Construction of transgenes
The human interferon α2a coding sequence was codon-optimized for chicken and synthesized with a Kozak consensus sequence, start codon and chicken lysozyme signal peptide at the 5′ end. The synthetic gene was ligated into a replication defective vector derived from equine infectious anemia virus (EIAV) containing a 4.4 kb modified form of the EREOVA promoter and regulatory elements (EREOVA2) previously shown to drive protein expression restricted to the hen oviduct . The original EREOVA promoter had 0.9 kb deleted between the estrogen-responsive enhancer element (ERE) and the steroid-dependent regulatory element (SDRE) that was preserved in EREOVA2. The new promoter was found to enhance protein expression in egg white at least 10-fold compared to EREOVA. The synthesized, codon-optimized gene for pCSF1-Fc (Entelechon, Germany) consisted of a Kozak sequence, start codon, lysozyme signal peptide and 154 amino acid active form of porcine CSF1 joined to the hinge-CH3 region of porcine IgG1a as previously described (13). This transgene was cloned using designed restriction sites AatII and NheI into a modified pLenti6/DEST Gateway vector, with the V5 tag removed and downstream oPRE added. This vector was derived from human immunodeficiency virus (HIV) and contained the EREOVA2 promoter.
Production of transgenic birds
The birds were derived from a flock of Novagen Brown commercial egg layer strain. Lentiviral vectors were packaged and injected into embryos from new-laid eggs to generate G0 transgenic birds . Cockerels estimated by PCR analysis to have vector sequence present in the germ line at frequency of > 1% were bred with wild-type hens to generate G1 birds, which were screened by PCR and Southern transfer analysis to confirm that the complete gene was present as a single copy. Egg white from G1 hens or female G2 offspring of G1 cockerels was tested for presence of transgene-derived protein. Lines were established from validated G1/G2 birds for each transgene.
Protein expression analysis
Protein expression was demonstrated by SDS-PAGE and western blot. Samples were prepared by addition of 4x NuPAGE LDS sample buffer, and 10x sample reducing agent for gels that were run under reducing conditions and heated at 70 °C for 10 min before gel electrophoresis using Novex NuPAGE 12% Bis-Tris/MOPS gels (Life Technologies). Proteins were transferred onto nitrocellulose membranes by use of an XCell II Blot Module (Life Technologies). Transfer was verified by Ponceau S staining and membranes were blocked overnight at 4 °C. For westerns imaged using the LI-COR scanner, blocking was performed with Odyssey Blocking Buffer, while for westerns visualized using HRP, blocking was performed with 5% (w/v) powdered milk in TBS. Interferon α2a was detected with monoclonal anti-interferon α (Sigma, UK, catalogue number SAB1409236) at 1:1000 dilution and sheep anti-mouse IgG conjugated HRP (GE Healthcare NXA931-1ML) or LI-COR IRDye 680RD Donkey anti-Rabbit IgG (H + L) at 1:20,000 dilution and imaged with a LI-COR Odyssey Imager according to manufacturer’s instructions. Concentration was estimated with a commercial ELISA kit according to manufacturer’s instructions (PBL Biomedical Laboratories 41,100). Due to a lack of antibody against porcine CSF1, CSF1-Fc was immunoblotted with anti-porcine Fc conjugated to HRP (Bethyl Laboratories A100-104P) at a 1:10,000 dilution in 3% (w/v) powdered milk in TBS 0.1% (v/v) Tween-20. Interferon concentration was determined by quantitative Western blot using LI-COR Image Studio software.
