Efficient production of human interferon beta in the white of eggs from ovalbumin gene–targeted hens

Transgenic chickens could potentially serve as bioreactors for commercial production of recombinant proteins in egg white. Many transgenic chickens have been generated by randomly integrating viral vectors into their genomes, but transgene expression has proved insufficient and/or limited to the initial cohort. Herein, we demonstrate the feasibility of integrating human interferon beta (hIFN-β) into the chicken ovalbumin locus and producing hIFN-β in egg white. We knocked in hIFN-β into primordial germ cells using a CRISPR/Cas9 protocol and then generated germline chimeric roosters by cell transplantation into recipient embryos. Two generation-zero founder roosters produced hIFN-β knock-in offspring, and all knock-in female offspring produced abundant egg-white hIFN-β (~3.5 mg/ml). Although female offspring of the first generation were sterile, their male counterparts were fertile and produced a second generation of knock-in hens, for which egg-white hIFN-β production was comparable with that of the first generation. The hIFN-β bioactivity represented only ~5% of total egg-white hIFN-β, but unfolding and refolding of hIFN-β in the egg white fully recovered the bioactivity. These results suggest that transgene insertion at the chicken ovalbumin locus can result in abundant and stable expression of an exogenous protein deposited into egg white and should be amenable to industrial applications.


Results
Production of the KI chickens. To abundantly, stably, and specifically produce recombinant hIFN-β in chicken egg white, we inserted hIFN-β at the translation initiation site of the chicken OVA locus using a CRISPR/ Cas9 system. We had previously constructed and evaluated four sgRNA/Cas9 (sg, single guide RNA) plasmids that could target OVA after cell transfection 38 . Based on those results, for the experiment reported herein, we employed pX330-Neo-OVATg1 that targets OVA near its transcription initiation codon (Fig. 1a). For insertion of hIFN-β, we constructed a donor vector that contained a 5′ OVA homology arm of 2.8 kb, the hIFN-β coding region fused to the OVA initiation codon, the bovine growth hormone polyadenylation signal, the puromycin resistance gene sequence, and a 3′ OVA homology arm of 3.2 kb. The donor and pX330-Neo-OVATg1 vectors were co-transfected into PGCs isolated from Barred Plymouth Rock chicken (BPR) male embryos. PGCs containing the donor plasmid were selected with puromycin (0.5 μg/ml). Then, these cells were expanded, and genomic DNA from a portion of the cells was subjected to PCR to confirm that the cells had been knocked in as designed. Using the 5′-and 3′-primer pairs, namely P1-4 and P5-8, located outside the homology regions but inside the transgene (Fig. 1a), the 2.8-kb and 3.2-kb fragments were amplified for the 5′ and 3′ assays, respectively ( Fig. 1b; see Supplementary Table S1). The results of these assays indicated that hIFN-β had been knocked in at the OVA locus in at least some of the cells.
We next determined whether the KI PGCs could become functional spermatozoa. For this purpose, we generated germline G0 chimeras by transplanting KI cells into the blood of recipient chick embryos at day 2.5 of development (Fig. 2a). To increase the contribution of the donor PGCs, endogenous PGCs in the recipient embryos were ablated by exposing them to 5-6 Gy of γ-radiation before transplantation 38 . After being injected with the KI cells, the recipient embryos were incubated until hatching. Four presumptive male germline G0 chimeras (#411-414) were raised to sexual maturity, and genomic DNA from their semen was subjected to PCR to determine whether their semen contained donor PGC-derived hIFN-β KI cells. The semen of all four roosters contained hIFN-β KI cells (Fig. 2b). The two roosters (#411 and #412) that showed relatively strong KI PCR products by both the 5′ and 3′ assays were crossed with wild-type (WT) White Leghorn (WL) hens. The genotypes of their G1 progeny were also assessed by PCR (Fig. 2b), and the results showed that 22.5% and 14.5% of the progeny from Scientific RepoRts | (2018) 8:10203 | DOI: 10.1038/s41598-018-28438-2 roosters #411 and #412, respectively, had the hIFN-β knock-in at the OVA locus (Table 1). We obtained 15 male and 16 female KI G1 chicks, and all were normal in appearance and had no discernable growth defects. Production of recombinant hIFN-β in the oviduct of KI hens and its deposition into egg white. Expression of endogenous OVA is restricted to the tubular gland cells in the oviduct magnum 39 . To determine the site of ectopic gene expression in our KI hens, we performed immunohistochemistry for hIFN-β on sections of the oviduct magnum (Fig. 3). Consistent with endogenous OVA expression, hIFN-β was found in the tubular gland cells of the KI hens but was absent in adjacent epithelial cells (Fig. 3c), indicating that hIFN-β expression was controlled by OVA regulatory mechanisms. Expression of hIFN-β was not detected in the oviduct magnum of WT WL hens ( Fig. 3e and f).
