Production of human erythropoietin by chimeric chickens

Kodama, Daisuke; Nishimiya, Daisuke; Iwata, Ken; Yamaguchi, Kyoji; Yoshida, Kazuhiro; Kawabe, Yoshinori; Motono, Makoto; Watanabe, Hiroyuki; Yamashita, Takashi; Nishijima, Ken ichi; Kamihira, Masamichi; Iijima, Shinji

doi:10.1016/j.bbrc.2008.01.020

Cited by 42 publications

(41 citation statements)

References 25 publications

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“…A variety of methods for generating genetically manipulated (GM) avians have been reported (Love et al 1994;Mizuarai et al 2001;Harvey et al 2002;Mozdziak et al 2003;McGrew et al 2004;Kamihira et al 2005;Zhu et al 2005; Van de Lavoir et al 2006;Lillico et al 2007). Our group has revealed the feasibility of using GM avians for the production of various recombinant proteins, such as a single-chain antibody fragment fused with the Fc region of human immunoglobulin G (Kamihira et al 2005;Kawabe et al 2006a), chimeric monoclonal antibodies (Kamihira et al 2009), Fc-fused extracellular domain of tumor necrosis factor receptor 2 (Kyogoku et al 2008) and human erythropoietin (hEpo; Kodama et al 2008).…”

Section: Introductionmentioning

confidence: 98%

Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens

et al. 2009

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We previously reported the production of human erythropoietin (hEpo) using genetically manipulated (GM) chickens. The recombinant hEpo was produced in the serum and egg white of the GM chickens, and the oligosaccharide chain structures of the serum-derived hEpo were more favorable than those of the egg white-derived hEpo. In the present study, a retroviral vector encoding an expression cassette for a fusion protein of hEpo and the Fc region of human immunoglobulin G (hEpo/Fc) was injected into developing chicken embryos, with the aim of recovering the serum-derived hEpo from egg yolk through the yolk accumulation mechanism of maternal antibodies. The GM chickens that hatched stably produced the hEpo/Fc fusion protein not only in their serum and egg white, but also in the egg yolk as expected. Lectin blot analyses revealed that significant amounts of the oligosaccharide chains of hEpo/Fc produced in the serum and eggs of GM chickens terminated with galactose, and that the oligosaccharide chains of the serum- and yolk-derived hEpo/Fc incorporated sialic acid residues. Moreover, biological activity assessment using Epo-dependent cells revealed that the yolk-derived hEpo/Fc exhibited a comparable performance to the serum- and CHO-derived hEpo/Fc. These results indicate that transport of Fc fusion proteins from the blood circulation to the yolk in chickens represents an effective strategy for the production of pharmaceutical glycoproteins using transgenic chicken bioreactors.

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Section: Introductionmentioning

confidence: 98%

Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens

et al. 2009

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“…Materials Genomic DNA extracted from magnum and heart from a chimeric chicken containing the hEpo gene 1) or from blood from a transgenic chicken containing green fluorescent protein (GFP) gene 13) was kindly provided by Dr. Nishijima. Six samples of raw chicken meat (breast, sasami [white chicken meat], liver, leg, wing, and minced) and six samples of processed foods containing chicken meat (fried chicken, oyakodon [a bowl of rice topped with boiled chicken and eggs], yakitori with liver, chicken cutlet, teriyaki chicken, and chicken curry) as ingredients were purchased at a market in Tokyo.…”

Section: Methodsmentioning

confidence: 99%

“…Literature on the development of GM chickens expressing the human erythropoietin (hEpo) gene has been reported in Japan 1,2) and Korea. 3) According to these publications, the recombinant hEpo produced in GM chickens was biologically active when examined in vitro.…”

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confidence: 99%

Method of Detecting Genetically Modified Chicken Containing Human Erythropoietin Gene

Nakajima

Nakamura

Kondo

et al. 2013

Biological & Pharmaceutical Bulletin

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Genetically modified (GM) chickens carrying the human erythropoietin (hEpo) gene have been developed to produce recombinant hEpo protein in eggs. However, such animals have not been approved as food sources in Japan. We developed a method for detecting the hEpo gene in chicken meat using a real-time polymerase chain reaction (real-time PCR). The hEpo gene was clearly detected in genomic DNA extracted from magnum and heart of a chimeric chicken containing the hEpo gene. A plasmid containing the hEpo gene was used as a standard reference molecule as well. The results clearly showed that our method was capable of detecting the hEpo gene contained in the plasmid in the presence of genomic DNA extracted from a raw chicken meat sample. We successfully used this method to test six samples of raw chicken meat and six samples of chicken meat in processed foods. This method will be useful for monitoring chicken meat that might have originated from GM chickens carrying the hEpo gene to assure food safety.

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“…To remove N-glycan, 500 ng TNFR/Fc was treated with 0.24 U PNGase in a 50 mM phosphate buffer (pH 7.5) supplemented with 0.72 % (v/v) Triton X-100 at 37°C for 20 h. An aliquot of the reaction mixture was further treated with 0.5 mU neuraminidase. Samples were analyzed by lectin blot as described previously (Kodama et al 2008;Mizutani et al 2012). In brief, samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to a polyvinylidene difluoride membrane.…”

Section: Detection Of Sialic Acid In Tnfr/fc By Lectin Blotmentioning

confidence: 99%

“…This is in contrast to glycans in mammalian species other than humans: N-glycolylneuraminic acid alone or both Nglycolyl-and N-acetylneuraminic acids have been added as the terminal residue of N-glycans. However, exogenously expressed antibodies and erythropoietin in the egg white of genetically manipulated chickens were shown to mainly possess the truncated forms of N-glycans without galactose and sialic acid (Kamihira et al 2009;Kodama et al 2008;Zhu et al 2005), which was partly improved by the genetic introduction of the galactosyltransferase gene (Mizutani et al 2012).…”

Section: Introductionmentioning

confidence: 99%

Recombinant proteins produced into yolk of genetically manipulated chickens are partly sialylated in N-glycan

et al. 2013

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The transgenic chicken is a candidate for the production of biopharmaceutical proteins with several economic superiorities. In general, the addition of sialic acid at the terminal of N-glycan is important for the bioactivity of biopharmaceuticals including plasma half-life; however, sialic acid has not been detected in the N-glycan of proteins produced in the egg white of genetically manipulated chickens. In this study, the extracellular domain of the TNF receptor and single chain Fv fused to Fc (referred to as TNFR/Fc and scFv/Fc, respectively) were purified from the egg yolk of genetically manipulated chickens and their sialylation in N-glycan was examined. In contrast to the glycan in egg white, yolk-derived proteins were partly sialylated. Lectin blot showed the existence of a2,6-sialic acid on TNFR/Fc, which disappeared with the removal of N-glycan by PNGase. In scFv/Fc, up to 7 % of N-glycan contained sialic acid. Disialyl glycans, which were detected in serumderived scFv/Fc in a previous study, were not found in the yolk sample. Ovarian follicular tissue, which surrounds growing yolk, expressed several neuraminidases, suggesting the partial truncation of glycan during the yolk transfer process from the blood.

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Production of human erythropoietin by chimeric chickens

Cited by 42 publications

References 25 publications

Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens

Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens

Method of Detecting Genetically Modified Chicken Containing Human Erythropoietin Gene

Recombinant proteins produced into yolk of genetically manipulated chickens are partly sialylated in N-glycan

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