Date of Award

5-2011

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 A. Molina

Committee Member

Ann Marie DiLorenzo

Committee Member

Kirsten Monsen

Abstract

Inducible cAMP Early Repressor (ICER) is an important regulator of folliculogenesis as it regulates the nuclear response to gonadotropins in ovarian tissue. Studies in mice demonstrated a 3.0 kb region of the alpha inhibin (INHa) promoter is a site of transcriptional activation in response to follicle-stimulating hormone (FSH). The purpose of this study was to isolate and construct an ICER transgene and characterize the putative 3.0 kb promoter region of the ovarian-specific INHa gene in Danio rerio (zebrafish).

The sequences for ICER and the INHa promoter in zebrafish were identified using BLAST searches with the known sequences from mouse and chicken. The putative sequences were subsequently isolated and amplified using RT-PCR and PCR. Successful isolation and amplification was confirmed with restriction enzyme mapping and DNA sequencing. ICER was cloned into pFLAG-CMV-2 plasmid to create the FLAG-dr- ICER Iy transgene construct. Expression of FLAG-ICER protein was confirmed by Western blot after transfection into HEK293T human kidney cells. The INHa promoter was cloned into a pGL3-Basic luciferase expression vector for characterization studies. The transgene containing the INHa promoter was transfected into AB9 zebrafish tailfm cells and embryonic PAC2 zebrafish cells. Transfection experiments in AB9 and PAC2 cells demonstrated promoter induction by follicle-stimulating hormone (FSH) and repression of promoter activity by luteinizing hormone (LH).

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