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.
Aladar A. Szalay
E. Clifford Herrmann
Robert L. Schultz
Doctor of Philosophy (PhD)
Year Degree Awarded
Date (Title Page)
Library of Congress/MESH Subject Headings
Gene expression; Cell Differentiation; Gene Therapy; Gene Expression Regulation; Cell Fusion.
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Yu, Yong, "Inducible Gene Expression in Cell Cultures and in Microencapsulated Cells" (2002). Loma Linda University Electronic Theses, Dissertations & Projects. 828.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives