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Advances in understanding the regulation of acvrl1 gene expression

Tran, Thanhlong (2019) Advances in understanding the regulation of acvrl1 gene expression. Master's Thesis, University of Pittsburgh. (Unpublished)

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Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease characterized by abnormal vascular structures known as telangiectases or arteriovenous malformations (AVMs). AVMs are direct connections between arteries and veins that may present as a tangle of abnormal, rupture-prone blood vessels, which can lead to hemorrhages, stroke, or high-output heart failure. HHT may be caused by haploinsufficiency due to the reduced levels of acvrl1(ALK1) or endoglin (ENG) on the endothelial cell surface, and it is well established that ALK1 signaling enhances ENG expression. Therefore, enhancing ALK1 gene expression is a reasonable approach to the development of targeted therapeutics for HHT patients. In order to understand how the alk1 gene is regulated, we generated a transgenic zebrafish model, Tg(alk1e5:egfp)pt517 , in which a 1910-bp DNA fragment from zebrafish alk1 intron 1 in conjunction with a basal promoter drives EGFP expression. In this model, egfp expression is similar to endogenous alk1, with blood flow-dependent expression in select arteries, suggesting the presence of important regulatory elements within this intronic fragment. However, because this transgene inserted near an endothelial gene, the goal of my thesis work was to demonstrate a requirement for the alk1 intron 1 element in driving the observed pattern of EGFP. To this end, I applied a genome editing technique, clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9, to delete this intronic element from Tg(alk1e5:egfp)pt517. Using paired single guide RNAs, I successfully generated large deletions at the targeted site. To date, the largest deletion detected in F1 offspring of P0 founders was 1243 bp, eliminating the 5’ end of the alk1 intron 1 fragment. Current efforts are underway to characterize effects of deletions on the spatiotemporal pattern of EGFP expression. If complete deletion of this alk1 intron 1 element eliminates arterial-specific and/or flow responsive transgene expression, future goals will be to identify specific cis-acting elements within this intron fragment that are responsible for this unique expression pattern. This work has public health significance because it is focused on validating a new model for understanding ALK1 regulation, which may aid in development of targeted therapeutics for patients suffering from HHT, an understudied rare disease.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Tran, Thanhlongtgt6@pitt.edutgt6
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairRoman, Bethromanb@pitt.eduromanb
Committee MemberDemirci, Yesimfyd1@pitt.edufyd1
Committee MemberRebeiz, Markrebeiz@pitt.edurebeiz
Date: 24 July 2019
Date Type: Publication
Defense Date: 4 June 2019
Approval Date: 24 July 2019
Submission Date: 21 June 2019
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 45
Institution: University of Pittsburgh
Schools and Programs: School of Public Health > Human Genetics
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: alk1 HHT Crispr/CAS9 zebrafish gene regulation
Date Deposited: 24 Jul 2019 16:42
Last Modified: 01 Jul 2021 05:15


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