Identification of new polyamine targets as stem cell regulators

• Main Author: Ng, Boon Kiat
• Other Authors: School of Biological Sciences
• Format: Final Year Project
• Language: English
• Published: 2014
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/59763
• Institution: Nanyang Technological University

Embryonic Stem Cells (ESCs) hold great therapeutic potential due to their self-renewal and pluripotent characteristics. The polyamine regulators adenosylmethionine decarboxylase 1 (AMD1) and ornithine decarboxylase 1 (ODC1) were shown to promote ESC self-renewal. In the absence of leukemia inhibitory factor (LIF), overexpression of Amd1 in ESCs could promote ESC self-renewal. Microarray was previously performed and various genes were found to be downregulated upon LIF withdrawal and rescued upon Amd1 overexpression. Nine of such genes were chosen to be investigated as potential polyamine targets that could promote ESC self-renewal. qRT-PCR primers for five candidates were successfully designed and qRT-PCR was performed to validate the microarray results. Subsequently, aquaporin 3 (Aqp3), KH domain containing 3 (Khdc3), and zinc finger matrin type 4 (Zmat4) variant 1 and 2 were chosen for cloning but the high GC content of Khdc3 impeded the cloning process. Aqp3 and Zmat4 were overexpressed and Aqp3 was knocked down in mouse ESCs. Zmat4 did not have self-positive feedback regulation. Although transfection was successful in the overexpression and knockdown studies, the ESCs did not respond to the positive controls, possibly due to cell death and cell density respectively. Therefore, the effect of Aqp3 and Zmat4 on ESC self-renewal was inconclusive.