Creating a versatile toolkit for transgene expression using BACS and for dissecting large-scale chromatin organization

Zhao, Binhui

Permalink

https://hdl.handle.net/2142/105909 Copy

Description

Title

Creating a versatile toolkit for transgene expression using BACS and for dissecting large-scale chromatin organization

Author(s)

Zhao, Binhui

Issue Date

2019-07-09

Director of Research (if dissertation) or Advisor (if thesis)

Belmont, Andrew S

Doctoral Committee Chair(s)

Belmont, Andrew S

Committee Member(s)

• Prasanth, Supriya
• Stubbs, Lisa J
• Freeman, Brian C
• Zhao, Humin

Department of Study

Cell & Developmental Biology

Discipline

Cell and Developmental Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(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.

Graduation Semester

2019-08

Type of Resource

text

Permalink

http://hdl.handle.net/2142/105909

Copyright and License Information

Copyright 2019 Binhui Zhao

Owning Collections