Next, we studied the effect of the hIFN-β knock-in on egg production. The average production rate for the G1 KI hens was about two-thirds that of the WT controls (BPR × WL) and was statistically significant (p < 0.05, Welch's t-test; Table 2). In addition, all G1 hens produced eggs, although their eggs appeared to be smaller than those of WT hens (Fig. 4a). Consistent with this observation, the average weight of the eggs from 10-month-old KI hens was 42.1 ± 3.1 g (n = 26), which was significantly less than that of WT (BPR × WL) (50.1 ± 3.4 g (n = 16); p < 0.05, Student's t-test). Unexpectedly, the portion of the KI egg white closest to the yolk was white and cloudy ( Fig. 4b and c), whereas the periphery portion of the egg white was clear and apparently of low viscosity. The volume of the clear region was relatively small compared with that of the cloudy region (4.3 ± 1.8 ml/egg vs. 14.4 ± 3.6 ml/egg, respectively, n = 41).
Next, we determined whether the KI egg white contained recombinant hIFN-β. The cloudy and clear parts of the KI egg white (from the #640 KI hen) as well as the thick albumen from the egg of a WT (BPR × WL) hen were subjected to SDS-PAGE followed by Coomassie Brilliant Blue staining (Fig. 4d). A Coomassie Blue-stained band for protein from the cloudy part of KI egg white was observed near that of the expected size (~23 kDa) for hIFN-β 40 . The same band was nearly absent in the clear part of the KI egg white and was not seen at all in WT egg white. We also subjected the egg-white samples to immunoblotting with an antibody against hIFN-β. Consistent The top diagram shows the WT chicken OVA locus. The target single-guide RNA (sgRNA) sequence that is part of exon 2 is denoted by the black bar above the nucleotide sequence. The protospacer adjacent-motif sequence is indicated by the red bar. The OVA initiation codon is shown in uppercase letters. The middle diagram shows the donor construct containing the 5′ and 3′ homology regions (HR), the hIFN-β -bovine growth hormone polyadenylation signal construct, and the PGK promoter that drives the puromycin resistance gene (PGK-Puro r ). The bottom diagram shows the KI allele along with PCR primers P1 to P9 that were used for 5′, 3′, and endogenous OVA assays in this study. (b) PCR amplification of the donor cassette knock-in at the OVA locus in the PGC genome. KI PGCs and their parental untransfected cells (UT) were subjected to nested PCR using primers P1-4 (5′ assay) and P5-8 (3′ assay). The middle lane, labeled M, contains DNA molecular mass markers (1-kbp DNA ladder, Nacalai). PCR amplicons of the expected sizes (2.8 kb for the 5′ assay, and 3.2 kb for the 3′ assay) are indicated by the arrows. with the Coomassie Blue staining results, substantial and relatively weak putative IFN-β signals were detected in the samples of the cloudy and clear portions of the KI egg white, respectively (Fig. 4e). In contrast, no obvious band of the same mass as hIFN-β was observed in the egg white from the WT hen. Based on results from both SDS-PAGE and immunoblotting, it was evident that hIFN-β was deposited in KI egg white, although not evenly distributed as it had accumulated in the cloudy part rather than in the clear part. The egg-white proteins from eggs of four additional KI G1 hens were also subjected to SDS-PAGE (Fig. 4f), and the cloudy portion of each KI hen egg white contained substantial and similar amounts of hIFN-β, which indicated that insertion of hIFN-β into the OVA locus was precisely controlled by the OVA transcriptional regulatory machinery and that hIFN-β was almost equally expressed in the oviduct of the KI hens and then secreted into their egg white.
