title: Differential in vitro and in vivo glycosylation of human erythropoietin expressed in adenovirally transduced mouse mammary epithelial cells.
abstract: The expression of human erythropoietin in the mammary gland is an attractive approach to diminish its current production cost. Previous attempts to produce erythropoietin in the milk of transgenic animals resulted in very low expression levels and in a detrimental effect in the health of the founder animals. Here, we show that the direct transduction of the mouse mammary gland with an adenoviral vector carrying the cDNA of erythropoietin promotes its expression in milk at a level as high as 3.5 mg/ml. The recombinant erythropoietin derived from mouse milk showed a different migration and distribution after SDS-PAGE electrophoresis as well as a low in vivo hematopoietic activity. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation compared to with its counterpart produced in CHO and HC11 cell lines. The difference between in vivo and in vitro glycosylation of human erythropoietin expressed in adenovirally transduced mammary epithelial cells suggests that key enzymes in the glycosylation pathway may be insufficient during lactation. Thus, the direct transduction of the mammary epithelium seems to be a powerful tool to express toxic proteins in milk at levels high enough for their physical, chemical and biological characterization before undertaking the generation of a transgenic mammal.
authors.affiliation: Department of Transgenesis and Animal Cloning, Animal Biotechnology Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba.
authors.forename: Jorge R
authors.initials: JR
authors.lastname: Toledo
authors.forename: Oliberto
authors.initials: O
authors.lastname: Sánchez
authors.forename: Raquel
authors.initials: R
authors.lastname: Montesino Seguí
authors.forename: Yaiza
authors.initials: Y
authors.lastname: Fernández García
authors.forename: María P
authors.initials: MP
authors.lastname: Rodríguez
authors.forename: José A
authors.initials: JA
authors.lastname: Cremata
mesh_terms: D000256:Adenoviridae; D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism*; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006031:Glycosylation; D006801:Humans; D008321:Mammary Glands, Animal / Q000166:cytology*; D051379:Mice; D008892:Milk / Q000378:metabolism*; D009844:Oligosaccharides / Q000302:isolation & purification; D011247:Pregnancy; D014161:Transduction, Genetic
chemical_list: D018076:DNA, Complementary; D009844:Oligosaccharides; D004921:Erythropoietin