Date of Award

1-2015

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Biology

Thesis Sponsor/Dissertation Chair/Project Chair

Carlos Molina

Committee Member

Kirsten Monsen-Collar

Committee Member

John Siekierka

Abstract

The cAMP response element modulator (CREM) regulates cAMP- responsive genes and is an important component of the cAMP mediated signal transduction in spermatogenesis. CREM has a regulatory relationship with Inducible cAMP early repressor (ICER) which acts as a tumor suppressor and is a dominant negative transcriptional repressor with gonadal specific properties. ICER is also known to moderate cAMP antiproliferative actions and has been targeted in the past for expression in the ovaries of transgenic mouse models to generate higher levels of ovulation. Earlier studies on mice lacking the CREM gene have shown severe impairment in spermatogenesis.

Enhanced Green Fluorescent Protein (EGFP) is a versatile protein often used as a biosensor to determine the expression of proteins in cells. This is accomplished by introducing EGFP along with the desired protein in a vector construct into the organism. Fluorescence microscopy can detect the presence of EGFP and thus the protein if it is successfully introduced into the genome of the organism. Previously in Dr. Molina’s laboratory, CYP19A1 a gonadal specific promoter, was injected into zebrafish eggs. CYP19A1 was chosen due to its promising results using a three fragment vector construction method where EGFP was used as a marker to monitor whether it was effectively and specifically expressed in the testes. Once the vector construction was completed, the CYP19A1: EGFP promoter construct was injected into the zebrafish eggs generating transgenic zebrafish.

The main objective of this project was to show that CYP19A1 can function as a testes specific promoter that can be used to successfully express transgenes in the testes, with the potential to affect spermatogenesis in zebrafish. Western blots, PCR reactions, and immunohistochemical stains were performed to detect over-expression of EGFP in the testis of Danio rerio. The results of the study show that CYP19A1 was successfully expressed in the testes. The implications of the study indicate that the selected promoter region of CYP19A1 can be a testes specific promoter. This promoter can then be used in future experiments for the expression of relevant genes such as ICER, in the testes. For instance, the over expression of these genes in the testes of zebrafish can affect spermatogenesis and therefore have considerable implications for future research in fertility treatments and boosting livestock production.

File Format

PDF

Download

Included in

Biology Commons

Share

COinS