Validation of candidate genes involved in X chromosome inactivation

• Main Author: Lim, Zhong Ri
• Other Authors: Zhang Li Feng
• Format: Final Year Project
• Language: English
• Published: 2015
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/63613
• Institution: Nanyang Technological University

One of the two female X chromosomes is silenced in mammals to compensate for X-linked gene dosage. For over half a century, this process, known as X chromosome inactivation (XCI), has been studied intensively and Xist was found to have a central role in it. Although Xist RNA is critical for establishing XCI during early embryonic development, it plays a dispensable role in maintaining XCI in soma. In order to discover unknown factors involved in XCI, our lab carried out a genome-wide shRNA library screen. In this study, three candidate genes, M3, Kdm5c and MUL1, were validated individually. Using CRISPR/Cas9 system, gene knockout was carried out on AV3.1.12 cell line, which is a male mouse ES cell line carrying an inducible Xist transgene along its single male X chromosome. Inducible Xist expression causes severe cell death of AV3.1.12 cells due to ectopic XCI. As a result, knocking out a gene involved in XCI would rescue the cells. While knocking out Kdm5c and MUL1 did not rescue the cells, knocking out M3 showed a significant rescuing effect on AV3.1.12 cells. Consequently, the role of M3 in XCI is being further confirmed and investigated.