2022 Fiscal Year Research-status Report
Functional elucidation of m6A mRNA modification in spatial map formation in hippocampal CA1 pyramidal cells
| Project/Area Number |
22K20702
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
Phasuk Sarayut 国立研究開発法人理化学研究所, 生命機能科学研究センター, 特別研究員 (70961580)
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| Project Period (FY) |
2022-08-31 – 2024-03-31
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| Keywords | m6A / YTHDF3 / YTHDF1 / Place cells / Calcium Imaging / Two photon microscopy / Spatial memory / Virtual reality |
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| Outline of Annual Research Achievements |
We successfully generated conditional knockout (cKO) mouse models in which a m6A reader protein, YTHDF1 or YTHDF3 is deleted from the excitatory neurons. These mice are able to perform the task as shown by increasing running period during the habituation to non-virtual linear track task. We also found increase in running speed, licking number and pre licking behavior during the training sessions. Adeno-associated virus (AAV) containing GCaMP6f, a green calcium fluorescence sensor was introduced into hippocampal CA1 prior to brain imaging of mice performing VR task. This allowed us to observe the neuronal activity in hippocampal CA1 of the cKO mice while performing VR task.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
The 2-photon microcopy and virtual reality system has been tested and is optimized. We can also generate enough mice with AAV injection for each batch. Although the production of AAV viruses takes time at first since there is no set up in the lab. I have been optimized the protocols from plasmid construction till viral production. And we can now produce AAV viruses in our lab.
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| Strategy for Future Research Activity |
1. I will increase sample size of VR task for head-fixed animals combined with 2-photon in vivo calcium imaging
2. Data analysis for behaviors and neuronal activity.
3. I am currently producing AAV virus containing either YTHDF1, 2, 3 or iCre under CaMKIIa promoter.
4. I will start injection of AAV virus carrying YTHDFs under CaMKIIa promotor into cKO mice for rescued function study.
5. Mice will be injected with AAV virus containing iCre under CaMKIIa promotor which is made in our lab for region specific functional study of YTHDFs on place cell activity and spatial learning and memory.
6. Gathering all results and discussing with the team and deciding journal for publishing this project.
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| Causes of Carryover |
We have planned to order reagents for AAV production. So, we want to keep this leftover for the next fiscal year.
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