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Title:Creating a versatile toolkit for transgene expression using BACS and for dissecting large-scale chromatin organization
Author(s):Zhao, Binhui
Director of Research:Belmont, Andrew S
Doctoral Committee Chair(s):Belmont, Andrew S
Doctoral Committee Member(s):Prasanth, Supriya; Stubbs, Lisa J; Freeman, Brian C; Zhao, Humin
Department / Program:Cell & Developmental Biology
Discipline:Cell and Developmental Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):transgene expression
episomes
gene amplification
human artificial chromosomes
DNA synthesis
chromatin conformation
transcription regulation
CRISPR/Cas
Abstract:In eukaryotes, the genetic material, DNA, is highly compacted with histone proteins to form chromatin in interphase nuclei. Both the higher levels of chromatin folding and the spatial organization of the chromatin, referred to as large-scale chromatin organization, have been shown to correlate with transcriptional activity. One example suggesting transcriptional regulation by large-scale chromatin organization is position effects and position effect variegations observed in transgene expressions, which, as well as other epigenetic silencing mechanisms, have been obstacles to achieving predictable and stable transgene expression. Molecular dissections of the determinants regulating large-scale chromatin organization would help to elucidate the real relationship between large-scale chromatin organization and transcriptional regulation, yet are difficult due to the complexity of the mammalian genome. Bacterial artificial chromosomes (BACs), containing 100-300 kb mammalian genomic regions have been shown to recapitulate the expression level and nuclear localization of the corresponding genomic regions, and to protect embedded reporter mini-genes from epigenetic silencing. Here in Chapter 2 we show that BACs could provide a versatile platform for achieving reproducible, stable simultaneous expression of multiple transgenes maintained either as episomes or stably integrated copies. Moreover, in Chapter 3 we show that BACs could be used as a powerful model system for dissecting mechanisms regulating large-scale chromatin organization, by demonstrating distinctive large-scale chromatin conformations formed by BAC transgene arrays and results indicating separation of large-scale chromatin compaction, nuclear localization and transcriptional activities.
Issue Date:2019-07-09
Type:Text
URI:http://hdl.handle.net/2142/105909
Rights Information:Copyright 2019 Binhui Zhao
Date Available in IDEALS:2019-11-26
Date Deposited:2019-08


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