東華大學圖書館 |

語系: 繁體中文

說明(常見問題)

回圖書館首頁

手機版館藏查詢

登入

回首頁

切換: 標籤 | MARC模式 | ISBD

Engineering Open Chromatin with Synt...

Barrett, Cassandra M.

FindBook

Google Book

Amazon

博客來

Engineering Open Chromatin with Synthetic Pioneer Factors: Enhancing Mammalian Transgene Expression and Improving Cas9-Mediated Genome Editing in Closed Chromatin.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Engineering Open Chromatin with Synthetic Pioneer Factors: Enhancing Mammalian Transgene Expression and Improving Cas9-Mediated Genome Editing in Closed Chromatin./
作者:	Barrett, Cassandra M.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
面頁冊數:	184 p.
附註:	Source: Dissertations Abstracts International, Volume: 80-11, Section: B.
Contained By:	Dissertations Abstracts International80-11B.
標題:	Molecular biology. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13862175
ISBN:	9781392139936

Engineering Open Chromatin with Synthetic Pioneer Factors: Enhancing Mammalian Transgene Expression and Improving Cas9-Mediated Genome Editing in Closed Chromatin.
Barrett, Cassandra M.

Engineering Open Chromatin with Synthetic Pioneer Factors: Enhancing Mammalian Transgene Expression and Improving Cas9-Mediated Genome Editing in Closed Chromatin. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 184 p.

Source: Dissertations Abstracts International, Volume: 80-11, Section: B.

Thesis (Ph.D.)--Arizona State University, 2019.

This item must not be sold to any third party vendors.

Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome editing efficiency by CRISPR/Cas9 systems. Many groups have attempted to de-silence chromatin to regulate genes and enhance DNA's accessibility to nucleases, but inconsistent results leave outstanding questions. Here, I test different types of activators, to analyze changes in chromatin features that result for chromatin opening, and to identify the critical biochemical features that support artificially generated open, transcriptionally active chromatin.I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions.SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug.Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells.

ISBN: 9781392139936Subjects--Topical Terms:

517296
Molecular biology.

Engineering Open Chromatin with Synthetic Pioneer Factors: Enhancing Mammalian Transgene Expression and Improving Cas9-Mediated Genome Editing in Closed Chromatin.
LDR:03594nmm a2200337 4500 001 2208872
005 20191025102432.5
008 201008s2019 ||||||||||||||||| ||eng d
020 $a 9781392139936
035 $a (MiAaPQ)AAI13862175
035 $a (MiAaPQ)asu:18820
035 $a AAI13862175
040 $a MiAaPQ $c MiAaPQ
100 1 $a Barrett, Cassandra M. $3 3435934
245 1 0 $a Engineering Open Chromatin with Synthetic Pioneer Factors: Enhancing Mammalian Transgene Expression and Improving Cas9-Mediated Genome Editing in Closed Chromatin.
260 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 2019
300 $a 184 p.
500 $a Source: Dissertations Abstracts International, Volume: 80-11, Section: B.
500 $a Publisher info.: Dissertation/Thesis.
500 $a Advisor: Haynes, Karmella A.
502 $a Thesis (Ph.D.)--Arizona State University, 2019.
506 $a This item must not be sold to any third party vendors.
520 $a Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome editing efficiency by CRISPR/Cas9 systems. Many groups have attempted to de-silence chromatin to regulate genes and enhance DNA's accessibility to nucleases, but inconsistent results leave outstanding questions. Here, I test different types of activators, to analyze changes in chromatin features that result for chromatin opening, and to identify the critical biochemical features that support artificially generated open, transcriptionally active chromatin.I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions.SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug.Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells.
590 $a School code: 0010.
650 4 $a Molecular biology. $3 517296
650 4 $a Biogeochemistry. $3 545717
650 4 $a Biomedical engineering. $3 535387
690 $a 0307
690 $a 0425
690 $a 0541
710 2 $a Arizona State University. $b Biological Design. $3 3435935
773 0 $t Dissertations Abstracts International $g 80-11B.
790 $a 0010
791 $a Ph.D.
792 $a 2019
793 $a English
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13862175