Title page for ETD etd-72398-115020


Type of Document Master's Thesis
Author McCourt, Shannon M.
Author's Email Address smccourt@vt.edu
URN etd-72398-115020
Title Lactogenesis Induction in Transgenic Virgin Pigs as a Model for Identifying Transgene Expression and Recombinant Protein Production
Degree Master of Science
Department Animal and Poultry Sciences
Advisory Committee
Advisor Name Title
Knight, James W. Committee Chair
Akers, Robert Michael Committee Member
Gwazdauskas, Francis C. Committee Member
Velander, William H. Committee Member
Keywords
  • Transgenic
  • Lactogenesis
  • Mammary Gland
  • Swine
Date of Defense 1998-07-07
Availability restricted
Abstract
The porcine mammary gland can be used for the production of recombinant proteins by directing a transgene to the mammary gland with a milk protein gene promoter. In order to determine whether or not the protein will be expressed, the animals must be maintained at least through their first lactation. An experiment was performed to determine if hormonal induction of lactogenesis in transgenic virgin pigs could be used as a method for identifying those gilts that are likely to express the recombinant protein during a natural lactation. Mammary development and lactogenesis were induced by administration of subcutaneous implants designed to release 7.1 mg of estradiol-17 beta and 18 mg of progesterone daily for 21 d. Histological analysis of tissue samples before and after the treatment period indicated that mammary secretory tissue underwent dramatic proliferation resulting in a greater degree of alveolar and individual epithelial cell differentiation. The presence of beta-lactoglobulin mRNA was detected in high levels in post-implant tissue samples, and minimally detected in samples cultured in media supplemented with insulin, hydrocortisone, and prolactin. However, protein expression was only detected in the post-implant samples, indicating that beta-lactoglobulin was not maintained well by in vitro culture. The transgene mRNA, recombinant human fibrinogen (A-alpha chain), was detected in all analyzed samples at varying levels. However, the corresponding protein was not detected in any sample, under either reduced or nonreduced conditions. These results indicate that lactogenesis was successfully induced using the hormonal implants. Also, the transgene was activated by the hormonal induction in vivo and in vitro, but the corresponding protein could not be detected. This study indicates that induction of lactogenesis can be used to detect the presence of transgene mRNA in mammary tissue of gilts. However, we cannot conclusively demonstrate that this procedure can be used to identify those gilts that are likely to express the recombinant protein during a natural lactation.
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