Characterization of E2F4 in human embryonic stem cells

• Main Author: Lim, Brenda Jun Fang
• Other Authors: Norris Ray Dunn
• Format: Final Year Project
• Language: English
• Published: Nanyang Technological University 2021
• Subjects: Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/153266
• Institution: Nanyang Technological University

E2F4 is a member of the E2F transcriptional family, which is known to play a part in the regulation of cellular proliferation and differentiation. However, there has not been any studies done on E2F4 and its effects on human embryonic stem cells (hESCs). Hence, this thesis aims to generate an E2F4 KO H9 hESC cell line to investigate the role of E2F4 in the context of pluripotency, proliferation, and differentiation. Results have shown that we have successfully generated the E2F4 KO hESC cell line through CRISPR/Cas9, validated by western blot and quantitative polymerase chain reaction (qPCR). Although the KO cells showed reduced proliferation rates compared to the WT cells and expressed lower levels of pluripotency markers, this did not have a significant impact on their ability to differentiate into definitive endoderm, which is similar to observations made by another group in mouse embryonic stem cells (mESCs). Future works should involve cell cycle progression analysis, cell death analysis via Trypan blue as well differentiation into other germ layer lineages to compare between hESCs and mESCs and obtain a more comprehensive understanding of the role of E2F4 in hESCs.