Egg white activity analysis
Whole egg white from transgenic hens’ eggs was tested for activity prior to purification. For interferon, the ability to stimulate interferon-stimulated response element (ISRE)-driven transcription of luciferase was used to determine initial whole egg white activity. The pGL4.45[luc2P/ISRE/Hygro] vector (Promega) was transfected in HEK293T cells seeded in white-walled 96-well plates at 1 × 104 cells/well using FuGENE HD (Promega) according to manufacturer’s instructions. 24 h post-transfection, the medium was replaced with antibiotic-free medium and either egg white from transgenic hens or wild type egg white with control interferon protein (Sigma) added at an estimated final concentration of 10 international units (IU), 102 IU and 104 IU, with diluted wild type egg white used as a negative control. After a further 24 h, the One Glo Luciferase assay kit (Promega) was used to measure luminescence according to manufacturer’s instructions. For pCSF1-Fc, a cell survival assay based upon the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed as previously described . Briefly, stable Ba/F3 cells expressing porcine CSF1 receptor were grown in complete RPMI (10% (v/v) fetal calf serum, penicillin, streptomycin, and GlutaMAX) and supplemented with 5% (v/v) X63 cell supernatant from X63 Ag8–653 myeloma cells carrying an expression vector for IL-3 (22). Cells were seeded in 96-well plates at a density of 2 × 104 cells/well in triplicate and treated with serial dilutions of egg or CHO cell-produced pCSF1-Fc instead of IL-3, with negative controls containing complete RPMI with no X63 or pCSF1-Fc. Plates were incubated at 37 °C, 5% (v/v) CO2 for 48 h and MTT (Sigma) at a final concentration of 0.5 mg/mL was added for a further 3-h incubation, followed by solubilization overnight with acidified isopropanol. Absorbance was measured at 570 nm and results normalized to the highest dose positive control.
Protein purification and analysis
For pCSF1-Fc and hCSF1-Fc purification, egg white was diluted 1:4 in 50 mM phosphate buffer pH 3, adjusted to pH 4, and stirred at room temperature for 10 min to precipitate ovomucin. The diluted egg white was centrifuged at 25,000 g for 45 min at 10 °C, the supernatant adjusted to pH 7 and centrifuged again at 25,000 g for 45 min at 10 °C to remove any further precipitates. The clarified egg white was diluted to a final volume of 10 times the original egg white volume and pCSF1-Fc was bound to a 5 mL HiTrap MabSelect SuRe column (GE Healthcare) pre-equilibrated with 50 mM phosphate buffer, pH 7, (PB) at 5 mL/min by use of an Äkta Prime Plus liquid chromatography system (GE Healthcare). The column was washed with 10 column volumes of PB and the protein was eluted with 3–5 column volumes of 100 mM citrate buffer (CB) pH 3, or until the UV peak had returned to baseline, into tubes containing 1 M Tris pH 9 at 30% of the final fraction volume. The column was regenerated with 3 column volumes of CB and 5 column volumes of PB, cleaned in place with 5 column volumes of 0.5 M NaOH, pH adjusted with 5 column volumes of PB and finally cleaned with 10 column volumes of 20% (v/v) ethanol for storage. The fractions containing eluted protein, as determined by SDS-PAGE, were pooled and concentrated to < 13 mL in an Amicon stirred cell with 10,000 Da ultrafiltration regenerated cellulose membrane and injected onto a Superdex 200 26/600 column (GE Healthcare) equilibrated with PBS at 2.5 mL/min by use of the Äkta Prime Plus. Fractions were collected manually to ensure that only the main portion of the correct peak was isolated. After use the column was cleaned with 1–2 column volumes of 20% (v/v) ethanol and stored.
For Interferon α2a, egg white was diluted 1:4 in 20 mM phosphate buffer pH 3, adjusted to pH 4, and stirred at room temperature for 10 min to precipitate ovomucin. The diluted egg white was then centrifuged at 25,000 g for 45 min at 10 °C, the supernatant adjusted to pH 7 and centrifuged again at 25,000 g for 35 min at 10 °C to remove any further precipitates. The clarified egg white was diluted to a final volume of 10 times the original egg white volume and interferon α2a was bound onto a 5 mL HiTrap Blue column (GE Healthcare) equilibrated with 20 mM PB pH 7 at 5 mL/min by use of an Äkta Prime Plus liquid chromatography system (GE Healthcare). The column was washed with 10 column volumes of PB and protein eluted with 20 mM PB containing 1 M NaCl. The fractions containing interferon α2a, as determined by SDS-PAGE, were pooled, diluted to 10 times the pooled volume in ddH2O to prevent salt-induced association of interferon and ovalbumin, concentrated to < 13 mL in an Amicon stirred cell with 10,000 Da ultrafiltration regenerated cellulose membrane and injected onto a Superdex 200 26/600 column (GE Healthcare) equilibrated with PBS at 2.5 mL/min by use of the Äkta Prime Plus. After use the column was cleaned with 1–2 column volumes of 20% (v/v) ethanol and stored. Interferon-containing fractions, as determined by SDS-PAGE, were concentrated by Amicon filtration. Protein concentration was determined by UV280. Identity was confirmed by SDS-PAGE and mass spectrometric analyzes (Roslin Proteomics and Metabolomics Facility) and quality was assessed by reversed phase chromatography and SEC-multi-angle light scattering at the Edinburgh Protein Production Facility (EPPF), Kings Buildings, Edinburgh.
