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CONTROL OF ENDOGENOUS GENE EXPRESSION IN THE MOUSE.
by
Kwang Ho Lee
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOCHEMISTRY & MOLECULAR BIOLOGY)
May 2009
Copyright 2009 Kwang Ho Lee
Object Description
| Title | Control of endogenous gene expression in the mouse |
| Author | Lee, KwangHo |
| Author email | kwangho@usc.edu; canary119@hotmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Biochemistry & Molecular Biology |
| School | Keck School of Medicine |
| Date defended/completed | 2009-03-26 |
| Date submitted | 2009 |
| Restricted until | Unrestricted |
| Date published | 2009-05-07 |
| Advisor (committee chair) | Laird, Peter W. |
| Advisor (committee member) |
Jones, Peter A. Maxson, Robert Comai, Lucio |
| Abstract | Several techniques have been developed to probe gene function through either loss or gain of function genetics in mice, such as conventional/conditional knock-outs and transgenics. However, cell lethality has limited the successful implementation of knockout technologies for some genes. In vivo knockdown approaches relying on tet-regulated siRNA expression may be able to address this problem, but siRNA development has proved challenging for some genes, and the degree of repression by RNA interference varies. Here, we present an alternative approach to achieving control of endogenous gene expression, and apply this system to the major DNA methyltransferase, Dnmt1.; Our approach to controlling endogenous gene expression employs a novel transcriptional regulatory system involving three binary systems, lacI, tetR and gal4, which allow for the reversible up- and down-regulation of a target gene from its endogenous locus. We engineered a number of modifications into both the lac operator and repressor, which significantly improved the repression as compared to wild type (WT) lac and tet systems. We introduced lac and tet operator sequences together with gal4 DBSs (DNA binding sequence) into the endogenous Dnmt1 promoter through gene-targeting, without noticeably altering promoter activity in the absence of the trans-elements. Endogenous Dnmt1 expression was successfully up and down regulated in a reversible temporo-spatial manner in mice. This technology was potent enough to reproduce the embryonic lethal phenotype of genetic knock-out and to attenuate transcription elongation, and the lethal phenotype was rescued by IPTG treatment. This establishes the first paradigm of experimental rescue of an embryonic lethal phenotype in loss-of-function genetics, and holds great promise for genes for which in vivo characterization have been limited due to a lethal phenotype.; The present study explores the possibility of controlling eukaryotic gene expression through the institution of a physical access to the endogenous promoter, and establishing a proof of principle for the methodology with Dnmt1, a gene for which the use of conventional/conditional knock-outs and transgenic approaches have been limited due to embryonic or cell lethality. In principle, this approach should be applicable to other genes and other organisms. |
| Keyword | LacI; control; gene expression; Dnmt1; transcription; tetVP16; lac repressor |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m2205 |
| Rights | Lee, KwangHo |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-Lee-2854 |
| Archival file | uscthesesreloadpub_Volume51/etd-Lee-2854.pdf |
Description
| Title | Page 1 |
| Full text | CONTROL OF ENDOGENOUS GENE EXPRESSION IN THE MOUSE. by Kwang Ho Lee A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (BIOCHEMISTRY & MOLECULAR BIOLOGY) May 2009 Copyright 2009 Kwang Ho Lee |
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