Title page for ETD etd-51897-141036


Type of Document Master's Thesis
Author Butler, Stephen P.
Author's Email Address stbutler@vt.edu
URN etd-51897-141036
Title Production and Secretion of Recombinant Human Fibrinogen by the Transgenic Murine Mammary Gland
Degree Master of Science
Department Dairy Science
Advisory Committee
Advisor Name Title
Akers, Robert Michael
Velander, William H.
Wong, Eric A.
Gwazdauskas, Francis C. Committee Chair
Keywords
  • Transgenic
  • Whey Acidic Protein
  • Mammary Gland
  • Fibrinogen
Date of Defense 1997-05-19
Availability unrestricted
Abstract
Production and Secretion of Recombinant Human Fibrinogen by the Transgenic Murine Mammary Gland

Stephen P. Butler

(ABSTRACT)

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 Alpha, Beta and gamma 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 ug/ml to 200 ug/ml. In one line of mice the total secretion of fibrinogen and unassembled subunits approached 700 ug/ml in milk. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the Beta and gammmma chains were rate limiting. Also, the subunit complexes gamma-gamma, alpha-gamma-gamma and the individual subunits alpha, Beta and gamma 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 Beta-chain contained a complex saccharide with low mannose. Glycosylation of the gamma-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.

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