Analysis of purified Interferon α2a activity
A dose-inhibition assay was performed to determine interferon activity compared to a commercially available control (Sigma SRP4594). HEK 293 T cells were used to produce influenza A/PR/8/34 (PR8) virus as described previously (17). A549 cells were pre-treated with varying concentrations of interferon for 24 h, then infected at low multiplicity (MOI 0.001). After 1 h of adsorption at 37 °C, virus-containing medium was replaced with virus growth medium (DMEM, 1 μg/mL TPCK-treated trypsin, 0.14% (w/v) BSA). After 48 h, virus was harvested and titer determined by plaque assay on MDCK cells with an Avicel overlay (virus growth medium supplemented with 1.2% (v/v) Avicel) followed by toluidine blue staining (17, 23). The virus titer was normalized to a no-interferon control to determine dose-inhibition.
Analysis of purified pCSF1-Fc activity
In vitro activity of purified egg pCSF1-Fc was tested by the MTT assay in Ba/F3 cells as described for whole egg white, and in pig bone marrow cells as previously described . Briefly, pig bone marrow cells were derived from pigs culled by captive bolt at Dryden farm as previously described [31, 38]. Cells were plated at 5 × 104 cells/well in a 96-well plate directly from frozen stocks and grown in complete RPMI supplemented with either CHO-derived or egg purified pCSF1-Fc at 10 ng/mL, 100 ng/mL or 1000 ng/mL. After 7 days, the culture medium was replaced with 50 μl of 1 mg/ml MTT solution and incubated for 1 h at 37 °C. The MTT solution was removed and tetrazolium salt solubilized with 100 μl of solubilization agent (0.1 M HCL, 10% Triton X-100 and isopropanol) followed by incubation at 37 °C with 5% CO2 for 10 min. Plates were read at 570 nm.
In vivo activity of pCSF1-Fc was tested in mice as previously described . Approval was obtained from The Roslin Institute’s and The University of Edinburgh’s Protocols and Ethics Committees. The experiments were carried out under the authority of a UK Home Office Project License under the regulations of the Animals (Scientific Procedures) Act 1986. Twelve male C57BL/6 mice aged 6–8 weeks were purchased from Charles River Laboratories (UK) and injected subcutaneously with either PBS, 1 mg/kg pCSF1-Fc from CHO cells or 1 mg/kg pCSF1-Fc from eggs (n = 4 per group) for 4 days and sacrificed on day 5 by rising levels of carbon dioxide. Liver and spleen were removed, weighed and fixed in 10% neutral buffered formalin for histological analysis. Blood was collected by cardiac puncture and transferred into EDTA-coated tubes, then prepared for flow cytometry using the Uti-Lyse kit (Dako, Ely, UK) and stained with PE anti-mouse F4/80 (Biolegend, CA, USA, catalogue number 122616, 1:200) and APC anti-mouse Mac1/CD11b (Abcam, Cambridge, UK, catalogue number ab25482, 1:2000) to identify the macrophage population. Results were analyzed with FlowJo software.
Analysis of purified hCSF1-Fc activity
Activity of purified egg hCSF1-Fc was tested by the bone marrow MTT assay as described for pCSF1-Fc. To test whether the protein could withstand drying for long-term storage and easier shipping, aliquots of protein were brought to dryness using a Savant SPD2010 Speedvac (Thermo) at a constant vacuum pressure of 5.1 Torr over 1.5-h without heating. The protein was reconstituted in the same volume of ddH2O and used as described for pCSF1-Fc in the bone marrow differentiation assay.
Analysis was performed with GraphPad Prism Version 6.04 and statistical significance was assessed using Student’s two-tailed t-test. Resulting values were considered statistically significant at p < 0.05.