The cattle were fed a standard commercial cow diet (Suwon Purina, Suwon, Kyunggi-Do, Korea) and given water ad libitum in accordance with the animal study guidelines of the SooAm Biotech Research Foundation for Accreditation for Laboratory Animal Care.

Mouse mammalian epithelial HC11 cells (ATCC, Manassas, VA, USA) were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS), 50 U/ml penicillin, 50 μg/ml streptomycin (all obtained from Welgene, Daejeon, South Korea), 5 μg/ml insulin (Gibco) and 10 ng/ml epidermal growth factor (Invitrogen, Carlsbad, CA, USA). Bovine fibroblasts were obtained from a miniature bovine fetus (Deutsche Schwarzbunte) on day 30 of pregnancy. Bovine fibroblasts were then cultured in DMEM (Gibco) containing 10% FBS, 50 U/ml penicillin and 50 μg/ml streptomycin. All cells were grown at 37°C in a humidified 5% CO₂ atmosphere.

Genomic DNA of the bovine fibroblasts was isolated with a G-DEX™ IIc Genomic DNA Extraction kit (Intron Biotechnology, Seoul, South Korea). Genomic DNA (1 μg) was amplified in a 20-μl PCR containing 1 unit LA-Taq polymerase (Takara, Otsu, Shiga, Japan) for long-range PCR or 1 unit i-star Taq polymerase (Intron) for the standard PCR, 2 mM dNTPs (Takara) and 10 pmol of each specific primer. Details of all primers used are presented in Table I. PCR was performed with the following program: denaturation at 95°C for 30 sec, annealing at 62°C for 30 sec and extension at 72°C for 1, 2 or 3 min. The PCR products were separated on a 0.7% agarose gel, stained with ethidium bromide and images were captured under UV illumination. Images were scanned using Gel Doc EQ (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The products were then used for cloning.

Total RNA from HC11 cells was extracted using TRIzol reagent (Invitrogen) according to the the manufacturer’s instructions. Concentration of total RNA was determined by measuring absorbance at 260 nm. First-strand cDNA was prepared by reverse transcription with total RNA (1 μg) using mMLV reverse transcriptase (Invitrogen) and random primers (9-mers; Takara). To determine the optimal conditions for logarithmic phase PCR amplification of the target cDNA, aliquots of total cDNA (1 μg) were amplified using various cycle numbers. Mouse cytochrome c oxidase subunit 1 (1A) gene was used as an internal control to eliminate the possibility of RNA degradation and to control for variations in mRNA concentration. A linear correlation between PCR product band visibility and the number of amplification cycles was observed for the target mRNA sequences. 1A and the target human IFN-α or EPO genes were quantified using 28 and 30 cycles, respectively. PCR was performed as follows: denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec and extension at 72°C for 30 sec. The PCR products were separated in a 2.3% agarose gel, stained with ethidium bromide and an image was captured under UV illumination. The image was scanned and band density was analyzed using Gel Doc EQ (Bio-Rad Laboratories, Inc.).

Restriction and ligase enzymes were obtained from Takara. Various regions of the milk protein gene promoter were prepared by long-range PCR using bovine genomic DNA of the bovine fibroblasts as a template and specific primers containing restriction enzyme sites (MluI at the 5′ end or XhoI at the 3′ end). Amplified fragments were digested with MluI and XhoI and ligated into the promoter-less luciferase expression plasmid pGL3-Basic (Promega, Madison, WI, USA). The human IFN-α or EPO expression cassette plasmid was produced in several steps. Human IFN-α or EPO cDNA was prepared by PCR using cDNA amplified from total human RNA (Clontech, Mountain View, CA, USA) as a template. Amplified fragments were ligated into the recombinant pGL3 construct containing the milk protein gene promoter. For cassette selection, the enhanced green fluorescent protein (EGFP) gene was amplified from pIRES2-EGFP (Clontech), digested with EcoRV and BamHI and inserted into the pIRES-Neo plasmid (Clontech). The EGFP and neomycin resistance (Neo^r) genes were amplified by PCR, digested with SalI and ligated into the recombinant pGL3 vector encoding human IFN-α or EPO controlled by the milk protein gene promoter. Sequences of the targeting vector were confirmed by nucleotide sequencing (Genotech Co. Ltd., Daejeon, South Korea).

