Transgenic Research 11: 257–268, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands. 257 Temporal and spatial expression of biologically active human factor VIII in the milk of transgenic mice driven by mammary-specific bovine α-lactalbumin regulation sequences Chuan-Mu Chen 1, ∗ , Chih-Hong Wang 1 , Shinn-Chih Wu 2 , Chih-Cheng Lin 1 , Shwu-Hwa Lin 3 & Winston T. K. Cheng 4 1 Department of Zoology, National Chung Hsing University, Taichung, Taiwan, ROC 2 Pig Research Institute Taiwan, Chunan, Taiwan, ROC 3 Institute of Medical Biotechnology, National Taiwan University, Taipei, Taiwan, ROC 4 Department of Animal Science, National Taiwan University, Taipei, Taiwan, ROC Received 21 March 2001; revised 2 October 2001; accepted 21 November 2001 Key words: α-lactalbumin promoter, hemophilia A, human factor VIII, mammary gland, transgenic animal Abstract Hemophilia A is one of the major inherited bleeding disorders caused by a deficiency or abnormality in coagulation factor VIII (FVIII). Hemophiliacs have been treated with whole plasma or purified FVIII concentrates. The risk of transmitting blood-borne viruses and the cost of highly purified FVIII are the major factors that restrict prophylaxis in hemophilia therapy. One of the challenges created by the biotechnology revolution is the development of methods for the economical production of highly purified proteins in large scales. Recent developments indicate that manip- ulating milk composition using transgenesis has focused mainly on the mammary gland as a bioreactor to produce pharmaceuticals. In the present study, a hybrid gene containing bovine α-lactalbumin and human FVIII cDNA was constructed for microinjection into the pronuclei of newly fertilized mouse eggs. The αLA-hFVIII hybrid gene was confirmed to be successfully integrated and stably germ-line transmitted in 12 (seven females/five males) lines. Western-blot analysis of milk samples obtained from eight of the transgenic founders and F1 offspring indicated that the recombinant hFVIII was secreted into the milk of the transgenic mice. The concentrations of rFVIII ranged from 7.0 to 50.2 μg/ml, over 35–200-fold higher than that in normal human plasma. Up to 13.4 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma. Introduction Hemophilia is the most common human bleeding disorder and affects approximately one in 5,000 males, causing lifelong, repeated, and potentially life- threatening hemorrhagic episodes. It results from a de- ficiency in functional blood coagulation factors, either in a serine protease called Factor IX (hemophilia B) or in its co-factor Factor VIII (hemophilia A) (Tud- denham et al., 1991). Because hemophilia is caused by defects in single X-linked genes that encode circu- ∗ Author for correspondence: E-mail: chchen1@dragon.nchu.edu.tw lating plasma proteins, the development of therapeutic strategies has focused on finding ways to replace the defective or deficient proteins. Human FVIII is synthesized primarily in the liver and secreted into the circulation at low plasma con- centrations, approximately 100–200 ng/ml (Hoyer, 1987). Analysis of the cloned cDNA for human FVIII (Gitschier et al., 1984; Toole et al., 1984) has provided more information about its gene and protein structure. The full length FVIII cDNA contains an open reading frame coding for a polypeptide of 2351 amino acids. This primary translation product contains a 19 amino acid signal peptides and six homologous domains in the order A1-A2-B-A3-C1-C2 (Vehar et al., 1984).