Correlative light and electron microscopy analysis of telomere ultrastructure in immortalised mouse embryonic fibroblasts

• Main Author: Agatha, Olivia
• Other Authors: Sara Sandin
• Format: Final Year Project
• Language: English
• Published: 2019
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/77824
• Institution: Nanyang Technological University

Telomeres, the ends of eukaryotic chromosomes, consist of TTAGGG repeats, with DNA region, as well as a complex of six proteins known as the shelterin complex. One of the components of the complex, telomeric repeat-binding factor 2 (TRF2), binds the double-stranded region of telomere and is shown to be the main factor contributing to the formation and maintenance of a structure called T-loop, which is thought to protect the ends of chromosome from DNA damage response (DDR). Many studies have been conducted to determine their structure in vitro. However, little is known of their ultrastructure in vivo. In this project, the ultrastructure of telomeres in Mouse Embryonic Fibroblasts (MEF) was analyzed. This study utilized Correlative Light and Electron Microscopy (CLEM) on cells transfected with GFP-APEX2-TRF2 construct and Electron Tomography (ET). In conclusion, telomeres in vivo appear round and those within one cell could differ in size but have similar compactness. Furthermore, a new construct of mCherry-APEX2-TRF2 was cloned due to the ability of mCherry to retain its fluorescence for a longer period inside the section than GFP.