We sequenced the first five N-terminal residues of hIFN-β from a KI egg white. Although the coding region of hIFN-β, including its signal sequence, was introduced into the OVA locus, the N-terminal sequence of hIFN-β from the KI egg white was that of mature hIFN-β expressed in mammalian cells (NH 2 -MSYNL), suggesting that the signal sequence had been properly cleaved 41 . The concentrations of hIFN-β in the KI egg white from four eggs each from four G1 hens was measured by an enzyme-linked immunosorbent assay (ELISA). The hIFN-β concentration in the cloudy part and clear part of the egg white ranged from 1.86 ± 0.34 to 3.52 ± 0.89 mg/ml and from Genomic DNA from the sperm of four chimeric roosters (411-414) and a WT rooster was PCR amplified with primer pairs P5/P8, P1/P4, and P1/P9 for the 3′ and 5′ assays and the endogenous OVA assay (O), respectively. Genomic DNA from transplanted PGCs containing hIFN-β KI cells (KI PGC) was also PCR amplified. The gels show the PCR-amplified products. (c) KI hIFN-β in the G1 chickens. Genomic DNA from the blood of the G1 progenies of #411 (left panel) and #412 (right panel) was PCR amplified for the 3′, 5′, and endogenous OVA assays using primer pairs P5/P8, P1/P4, and P1/P9, respectively. The genomic DNA from the blood of WT chickens and from the transplanted PGCs (KI PGC) was also PCR amplified. The gels show the PCR-amplified products. The lanes at the left of each gel panel are the DNA molecular mass markers as described in Fig. 1. 0.14 ± 0.05 to 0.68 ± 0.19 mg/ml, respectively (Fig. 4g). We also measured the hIFN-β concentration in chicken serum. As shown in Supplementary Table S2, hIFN-β was detected in serum from KI hens (from 0.23 ± 0.04 to 1.75 ± 0.12 ng/ml) but not in serum from a KI rooster, suggesting that portion of the hIFN-β expressed in the oviduct was transferred into the bloodstream. Next we assessed the fertility of the G1 KI hens. These hens were crossed with a WT WL rooster, and the resulting eggs were incubated; however, all fertilized eggs did not develop past day 8 so that no hatched chicks were obtained (n = 34). To determine why the eggs from hIFN-β KI hens were sterile, we transferred a portion of the KI or WT egg white into WT fertilized eggs and incubated them. Indeed, the presence of hIFN-β KI egg white significantly disrupted the embryonic development of recipient eggs (Supplementary Fig. 1; p < 0.05, chi-square test). This result indicated that the hIFN-β KI egg white was detrimental to the development of the chicken embryo, although it was unclear whether this was the only underlying cause of the egg sterility.
To obtain G2 offspring KI hens, we crossed a G1 KI rooster with WT WL hens. Unlike crossing the G1 KI hens with WT roosters, the crosses involving the KI roosters and WT hens produced male and female G2 KI progeny. The G2 KI hens were raised to sexual maturity, and their ability to lay eggs was assessed. Although, the G1 and G2 hens did not share exactly the same genetic background, the egg production rate and age at first egg laid did not differ significantly between the G1 and G2 groups (Table 2). Furthermore, the hIFN-β concentration in the cloudy part of the egg white was an inheritable feature (2.70 ± 0.59 to 4.42 ± 1.40 mg/ml, Fig. 4h). Both the efficiency of egg production and hIFN-β concentration in cloudy part of the egg white were similar in the G1 and G2 hens, which suggested that the average hIFN-β productivity of the KI hens was not drastically affected by the generation, at least if they had similar genetic backgrounds. Consistent with this idea, the mean values for egg Panels d-f and j-l are magnified views of the enclosed rectangular sections in panels a-c and g-i, respectively. The presence of hIFN-β is apparent in the oviduct magnum section from the hIFN-β KI hen (b,e) with its expression restricted to the tubular glands (TG). Ep, epithelial cells. All sections were counterstained with hematoxylin. Scale bars, 500 μm (a-c,g-i); 50 μm (d-f,j-l).
