Transgenic animals secreting individual chains and assembled fibrinogen were produced to evaluate the capacity of the mammary gland for maximizing assembly, glycosylation and secretion of recombinant human fibrinogen (rhfib). Transgenes were constructed from the 4.1 kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the Aalpha, Bbeta and gamma fibrinogen chains. Transgenic mice secreted fully assembled fibrinogen into milk at concentrations between 10 and 200 microg/ml, with total secretion of subunits approaching 700 microg/ml in milk. Partially purified fibrinogen was shown to form a visible and stable clot after treatment with human thrombin and factor XIII. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the Bbeta and gamma chains were rate limiting. Both the Bbeta and gamma chains were glycosylated when co-expressed and the degree of saccharide maturation was dependent on expression level, with processing preferred for gamma chains over Bbeta chains. Also, the subunit complexes gamma2, Aalphagamma2 and the individual subunits Aalpha, Bbeta and gamma were found as secretion products. When the Bbeta was secreted individually, the glycosylation profile of the molecule was of a mature complex saccharide indicating recognition of the molecule by the glycosylation pathway without association with other fibrinogen chains. To date secretion of Bbeta chain has been not observed in any cell type, suggesting that the secretion pathway in mammary epithelia is less restrictive than that occurring in hepatocytes and other cells previously used to study fibrinogen assembly.

The mammary gland of lactating transgenic animals has several advantages for production of heterologous proteins including a high cell density that results in high concentrations of secreted protein and the ability to perform several types of post-translational modifications. Transgenes were constructed from the 4.1 kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the A", B$ and ( fibrinogen chains to evaluate the requirements of the transgenic murine mammary gland for high level secretion of fully assembled human fibrinogen. After introducing the constructs into the murine zygotes by microinjection, secretion of fully assembled fibrinogen into milk was measured at concentrations between 10 :g/ml to 200 :g/ml.In one line of mice the total secretion of fibrinogen and unassembled subunits approached 700 :g/ml in milk. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the B$ and ( chains were rate limiting. Also, the subunit complexes ( 2 , A"( 2 and the individual subunits A", B$ and ( were found as secretion products. This is the first time that secretion of individual B$-subunits by any cell type has been reported and suggests the organization of the secretion pathway in mammary epithelia is different from that in liver. Glycosylated forms of individual B$-chain contained a complex saccharide with low mannose. Glycosylation of the (-chain was also observed. These results suggest the 4.1 mWAP promoter can drive expression of fibrinogen cDNAs to high levels and that the amount of fully assembled fibrinogen secreted is equal to the level of the lowest expressing chain.iii