategies. − Since screening is limited for the establishment of transgenic mice, the specific targeting of cassettes into predefined chromosomal loci is a favorable strategy for prediction of expression properties in tissues.…”