title: Expression of recombinant human factor VIII in milk of several generations of transgenic rabbits.
abstract: Transgenic founder rabbits carrying a gene construct consisting of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb of the human clotting factor VIII (hFVIII) cDNA and 4.6 kb of 3' flanking sequences of mWAP gene were crossed for three generations. All transgenic animals showed stable transgene transmission. Transgenic females showed high level of recombinant hFVIII (rhFVIII) mRNA expression in biopsed mammary gland tissues, while marginal expression of rhFVIII mRNA was observed in the spleen, lung and brain. No adverse effects of ectopic expression on the physiology of the rabbits were observed. Expression was not detected in the liver, kidney, heart and skeletal muscle. In transgenic females derived from three generations, rhFVIII protein was secreted from the mammary gland of lactating females, as shown by Western blotting. Biological activity of rhFVIII protein, as revealed in clotting assays was ranged from 0.012 to 0.599 IU/ml corresponding to 1.2% and 59.9% of the hFVIII level in normal human plasma. No apparent effect of secreted rhFVIII on the milk performance of rabbits was observed. Our results confirm the possibility of producing a significant amount of a biologically active rhFVIII in the mammary gland of established transgenic rabbit lines.
authors.affiliation: Slovak Agricultural Research Centre, Nitra, Slovak Republic. chrenekp@yahoo.com
authors.forename: Peter
authors.initials: P
authors.lastname: Chrenek
authors.forename: Lubos
authors.initials: L
authors.lastname: Ryban
authors.forename: Helga
authors.initials: H
authors.lastname: Vetr
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Makarevich
authors.forename: Pavel
authors.initials: P
authors.lastname: Uhrin
authors.forename: Rekha K
authors.initials: RK
authors.lastname: Paleyanda
authors.forename: Bernd R
authors.initials: BR
authors.lastname: Binder
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D005169:Factor VIII / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics; D005260:Female; D006801:Humans; D007774:Lactation; D008321:Mammary Glands, Animal / Q000378:metabolism; D051379:Mice; D008892:Milk / Q000737:chemistry* / Q000378:metabolism; D008894:Milk Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution; D019076:Transgenes
chemical_list: D008894:Milk Proteins; D012333:RNA, Messenger; C024471:whey acidic proteins; C078147:F8 protein, human; D005169:Factor VIII