Characterization of hIFN-β produced by the KI hens. Given the abundant deposition of hIFN-β in the KI egg white, we next examined whether the deposited hIFN-β had biological activity. The cloudy part of the egg white from three eggs produced by each of three different KI hens and the thick albumen from an egg of a WT hen were sonicated, serially diluted with phosphate-buffered saline (PBS), and assayed for hIFN-β bioactivity by a reporter assay that used HEK-Blue IFN-α/β reporter cells to induce production of secreted alkaline phosphatase following stimulation with human type-I interferon 42 . The secreted alkaline phosphatase was quantified for each sample, and their half-log dose response curves were plotted (Fig. 5a, top panel). All KI egg white exhibited IFN bioactivity, but the egg white from the WT hen did not. However, the relative bioactivity of IFN-β in the KI egg whites was 4.2-5.7% that of commercially available, purified recombinant hIFN-β produced in mammalian cells (Fig. 5a, bottom table). A possible reason for this reduced bioactivity was that the majority of the protein was aggregated and/or misfolded. To address this possibility, we treated KI egg white of the KI hen #3614 with 6 M guanidine hydrochloride to ensure that the hIFN-β was unfolded; we then refolded the protein by dilution into a solution containing an artificial chaperone, namely highly polymerized cycloamylose 43 . The bioactivity of hIFN-β in both untreated and treated KI egg white was then examined. As shown in Fig. 5b, the hIFN-β activity after unfolding and refolding was >28-fold greater than that in the untreated egg white and was regarded as fully recovered to the level of that measured for commercially produced hIFN-β. This result indicated that the majority of hIFN-β in the KI egg white was misfolded and/or aggregated and hence was inactive, but activity could be restored upon refolding.

Discussion
This study reports the first successful knock-in of a gene in chickens for the production of an encoded recombinant protein in their eggs. All analyzed G1 and G2 KI hens produced large amounts of recombinant hIFN-β in the white of their eggs (1.86-4.42 mg/ml; ~30-60 mg (~1.3-2.7 μmol) per egg), demonstrating that gene targeting in chickens represents a potentially powerful means of producing recombinant proteins. Average egg production was ~70 per hen among the KI hens within the first 150 days ( Table 2), implying that <500 KI hens could provide a kilogram of recombinant hIFN-β in this time frame.
To date, lentiviral vectors have been the preferred transgene vehicle for the production of transgenic chickens because they efficiently modify germlines 22 . Lillico and colleagues generated a transgenic hen line using a lentiviral vector containing the 5′ regulatory sequence of OVA that included putative regulatory elements and hIFN-β 16 . As with our KI hens, the lentivirus-mediated G1 and G2 transgenic hens specifically expressed hIFN-β in their oviduct and secreted bioactive recombinant hIFN-β into the egg white. Unlike our system, however, the average concentration of hIFN-β in the white of eggs from the G1 and G2 lentiviral transgenic hens (3.5-426 μg/ ml; the range is the largest and smallest mean value calculated for six individual hens) varied greatly and was relatively less than that from our hens (1.86-4.42 mg/ml). The different results for the two hIFN-β systems may be a consequence of position effects, although other factors, e.g., genetic background and the physical conditions of the hens, may also have contributed. In addition, the difference between endogenous OVA regulatory elements in the KI hens and the short, truncated OVA regulatory elements introduced into the lentiviral transgenic hens may have differentially affected transgene expression. Although the precise regulatory mechanism(s) controlling expression remains to be clarified, our knock-in system resulted in stable expression of hIFN-β and production of large amounts of the recombinant protein, which suggests that ours would be the preferred system for commercial production of recombinant proteins. The eggs laid by our KI hens included cloudy egg white (Fig. 4c). Given that hIFN-β accumulated in the cloudy part of the egg white, it is probable that hIFN-β had aggregated (possibly in association with other proteins). Protein aggregation is caused by various factors including misfolding, an excessive concentration, and physical parameters such as temperature, pH, and ionic strength 44 . The observed reduced bioactivity of hIFN-β in the KI egg white suggests that most of the hIFN-β molecules had misfolded and/or aggregated (Fig. 5a). Consistent with this idea, the specific bioactivity of hIFN-β in the egg white was drastically increased (and appeared to be completely recovered) after subjecting the KI egg white to the unfolding-refolding procedure (Fig. 5b). Because refolding of hIFN-β in the egg white was easily achieved, aggregation could be an advantageous property of recombinant proteins produced in our KI chicken system, i.e., it could be used to at least partially isolate hIFN-β or another aggregated recombinant protein by centrifugal enrichment of the cloudy aggregate for subsequent refolding and activation.