Transient transfection was performed using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s instructions. To normalize transfection efficiencies of the luciferase constructs, the Rous sarcoma virus (RSV)-lacZ plasmid was used to co-transfect the HC11 cells (34,35). Briefly, 3×10⁵ cells were seeded in 6-well tissue culture plates (Corning Incorporated, Corning, NY, USA) 1 day prior to transfection. Constructs (4 μg) containing the milk protein gene promoter and 0.5 μg of the RSV-lacZ plasmid were used to co-transfect the cells in serum-free DMEM (Gibco). Following incubation for 4 h, the medium was replaced with DMEM containing 10% FBS, 5 μg/ml PRL (Sigma-Aldrich, St. Louis, MO, USA) and 0.1 μM dexamethasone (DEX; Sigma-Aldrich) and the cells were incubated for an additional 48 h. Luciferase activity in the cell lysates was assayed using a Luciferase Assay System. Luminescence was measured with a GloMax 20/20 Luminometer. β-galactosidase activity was measured using a β-galactosidase Enzyme Assay System (all purchased from Promega). The relative luciferase activity was calculated as luciferase activity/β-galactosidase activity (%).

Bovine fibroblasts were transfected with the linearized construct using Lipofectamine 2000. After 24 h, the transfection medium was replaced with DMEM supplemented with 10% FBS and 500 μg/ml G-418 (Roche, Indianapolis, IN, USA) for 4 weeks. Antibiotic-resistant colonies were selected according to EGFP expression observed with a fluorescence microscope (Nikon, Tokyo, Japan). The selected colonies were subjected to PCR-based genotyping, using the confirming primer sets presented in Table I.

Data are presented as the mean ± standard error of the mean (SEM) and were analyzed with Student’s t-test and two-pair comparisons. P<0.05 was considered to indicate a statistically significant difference.

We monitored the expression of the reporter luciferase (Luc) gene controlled by bovine milk protein gene promoters in mouse mammary epithelial HC11 cells. Various bovine milk protein gene promoters were linked to the Luc gene in a promoter-less pGL3-Basic vector. These constructs were used to transfect HC11 cells and the relative promoter activities were measured with a Luc reporter gene assay. A previous study demonstrated that the expression of milk protein is increased by DEX with PRL in HC11 cells (36). Therefore, we measured bovine milk protein gene promoter activity in the HC11 cells following treatment with DEX and PRL. As demonstrated in Fig. 1, the αS1-casein promoter (−175 to +796 nt) resulted in a 16-fold increase of luciferase activity compared with that of the promoter-less construct. However, activities of the κ-casein, α-lactalbumin and β-lactoglobulin promoters were not identified as significantly altered in the HC11 cells following treatment with DEX and PRL. These results indicate that the αS1-casein promoter is an ideal candidate for inducing the secretion of recombinant human IFN-α and EPO into bovine milk.

We isolated human IFN-α and EPO cDNA sequences which were sub-cloned into the αS1-casein promoter. RT-PCR was used to measure the levels of human IFN-α and EPO mRNA produced by our recombinant system in HC11 cells. The expression of human IFN-α and EPO mRNA was evaluated following transient transfection with the two recombinant constructs. As demonstrated in Fig. 2, levels of the transcripts were identified as significantly increased in the transfected HC11 cells. These results demonstrate that the αS1-casein promoter is suitable for the production of recombinant human IFN-α and EPO in bovine milk.

The constructs in the present study were used to express human IFN-α and EPO under the control of the αS1-casein promoter, with two selection markers, EGFP and Neo^r (Fig. 3A and D). These constructs were linearized and used to transfect bovine fibroblasts using a liposomal-mediated gene delivery system. The fibroblasts were incubated in medium containing G-418 for 4 weeks to select for stable transfectants. Following this, expression of the constructs in the clones was confirmed by EGFP expression observed with fluorescent microscopy (Fig. 4). In addition, the clones were identified by PCR-based methods using primers specific for the constructs. Genomic DNA from clones expressing human IFN-α was analyzed with a, b primers (amplicon size, 914 bp; Fig. 3B) or with e, f primers (amplicon size, 1 kb; Fig. 3C). Additionally, genomic DNA from clones expressing human EPO was evaluated with c, d primers (amplicon size, 965 bp; Fig. 3E) or with e, f primers (amplicon size, 1 kb; Fig. 3F). We verified that the transfection efficiency was ~90% (Table II). Since our constructs included EGFP and Neo^r selection markers, positive recombination of our target genes into the bovine fibroblast genome was successfully monitored.