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Insulin-like growth factor I (IGF-I) has acute insulin-like metabolic effects and long-term anabolic actions offering a range of important therapeutic applications. To evaluate a system for large-scale production of this peptide in the mammary glands of transgenic livestock, we generated transgenic rabbits carrying fusion genes in which a synthetic DNA coding for human IGF-I (hIGF-I) was placed under the transcriptional control of regulatory elements isolated fi om the bovine alpha(S1)-casein (alpha(S1)-cas) gene. Western blot analysis of milk from alpha(S1)-cas-hIGF-I transgenic rabbits demonstrated production of high amounts of mature hIGF-I peptide (7.6 kDa). Quantitative analysis by RIA revealed hIGF-I levels between 50 and 300 mu g/ml milk. Recombinant hIGF-I purified from the milk of alpha(S1)-cas-hIGF-I transgenic rabbits bound to IGF-I receptors on human IM-9 lymphoblasts and stimulated DNA synthesis by growth-arrested MG-63 human osteosarcoma cells as efficiently as hIGF-I produced in Escherichia coli. Ligand blot analysis of milk serum revealed the presence of 45-kDa, 30-kDa, and 23-kDa IGF-binding proteins (IGFBPs). The 30-kDa IGFBP was shown to be IGFBP-2 by immunoprecipitation using an antiserum raised against human IGFBP-2. Secretion of IGFBP-2 was markedly stimulated by hIGF-I overproduction in alpha(S1)-cas-hIGF-I transgenic rabbits. The latter displayed slightly increased milk yield, but no significant changes in total protein content or overall milk protein composition, and reared their offspring without any problems or clinical signs of impaired welfare, even after multiple lactations. Our results indicate that high amounts of biologically active hIGF-I can be produced in the mammary glands of alpha(S1)-cas-hIGF-I transgenic rabbits. Local production of hIGF-I in mammary tissue is associated with increased secretion of IGFBP-2, which may prevent major biological effects: by high levels of hIGF-I on the mammary gland.