The observed aggregation of hIFN-β may have been a consequence of its high concentration in the egg white. Various exogenous proteins have been expressed in transgenic chickens; however, aggregation of these proteins in egg white has neither been reported nor, apparently, studied in depth. Even for the scFv-Fc transgenic hen system, for which scFv-Fc was produced in greater quantities than hIFN-β in our system, the physical state of the egg white containing scFv-Fc was not reported 21 . Therefore, it remains to be determined if aggregation of a protein in the white of eggs from transgenic hens can be fully attributed to the concentration of the protein or, at least in part, to the specific characteristics of the protein. To differentiate among the underlying physical properties that can induce aggregation, in addition to hIFN-β, the folding of other proteins in egg white should be analyzed by generating oviduct-specific gene-targeted hens. These studies would increase our understanding of the mechanism underlying the control of secretion of an expressed foreign protein into KI egg white. Moreover, methods must be developed to avoid protein aggregation and misfolding for the production of large-sized proteins and protein complexes, each of which is not suited to protein refolding. The cell culture supernatants were assayed for induction of secreted alkaline phosphatase reporter activity using a colorimetric assay. Plots show the mean ± standard deviation values for three independent assays. A four-parameter logistic curve was fit to each dataset, and the EC 50 values (median effective concentrations) were calculated for each curve (upper panel). By using the EC 50 of a commercially available, purified recombinant hIFN-β as the standard, the absolute IFN-β bioactivities in the KI egg white were calculated [shown as "Concentration (reporter assay)" in the table at the bottom panel]. For comparison, the concentration of hIFN-β in each egg white was determined by ELISA (Fig. 4h), and the relative hIFN-β bioactivities were calculated as the percentage of active hIFN-β in the total amount of IFN-β. (b) Egg white from the hIFN-β KI hen #3614 was denatured in 6 M guanidine hydrochloride and then renatured in the presence of the artificial chaperone, highly polymerized cycloamylose (see Methods). hIFN-β bioactivity in the untreated and renatured egg white was analyzed by the HEK-Blue IFN-α/β reporter assay. Plots show the mean ± standard deviation values for three independent assays. The data are reported as in Fig. 5a Protein N-glycosylation plays important roles in protein folding, stability, and function 45 . Human IFN-β is an N-glycosylated protein, and a non-glycosylated form produced by Escherichia coli was found to have substantially less antiviral activity and indeed formed inactive dimers and oligomers 46 . It has been reported that the recombinant proteins produced in egg white are N-glycosylated, but they lack galactose and sialic acid in the N-glycans 13,14,47 . This glycosylation pattern is mainly attributable to low expression of galactosyltransferase in the oviduct magnum 48 , and thus the N-glycans of hIFN-β deposited in egg white may have lacked the terminal galactose and sialic acid. Although a detailed analysis of glycosylation of hIFN-β in KI egg white would be required to address this issue, the lack of addition of these two terminal sugars may underlie our observed aggregation of hIFN-β in egg white as well as other possible alterations such as a reduction in protein half-life 13 . In this respect, transgenic chickens that ectopically express galactosyltransferase may constitute a platform to generate a bioreactor system based on KI hens 49,50 . Hence, proper galactosylation followed by sialyation in the oviduct magnum might resolve the issue of protein aggregation in egg white.
