Virginia Tech
Production and Secretion of Recombinant Human Fibrinogen by the Transgenic Murine Mammary Gland
Abstract
dc:description.abstractThe 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 Aα, Bβ and γ 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 Bβ and γ chains were rate limiting. Also, the subunit complexes γ₂, Aαγ₂ and the individual subunits Aα, Bβ and γ were found as secretion products. This is the first time that secretion of individual Bβ-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 Bβ-chain contained a complex saccharide with low mannose. Glycosylation of the γ-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.
Degree
thesis:*- Name thesis:degree_name
- Master of Science
- Level thesis:degree_level
- masters
- Discipline thesis:degree_discipline
- Dairy Science
- Department dc:contributor.department
- Dairy Science
- Grantor dc:publisher
- Virginia Tech
- Year dc:date.issued
- 1997
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Butler, Stephen P.
- Chair dc:contributor.committeechair
-
- Gwazdauskas, Francis C.
- Committee members dc:contributor.committeemember
-
- Velander, William H.
- Akers, Robert Michael
- Wong, Eric A.
Subjects
dc:subject × 4Rights
dc:rights- Statement dc:rights
-
- In Copyright
- Licence dc:rights.uri
Identifiers
dc:identifier.*- Dc Identifier Other
- etd-51897-141036
- OAI identifier oai:identifier
- oai:vtechworks.lib.vt.edu:10919/36776