Journal: Scientific Reports

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

doi: 10.1038/s41598-018-28438-2

Figure Lengend Snippet: CRISPR/Cas9-mediated human IFN- β knock-in at the OVA locus in chicken PGCs. ( a ) Schematic of the knock-in strategy. 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.

Article Snippet: Chinese hamster ovary cell–derived recombinant hIFN-β (Wako) suspended in 62.5 mM Tris-HCl, pH 6.8, 1% (w/v) SDS served as the standard.

Techniques: CRISPR, Knock-In, Sequencing, Construct, Amplification, Nested PCR, Labeling

Journal: Scientific Reports

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

doi: 10.1038/s41598-018-28438-2

Figure Lengend Snippet: CRISPR/Cas9-mediated knock-in of hIFN- β at the chicken OVA locus. ( a ) Schematic of the experimental procedure that generated hIFN- β KI chickens. ( b ) KI hIFN- β in semen of chimeric G0 roosters. 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.

Article Snippet: Chinese hamster ovary cell–derived recombinant hIFN-β (Wako) suspended in 62.5 mM Tris-HCl, pH 6.8, 1% (w/v) SDS served as the standard.

Techniques: CRISPR, Knock-In, Generated, Amplification

Journal: Scientific Reports

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

doi: 10.1038/s41598-018-28438-2

Figure Lengend Snippet: Immunohistochemistry of hIFN-β in the oviduct magnum sections. Oviduct sections from hIFN- β KI ( a – f ) and WT ( g – l ) hens were stained with hematoxylin and eosin ( a , d , g and j ), immunohistochemically stained for hIFN-β and counterstained with hematoxylin ( b , e , h and k ), or stained with Giemsa ( c , f , i and l ). 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) .

Article Snippet: Chinese hamster ovary cell–derived recombinant hIFN-β (Wako) suspended in 62.5 mM Tris-HCl, pH 6.8, 1% (w/v) SDS served as the standard.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Scientific Reports

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

doi: 10.1038/s41598-018-28438-2

Figure Lengend Snippet: Egg production by hIFN-β KI G1, G2, and G3 hens and by WT (G1) hens.

Article Snippet: Chinese hamster ovary cell–derived recombinant hIFN-β (Wako) suspended in 62.5 mM Tris-HCl, pH 6.8, 1% (w/v) SDS served as the standard.

Techniques: Knock-In

Journal: Scientific Reports

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

doi: 10.1038/s41598-018-28438-2

Figure Lengend Snippet: Production of recombinant hIFN-β in KI egg white. ( a–c ) Eggs from a hIFN- β KI and a WT hen. ( a ) Eggs from WT and KI hens. ( b , c ) The white and yolk from an egg of a WT ( b ) or KI ( c) hen. Most of the KI egg white appeared to be white and cloudy (arrow in c ). The arrowhead in ( c ) indicates a clear section of the egg white. ( d ) Production of hIFN-β in the white of eggs from a KI (#640) and WT hen as assessed by SDS-PAGE. The arrowhead indicates the position of hIFN-β. Wc, white, cloudy part of the KI egg white; C, clear part of the KI egg white. ( e ) Immunoblotting of KI egg-white proteins with anti-hIFN-β. rhIFN-β, recombinant hIFN-β (40 ng); WT, Wc, and C have the same meanings as in Fig. 4d. For each lane, 3 μg of the egg white was electrophoresed. ( f ) SDS-PAGE showing similar production levels of hIFN-β in the white of eggs laid by the four KI hens (#771, #773, #779, #642). The two parental G0 KI roosters (#411, #412) of the hens are identified in the panel. ( g, h ) Concentration of recombinant hIFN-β in KI egg white from G1, G2, and G3 KI hens. Solutions of the cloudy part of the KI egg white from four G1 hens ( g ), three G2 hens, and two G3 hens ( h ) were subjected to ELISA for the presence of recombinant hIFN-β. Bars show the mean ± standard deviation of the concentration of recombinant hIFN-β in each KI egg white. Four (G1) and three (G2 and G3) eggs from individual KI hens were subjected to ELISA.

Article Snippet: Chinese hamster ovary cell–derived recombinant hIFN-β (Wako) suspended in 62.5 mM Tris-HCl, pH 6.8, 1% (w/v) SDS served as the standard.

Techniques: Recombinant, SDS Page, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Scientific Reports

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

doi: 10.1038/s41598-018-28438-2

Figure Lengend Snippet: Detection of bioactive hIFN-β in KI egg white. ( a ) HEK-Blue IFN-α/β reporter cell samples were individually stimulated with five-fold serial dilutions of egg white from three hIFN- β KI hens (#3607, #3614, #3619) and a WT hen. 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. ), 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.

Article Snippet: Chinese hamster ovary cell–derived recombinant hIFN-β (Wako) suspended in 62.5 mM Tris-HCl, pH 6.8, 1% (w/v) SDS served as the standard.