Our KI chickens were normal in appearance and had no obvious growth or health problems. In addition, total life span did not differ significantly between KI and control hens ( Table 2, G1). Conversely, both the number and size of the KI hen eggs were reduced compared with WT (Table 2 and Fig. 4a). Because IFN-β induces various biological reactions such as antiviral and antiproliferative responses and may cause toxicity and infertility 51,52 , and thus it is plausible that a large amount of hIFN-β in the oviduct gland cells and/or the oviduct lumen negatively affected egg production. Although we did not observe any apparent histological abnormalities in the oviduct of the KI hens (Fig. 3), it is possible that the oviduct cells experienced damage and/or malfunction. The presence of hIFN-β in blood of KI hens (Supplementary Table S2) may also have contributed to the reduction in egg production. Moreover, heterozygous mutation of OVA might have negatively impacted egg production owing to the reduced production of OVA protein. In this respect, employment of a targeting vector, i.e., one designed to not affect endogenous OVA expression using a self-cleaving peptide or bicistronic expression system, might result in normal egg production as well as an abundance of recombinant protein.
As shown in this study, even though the KI hens were sterile possibly owing to foreign protein expression, the fertility of the KI roosters was hardly affected. Roosters are very fertile; therefore, in principle, the sperm from a KI rooster could easily be used to generate >1000 KI male and female offspring by artificial insemination. Each male offspring could then act as a new founder, and the female offspring would stably produce large quantities of a recombinant protein in egg white, allowing for large-scale production of the protein within a few generations of the first KI rooster. This scalability and time-efficient expansion of a KI bioreactor are advantages that chickens have compared with other types of livestock (i.e., goats and cows) and plant bioreactor systems. In addition, because transgenic hen systems can be developed easily, expression of a foreign protein in the eggs of transgenic hens is expected to be stable over time, gene targeting at the OVA locus has the potential to be the preferred technique to establish transgenic chicken bioreactors and the key driver for mass production of recombinant proteins using chickens. Plasmid construction. The plasmid expressing hCas9 and sgRNA targeted to OVA (px330-Neo-OVATg1) were generated as described elsewhere 38 . The donor vector for hIFN-β was generated by ligating a PCR-amplified, 2.8-kb DNA OVA fragment (upstream of the ATG initiation codon) as the 5′ homology arm, the cDNA containing the encoded hIFN-β sequence, the bovine growth hormone polyadenylation and puromycin resistance gene sequences, and a PCR-amplified, 3.2-kb DNA an OVA 3.2-kb sequence as the 3′ homology arm at the SalI and BamHI sites in pBluescript II SK(+). Primers for PCR amplification are shown in Supplementary Table S1, and PCR was carried out with PrimeSTAR HS DNA polymerase (TaKaRa, Otsu, Japan).

Targeted gene knock-in of cultured PGCs. PGCs derived from the blood of BPR embryos at
Hamburger-Hamilton stages 14 to 16 53 were cultured and transfected with px330-Neo-OVATg1 and the hIFN-β donor vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), as described previously 38 . Briefly, 0.8 μg each of px330-Neo-OVATg1 and the hIFN-β donor vector diluted with 50 μl OPTI-MEM (Thermo Fisher Scientific, Waltham, MA) was mixed with 50 μl OPTI-MEM containing 3 μl Lipofectamine 2000 reagent and then incubated with 0.5-1 × 10 5 PGCs for 5 min. Subsequently, the cells were suspended in 400 μl of an antibiotic-free KO-DMEM-type culture medium (Thermo Fisher Scientific) and incubated for 2 h at 37 °C 38 . PGCs were the cultured with Buffalo rat liver feeder cells for 4 days and then selected with puromycin (0.5 μg/ml; InvivoGen, San Diego, CA) for 3 days. The selected PGCs were expanded for 2 to 3 weeks and then selected again with puromycin under the same conditions. Finally, integration of hIFN-β at the OVA locus was confirmed by PCR using genomic DNA isolated from a proportion of the cells (see below).
Detection of hIFN-β in the OVA locus. Genomic DNA was extracted from PGCs, from the semen of G0 roosters, and blood from G1 and G2 roosters and hens using reagents of the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA). Gene-targeting events were detected by single or nested PCR using MightyAmp DNA polymerase Ver.2 (TaKaRa) with the primers shown in Supplementary Table S1. Generation of germline G0 chimera. Fertilized eggs for recipient embryos were irradiated with γ-rays at 5 to 6 Gy using a Gammacell 40 irradiator (Atomic Energy of Canada Ltd., Chalk River, ON, Canada). PGC samples (1000-2000 cells) that included the transgenic cells, were injected into the bloodstream of the recipient embryos (Hamburger and Hamilton stages [13][14][15]. The male embryos, identified by PCR 54,55 , were incubated until they hatched as described elsewhere 38 and then were raised to sexual maturity, after which genomic DNA from sperm was analyzed with PCR.

