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|
| Type |
Poster Presentation |
| Area |
Life Chemistry |
| Room No. |
Event Hall |
| Time |
4월 20일 (금요일) 11:00~12:30 |
| Code |
BIO.P-267 |
| Subject |
Transcriptional regulation of endogenous polyubiquitin genes using a modified CRISPR-Cas9 system |
| Authors |
Seung-Woo Han, Byung-Kwon Jung, Kwon-Yul Ryu*
Department of Life Science, University of Seoul, Korea
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| Abstract |
Ubiquitin (Ub) is a small globular protein which is highly conserved in eukaryotes and involved in posttranslational modification of other proteins. In mammals, Ub is encoded by two monoubiquitin genes (Uba52 and Uba80) and two polyubiquitin genes (Ubb and Ubc). Expression of these genes produces freely available cellular Ub pools. We have generated and studied diverse effects of Ubb or Ubc knockout (KO) in mice, and consistently demonstrated the negative outcomes of disrupted Ub homeostasis: the neurodegenerative phenotypes in Ubb KO mice and the developmental defects of fetal liver cells in Ubc KO embryos. However, it is currently unknown whether the positive outcomes could be induced by upregulation of endogenous polyubiquitin genes. To investigate this, we introduced CRISPR-Cas9 system which was originated from the bacterial immune response and can also be used to upregulate specific gene expression when its nuclease activity is removed (dCas9) and it is fused with the transcriptional activator domain VP64. By using dCas9-VP128 (2xVP64) and Ubb+/-(eGFP) or Ubc+/-(eGFP) mouse embryonic fibroblasts (MEFs) which express enhanced green fluorescence protein (eGFP) endogenously via Ubb or Ubc promotor, we identified the guide RNA targeting sequences in the promoter region of Ubb or Ubc through monitoring of the fluorescence increase by flow cytometry analysis. We then generated cell lines in which Ubb or Ubc is consistently upregulated and tested whether they acquired increased stress tolerance under oxidative or proteotoxic stress conditions. |
| E-mail |
eipot@naver.com |
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121st General Meeting of the KCS