Validation of candidate genes involved in X chromosome inactivation
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 establis...
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| Format: | Final Year Project |
| Language: | English |
| Published: |
2015
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| Online Access: | http://hdl.handle.net/10356/63613 |
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| Institution: | Nanyang Technological University |
| Language: | English |
| Summary: | 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. |
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