Generation of an FSH Inducible Inh-[alpha]-FLAG-gg-ICER I[gamma] Ovarian Specific Gene Construct
Date of Award
Master of Science (MS)
College of Science and Mathematics
Thesis Sponsor/Dissertation Chair/Project Chair
Carlos A. Molina
Quinn C. Vega
Avian animal models have led to many essential discoveries in molecular biology and biochemistry, including the discovery of vitamins, the chemistry behind vision and the development of transgenic hens as natural bioreactors for pharmaceutically relevant proteins. Hyperovulation in domesticated hens is considered a highly desirable genetic trait, a feature that has been explored in multiple studies and remains the focus of many biotech companies. Unlocking the molecular mechanism that controls ovulation may provide a basis for development in new reproductive technologies.
Previous research done on rodents provided data and identified a single transcription factor, the Inducible cAMP Early Repressor (ICER), found to directly mediate the nuclear response to gonadotropins and trigger hyperovulation. Transgenic mice carrying tissue-specific FSH-inducible ICER construct showed two-fold ovulation rate increase following hormonal activation of the cAMP second messenger relay.
In this study, I focus on generation of a transgenic construct derived and specific for the domesticated hen, Gallus gallus. For this purpose, I amplified the 3kb promoter sequence of the Inhibin-a subunit gene. I also subcloned the avian homolog of the ICER I[gamma] isoform to the pFLAG-CMV-2 plasmid and created the FLAG-gg-ICER I[gamma] transgene construct. The DNA sequencing analysis showed successful amplification of gg-ICER I[gamma] Following transfection into 293T (Human Kidney Carcinoma cells), the Western Blot analysis identified gg-ICER I[gamma] expression under control of CMV promoter.
I am now focusing on subcloning the Inh-a-FLAG-gg-ICER I[gamma] transgenic construct onto an expression vector and perform functional analysis in cell culture. Last, my goal would be to utilize a lenti-viral delivery vector to infect fertilized Gallus gallus eggs. Using a well established signaling pathway, my hypothesis is that the extracellular signals of the Follicle Stimulating Hormone (FSH) will trigger changes in gene expression that will induce hyperovulation. This molecular mechanism would provide evidence for the complex interaction between the reproductive tract and the central nervous system.
Bogdanoski, Goce, "Generation of an FSH Inducible Inh-[alpha]-FLAG-gg-ICER I[gamma] Ovarian Specific Gene Construct" (2009). Theses, Dissertations and Culminating Projects. 1102.