Techniques: Cell Culture, Activity Assay, Colorimetric Assay, Standard Deviation, Purification, Recombinant, Concentration Assay, Reporter Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

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

doi: 10.1038/s41598-018-28438-2

Figure Lengend Snippet: CRISPR/Cas9-mediated human IFN- β knock-in at the OVA locus in chicken PGCs. ( a ) Schematic of the knock-in strategy. 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.

Article Snippet: For immunoblotting, egg-white proteins (3 μg per lane) and recombinant hIFN-β (40 ng per lane, Wako, Osaka, Japan) were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA).

Techniques: CRISPR, Knock-In, Sequencing, Construct, Amplification, Nested PCR, Labeling

Journal: Scientific Reports

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

doi: 10.1038/s41598-018-28438-2

Figure Lengend Snippet: CRISPR/Cas9-mediated knock-in of hIFN- β at the chicken OVA locus. ( a ) Schematic of the experimental procedure that generated hIFN- β KI chickens. ( b ) KI hIFN- β in semen of chimeric G0 roosters. 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.

Article Snippet: For immunoblotting, egg-white proteins (3 μg per lane) and recombinant hIFN-β (40 ng per lane, Wako, Osaka, Japan) were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA).

Techniques: CRISPR, Knock-In, Generated, Amplification

Journal: Scientific Reports

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

doi: 10.1038/s41598-018-28438-2

Figure Lengend Snippet: Immunohistochemistry of hIFN-β in the oviduct magnum sections. Oviduct sections from hIFN- β KI ( a – f ) and WT ( g – l ) hens were stained with hematoxylin and eosin ( a , d , g and j ), immunohistochemically stained for hIFN-β and counterstained with hematoxylin ( b , e , h and k ), or stained with Giemsa ( c , f , i and l ). 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) .

Article Snippet: For immunoblotting, egg-white proteins (3 μg per lane) and recombinant hIFN-β (40 ng per lane, Wako, Osaka, Japan) were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA).

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Scientific Reports

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

doi: 10.1038/s41598-018-28438-2

Figure Lengend Snippet: Egg production by hIFN-β KI G1, G2, and G3 hens and by WT (G1) hens.

Article Snippet: For immunoblotting, egg-white proteins (3 μg per lane) and recombinant hIFN-β (40 ng per lane, Wako, Osaka, Japan) were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA).

Techniques: Knock-In

Journal: Scientific Reports

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

doi: 10.1038/s41598-018-28438-2

Figure Lengend Snippet: Production of recombinant hIFN-β in KI egg white. ( a–c ) Eggs from a hIFN- β KI and a WT hen. ( a ) Eggs from WT and KI hens. ( b , c ) The white and yolk from an egg of a WT ( b ) or KI ( c) hen. Most of the KI egg white appeared to be white and cloudy (arrow in c ). The arrowhead in ( c ) indicates a clear section of the egg white. ( d ) Production of hIFN-β in the white of eggs from a KI (#640) and WT hen as assessed by SDS-PAGE. The arrowhead indicates the position of hIFN-β. Wc, white, cloudy part of the KI egg white; C, clear part of the KI egg white. ( e ) Immunoblotting of KI egg-white proteins with anti-hIFN-β. rhIFN-β, recombinant hIFN-β (40 ng); WT, Wc, and C have the same meanings as in Fig. 4d. For each lane, 3 μg of the egg white was electrophoresed. ( f ) SDS-PAGE showing similar production levels of hIFN-β in the white of eggs laid by the four KI hens (#771, #773, #779, #642). The two parental G0 KI roosters (#411, #412) of the hens are identified in the panel. ( g, h ) Concentration of recombinant hIFN-β in KI egg white from G1, G2, and G3 KI hens. Solutions of the cloudy part of the KI egg white from four G1 hens ( g ), three G2 hens, and two G3 hens ( h ) were subjected to ELISA for the presence of recombinant hIFN-β. Bars show the mean ± standard deviation of the concentration of recombinant hIFN-β in each KI egg white. Four (G1) and three (G2 and G3) eggs from individual KI hens were subjected to ELISA.

Article Snippet: For immunoblotting, egg-white proteins (3 μg per lane) and recombinant hIFN-β (40 ng per lane, Wako, Osaka, Japan) were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA).

Techniques: Recombinant, SDS Page, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Scientific Reports

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

doi: 10.1038/s41598-018-28438-2

Figure Lengend Snippet: Detection of bioactive hIFN-β in KI egg white. ( a ) HEK-Blue IFN-α/β reporter cell samples were individually stimulated with five-fold serial dilutions of egg white from three hIFN- β KI hens (#3607, #3614, #3619) and a WT hen. 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. ), 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.

Article Snippet: For immunoblotting, egg-white proteins (3 μg per lane) and recombinant hIFN-β (40 ng per lane, Wako, Osaka, Japan) were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA).

Techniques: Cell Culture, Activity Assay, Colorimetric Assay, Standard Deviation, Purification, Recombinant, Concentration Assay, Reporter Assay, Enzyme-linked Immunosorbent Assay