Development of an Immortalized Bovine Uterine Epithelial Cell Line
Open Access
- Author:
- Galli, Emily Nicole
- Area of Honors:
- Animal Sciences
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Troy Ott, Thesis Supervisor
Troy Ott, Thesis Honors Advisor
Joy Lee Pate, Faculty Reader - Keywords:
- immortalized
cell line
bovine
uterine
hTERT - Abstract:
- Bovine uterine epithelial cells are of interest to scientists because they play a vital role in interacting with the conceptus during early pregnancy. In addition, their function is regulated by hormones that vary during the estrous cycle. Moreover as these cells are part of the mucosal immune system of the genital tract, they are integral first responders to immune attack. The mechanisms behind these phenomena are difficult to study in vivo. Furthermore, primary cells have a limited lifespan and require repeated isolation, which limits their usefulness. Epithelial cells that do not undergo replicative senescence could prove to be useful tools in studying the physiology of early pregnancy. Currently, there are no immortalized bovine uterine epithelial cells available. Thus the objective of this research is to generate an immortalized cell line that will serve as a useful model of the uterine epithelium. The method chosen herein utilizes a retroviral vector and the hTERT catalytic subunit of telomerase. It is hypothesized that bovine uterine epithelial cells can be isolated and immortalized using HPV E6/E7 and hTERT, while still maintaining primary cell characteristics. To address this objective we developed methods for isolating uterine epithelial cells from bovine uteri collected at slaughter. Following enzymatic digestion, endometrial epithelial sheets were isolated. These cells were passaged until pure epithelial cell populations were obtained. The epithelial cells were then infected with a modified human papilloma virus (HPV), containing E6 and E7 viral oncogenes, and then transfected with a plasmid construct, containing the human telomerase reverse transcriptase (hTERT) gene. The E6/E7 oncogenes inhibit tumor suppressor genes that induce apoptosis and the hTERT construct maintains telomere length and inhibits cellular senescence. Combined these approaches have been demonstrated to induce a stable, immortal phenotype in a number of cell lines, including epithelial cells, which are particularly difficult to immortalize. These cell lines will be used to test hypotheses related to the physiology behind establishment and maintenance of pregnancy.