2015 Fiscal Year Research-status Report
Identification of Genes Controlling Treg Development by Combining the CRISPR-Cas9 System and Bone Marrow Chimeric Mouse Model
| Project/Area Number |
15K15154
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| Research Institution | Osaka University |
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Principal Investigator |
Jun Huang 大阪大学, 免疫学フロンティア研究センター, 特任研究員(常勤) (00751207)
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| Project Period (FY) |
2015-04-01 – 2018-03-31
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| Keywords | Self tolerance / Autoimmunity / Epigenetic regulation / Treg development |
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| Outline of Annual Research Achievements |
We identified that a gene (thereafter called X), one component of a protein complex implicated in multiple epigenetic mechanisms, plays a crucial role in thymic regulatory T cell (tTreg) development. By performing function assays both in vitro and vivo and analyzing the mice with Treg lineage-specific X depletion, we demonstrated that the role of X in Tregs is developmentally restrictive, as X deletion in differentiated Tregs has no effects on Tregs function nor their stability. By further characterization of tTreg development in the absence of X, we demonstrated that X is crucial for thymic CDSP cells to differentiate into Treg precursors, but dispensable for later steps of tTreg development.
In order to understand the underlying mechanism of our discovery, we carried out several experiments and had gained several crucial insights: 1) X contributes to Treg development in a cell-intrinsic manner rather than through affecting other T cells, as demonstrated by bone marrow co-transfer experiment. 2) By bisulfite sequencing, we demonstrated that tTregs developed in the absence of X display enhanced Foxp3 CNS2 demethylation. Thus X does not contribute to tTreg development via promoting demethylation of Treg signature genes.3) we got several lines of evidences from global transcriptome analysis and flow cytometric analysis suggesting that X might contribute to tTreg (precursors) development through modulate T-Cell Receptor (TCR) Signaling.
Results so far set the stage for further comprehensive studies to fully elucidate mechanistic role of X during Treg development.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
We had narrowed down candidate genes for further functional interrogation and obtained CRISPR/dCas9 lentiviral activation particles targeting those genes, which are ready for bone marrow cells transduction.
The order and delivery of experimental mice were slightly delayed.
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| Strategy for Future Research Activity |
In particular, we observed that several genes significantly downregulated in the absence of X, but it is still not clear whether these genes functionally mediate the downstream effects of X. Next, we will apply lentivirus-based CRISPR/Cas9 system, including CRISPR/dCas9 lentiviral activation system which we did not envisage its application previously, to functionally interrogate the role of these genes in the process of Treg development.
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| Causes of Carryover |
The order and delivery of laboratory mice were slightly delayed.
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| Expenditure Plan for Carryover Budget |
Incurring amount will be mainly used for the purchase of laboratory mice.
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