KI egg analyses.
A disposable medical dropper was used to collect samples of the clear and cloudy parts of the KI egg white based on their apparent viscosities and appearances. The volumes of the samples were then measured using the scales on the sides of the tubes. The viability of the embryos derived from the KI hens was assessed by candling the eggs on days 5 and 10. Dead embryos at the various developmental stages were confirmed by breaking eggs and examining the embryos.
Analysis of hIFN-β in the KI egg white. Sonicated samples of KI and WT egg white were individually suspended in 62.5 mM Tris-HCl, pH 6.8, 1% (w/v) SDS at a ratio of 1:20 for SDS-PAGE. Samples were each mixed with an equal volume of 2× Laemmli SDS-PGE sample buffer and subjected to gradient SDS-PAGE (5-20% w/v acrylamide; Oriental Instruments Ltd. Tokyo, Japan). Each gel was then stained overnight with Coomassie Brilliant Blue R-250 (Nacalai, Kyoto, Japan). The stained band from each lane corresponding to the molar mass of hIFN-β was excised and subjected to N-terminal sequencing (Hokkaido System Science Co., Ltd., Sapporo, Japan).
hIFN-β reporter assay. Bioactive hIFN-β produced in KI egg white was detected using HEK-Blue IFN-α/β reporter cells (InvivoGen). In brief, sonicated egg white was serially diluted with PBS, and then 20 μl of each dilution was added onto HEK-Blue IFN-α/β reporter cells: 5 × 10 4 cells per well in 180 μl of a DMEM-based culture medium (Thermo Fisher Scientific) in 96-well plates. After culture overnight at 37 °C, 20 μl of the culture supernatant from each sample was added into 180 μl Quanti-Blue reagent (InvivoGen) and then incubated for 1 h at 37 °C. The activity of the secreted alkaline phosphatase was measured as a colorimetric reaction at 650 nm using a microplate reader (Emax plate reader, Molecular Devices, Sunnyvale, CA). Results were analyzed using the Curve Fitter program of ImageJ (NIH, Bethesda, MD) to calculate half-maximal effective concentration (EC 50 ) values for hIFN-β from egg white and for the Chinese hamster ovary cell-derived recombinant hIFN-β (Wako).
Unfolding and refolding of hIFN-β. Unfolding and refolding of recombinant hIFN-β in KI egg white was performed with the Refolding CA kit (Takara). In brief, the egg white was sonicated and diluted 1:10 with PBS, then unfolded in 6 M guanidine hydrochloride with 40 mM dithiothreitol (final concentrations) for 1 h. The egg-white samples were then suspended in a 70-fold volume of surfactant solution (0.05% v/v Tween 40 and 2 mM dl-cystine in PBS) and incubated for 1 h at room temperature. Proteins were then refolded by adding 0.6% (v/v) of highly polymerized cycloamylose at a final concentration, followed by an 8-h incubation at room temperature. Samples were centrifuged at 20,000 × g for 10 min, and the supernatant was used as the refolded protein solution.

Immunohistochemistry.
A KI hen and a WT WL hen were sacrificed when they were 306 and 294 days old, respectively. The middle parts of their oviduct magnum were collected, fixed in 4% (w/v) paraformaldehyde, and then embedded in paraffin wax. Serial sections 5-μm thick were cut. The sections were deparaffinized in xylene, dehydrated through a graded series of ethanol, and treated with 0.3% (v/v) H 2 O 2 in methanol to inactivate endogenous peroxidase. After washing in PBS, sections were blocked in 5% (v/v) normal goat serum in PBS containing 0.1% (v/v) Tween-20 for 20 min and then incubated overnight at 4 °C with anti-hIFN-β (ab91245, Abcam). The sections were then rinsed three times in the same buffer, incubated with a peroxidase-conjugated anti-rabbit IgG (Histofine Simplestain Max PO; Nichirei, Tokyo, Japan) and then reacted with diaminobenzidine (Nichirei). Sections were counterstained with hematoxylin.