Generating fluorescent reporter constructs to study cardiovascular development and disease

Khizer, Amal (2023) Generating fluorescent reporter constructs to study cardiovascular development and disease. Doctoral thesis, University of East Anglia.

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Abstract

The cardiovascular system consists of the heart and its blood vessels, and a wide array of problems can arise within the cardiovascular system. The heart consists of three layers: the epicardium (outer layer), the myocardium (middle layer) which forms the muscular walls of the heart and the endothelial layer (inner layer) which comprises the endocardium. Endocardial cells are specialised endothelial cells which form a lining on the inside of the heart. They are an essential source for several lineages of the cardiovascular system including the coronary endothelium, cardiomyocytes, fibroblasts, endocardial cushion mesenchyme, and can also undergo endothelial to mesenchymal transition, to give rise to cells that can form mature valves. The endocardium has also been seen to contribute to trabeculation and revascularisation post myocardial infarction (MI), therefore by understanding the biology of endocardial cells better, we might be able to manipulate and harness its potential regenerative capacity. However, studying endocardial cell biology has been made difficult due to a lack of adult endocardial cell lines. In previous work, our lab, defined induced pluripotent stem cell (iPSC) differentiation protocols to generate endocardial- and coronary endothelial-like cells. To further utilise these cells better, in this project we aimed to generate reporter gene constructs that could be expressed in endocardial and coronary endothelial cells (cEC) to help mark specific stages of differentiation and thus allow us to optimise our current endocardial differentiation and cEC transdifferentiation protocols, along with, helping us model how endocardial cells and their derivatives can contribute to heart regeneration.
Studies have shown genetic factors that control the development of endothelial cells residing in the endocardium are highly related but still distinct from the endothelial cells located elsewhere in the embryo. For example, CDH5 is a pan-endothelial cell marker, the expression of NPR3 is restricted to endocardial cells, and APLN is expressed in cEC. The aim of my study is to focus on NPR3, APLN, and CDH5, and generate fluorescent reporter constructs for them. Our reporter constructs were made by isolating the promoter regulatory regions of NPR3, APLN, and CDH5, and cloning these into a vector, into which I also cloned either a green fluorescent protein or mCherry (red fluorescent protein). Due to time constraints, I only managed to test the CDH5:GFP reporter in adult endothelial progenitor cells, and the cells carrying the reporter emitted fluorescence when imaged. In future work, the remaining reporters will be tested in adult cells and later be used to produce an iPSC reporter line that will stably express NPR3 in iPSC-endocardial cells (iPSC-Endocs), and APLN and CDH5 in iPSCcoronary endothelial cells, which will then be used in studies as outlined above.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Kitty Laine
Date Deposited:	27 Jun 2023 11:16
Last Modified:	27 Jun 2023 11:16
URI:	https://ueaeprints.uea.ac.uk/id/eprint/92508
DOI:

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