2016 Fiscal Year Annual Research Report
| Project/Area Number |
15F15780
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
Carninci Piero 国立研究開発法人理化学研究所, ライフサイエンス技術基盤研究センター, 副センター長 (10333296)
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| Co-Investigator(Kenkyū-buntansha) |
BUDIC MARUSKA 国立研究開発法人理化学研究所, ライフサイエンス技術基盤研究センター, 外国人特別研究員
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| Project Period (FY) |
2015-11-09 – 2018-03-31
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| Keywords | SINEUP / upregulation / transcription factor / iPS / reprogramming |
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| Outline of Annual Research Achievements |
We designed SINEUPs for reprogramming human iPS cells (hiPSCs) into neuron cells. After the bioinformatic analysis of neuron differentiation time course data from the international research consortium FANTOM5 (http://fantom.gsc.riken.jp/5/), we selected five specific transcription factors (TF) namely NGN2, NGN3, OTX2, TLX3, PAX6, expressed in differentiated neurons, and engineered synthetic SINEUPs that overlap the 5’ ends of the selected TF mRNAs. We optimized the synthetic TF specific SINEUPs and tested their activity in HEK293T/17 cells. We obtained up to 100% increase in TF levels without variations of mRNA quantities.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
We performed bioinformatic analysis of neuron differentiation time course data from the international research consortium FANTOM5 (http://fantom.gsc.riken.jp/5/). We selected five specific transcription factors (TF), namely NGN2, NGN3, OTX2, TLX3, PAX6, expressed in differentiated neurons, and engineered synthetic SINEUPs that overlap the 5’ ends of the selected TF mRNAs. We designed synthetic SINEUPs for reprogramming human induced pluripotent stem cells (hiPSCs) into neuron cells, optimized them and tested their activity HEK293T/17 cells because of the cell line’s reliability and high transfection propensity. We co-transfected the cells with plasmid expressing target TF and plasmid expressing TF specific SINEUP. We harvested cells twenty-four hours after transfection and processed them for protein and RNA levels.
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| Strategy for Future Research Activity |
Efficient differentiation of hiPSCs into neurons is crucial for disease modeling, drug screening, and cell transplantation therapy in regenerative medicine. The use of long non-coding RNAs in biotechnology and therapy is still new. SINEUPs require the activity of an embedded inverted SINEB2 sequence to upregulate translation. They are the first example of gene-specific inducers of protein synthesis. Their binding domain can be engineered to specifically enhance translation of any target gene of interest. We designed and optimized the synthetic SINEUPs for reprogramming hiPSCs into neuron cells and tested their activity in HEK293T/17 cells. Lipid-mediated plasmid delivery has low efficiency in hiPSCs compared to viral delivery methods. We will construct lentiviral vectors to express TF specific SINEUPs to enhance translation of endogenous TF mRNA in iPSCs in order to reprogram them into neurons.
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