Yong Yu

Abstract

The mifepristone-regulated transcriptional activation system provides a means to exogenously control gene expression in transformed mammalian cells in a temporal and spatial fashion. Such a promoter activation system consists of three components: The first of these is a steroidal inducer drug, mifepristone. This drug binds to the second component, a chimeric transcription factor complex, consisting of the mutant human progesterone receptor fused to the yeast GAL4 DNA-binding domain and the Herpes simplex virus protein VP 16 activation domain. The third component is a synthetic promoter, consisting of a series of GAL4 recognition sequences upstream of the adenovirus major late E1B TATA box, linked to a Renilla luciferase - Aequoreagreen fluorescent protein (GFP) fusion gene (ruc-gfp). The transcription of the promoter-marker gene cassette is activated by the mifepristone-bound chimeric transcription factor complex. The mifepristone-activated gene expression of luciferase and GFP was monitored by a low light video camera and a UV microscope respectively. Using this activation system in transiently transfected cells, we observed a 10-25 fold mifepristone-dose-dependent activation of both luciferase and GFP expression which lasted up to 38 hours after the initial addition of the inducer drug, but no induction in cells not treated by mifepristone. Further, to facilitate the development of the mifepristone-induced transcriptional activation system in cellular implants for future in vivo studies, we used biocompatible calcium alginate/poly-Llysine (PLL) beads to encapsulate cells, which may provide immune protection for cells during prolonged cellular transplantation. Using GFP as a marker, we monitored the growth and proliferation of encapsulated cells in culture dishes over a long period of time, up to two and a half months. We also showed that the ruc-gfp fusion gene expression in encapsulated mammalian cells could be regulated in a mifepristone dose-dependent manner, with induction kinetics similar to those in nonencapsulated cells in culture. From these results, we conclude that the mifepristoneregulated ruc-gfp fusion gene expression system may facilitate studies answering fundamental biological and therapeutic questions experimentally. In addition, realtime monitoring of regulated gene expression from alginate-encapsulated cells may provide a useful tool for designing cellular implantation-based protein therapy for clinical applications.

LLU Discipline

Anatomy

Department

Anatomy

School

Graduate School

First Advisor

Aladar A. Szalay

Second Advisor

E. Clifford Herrmann

Third Advisor

Robert L. Schultz

Fourth Advisor

Charles Slattery

Fifth Advisor

Kenneth Wright

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

2002

Date (Title Page)

6-2002

Language

English

Library of Congress/MESH Subject Headings

Gene expression; Cell Differentiation; Gene Therapy; Gene Expression Regulation; Cell Fusion.

Type

Dissertation

Page Count

xiv; 168

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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