Expression of human blood clotting factor VIII in the mammary gland of transgenic sheep

By targeting the expression of sequences encoding non-milk proteins to the mammary gland of transgenic farm animals, the organ could serve as a 'biore actor' for producing pharmacologically active proteins on a large scale. He re we report the generation of transgenic sheep bearing a fusion gene const ruct with the human blood clotting factor VIII (hFVIII) cDNA under the tran scriptional control of a 2.2 kb fragment of the mammary gland specific prom oter of the ovine ss-Lactoglobulin (ss-Lac) gene. Six founder animals were generated bearing a hFVIII cDNA construct with the introns of the murine me tallothionein (MtI) gene (ss-Lac/hFVIII-MtI). Founders transmitted the tran sgene in a Mendelian fashion and two transgenic lines were generated. Ten o ut of 12 transgenic F1-females expressed rhFVIII mRNA in exfoliated mammary epithelial cells isolated from the milk. But only in transgenic F1 ewes 40 10 and 603 hFVIII clotting activity estimated at 4-6 ng/ml was detected in defatted milk. Furthermore, the presence of rhFVIII-protein in ovine milk w as demonstrated by a specific band at approximately 190 kD following immuno precipitation and immunoblotting. Transgenic founder 395 expressed rhFVIII mRNA in biopsied mammary gland tissue, in exfoliated mammary cells as well as ectopically in brain, heart, spleen, kidney and salivary gland, suggesti ng that the employed ss-Lac promoter fragment lacks essential sequences for directing expression exclusively to the mammary gland. A rhFVIII standard preparation (rhFVIII(std)) was rapidly sequestered in a saturable fashion i n ovine milk, thus rendering it largely inaccessible to immunoprecipitation although its biological activity was retained. Recovery of hFVIII(std) was dependent on milk donor, storage temperature and dilution of milk sample.