2018 Fiscal Year Research-status Report
The role of ROGDI during enamel formation
| Project/Area Number |
18K17261
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| Research Institution | The University of Tokushima |
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Principal Investigator |
MITSUI S.NAOMI 徳島大学, 大学院医歯薬学研究部(歯学域), 助教 (70786152)
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| Project Period (FY) |
2018-04-01 – 2020-03-31
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| Keywords | Amelogenesis imperfecta / Genome Editing / Tooth development |
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| Outline of Annual Research Achievements |
Kohlschutter-Tonz syndrome (KTS) is an autosomal-recessive disorder characterized by epilepsy, psychomotor regression and amelogenesis imperfecta. Although it has been reported that KTS is caused by mutation in the ROGDI, the role of this gene during amelogenesis is poorly understood. The present study is focused in elucidate the role of ROGDI during enamel formation through forward genetics using newly created disease animal model.
In this fiscal year, Rogdi deficient mice created using genome editing technologies were analyzed. Rogdi deficient mice showed chalky white appearance with partial loss of enamel in the occluding surface. Micro-CT and SEM analysis revealed that although enamel thickness was not altered, enamel mineral density was significantly decreased and the enamel structure was altered compared to wild-type mice. qPCR results revealed no significant difference in the expression of the enamel proteins and proteinases between wild-type and Rogdi deficient mice. However, calcein labeling of the Rogdi deficient mice failed to show the bands of smooth ended ameloblasts, observed in wild-type mice.
The obtained results suggest that Rogdi deficiency cause hypocalcified type of amelogenesis imperfecta as a consequence of disruption in ameloblasts modulation during enamel maturation.
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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
Due to the absence of prior publication regarding Rogdi knockout mice, a disease animal model was created targeting Rogdi using CRISPR/Cas system in mice. Rogdi mutations in humans cause epilepsy and other neurological phenotypes. Howerver, the breeding went smoothly and althoght unknown dead was verified in few Rogdi deficient mice, most of the mice lived long enough for analysis. Comprehensive analysis of gene expression has been started, and the analysis scheduled for the current fiscal year has been almost completed.
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| Strategy for Future Research Activity |
The results obtained suggest that Rogdi defficiency cause disruption in the ameloblast modulation durin enamel maturation. However, the clear mechanism remains unknown. Therefore, from this fiscal year, analiysis of gene expression pattern in ameloblasts of Rogdi mutant mice by RNA sequencing will be performed in ortder to elucidate the molecular mechanism involved in the amelogesis imperfecta caused by deficiency of Rogdi. Based in the results obtained in RNA sequencing, down or up-regulated genes will be target using CRISPR/Cas system to obtain mice to further verify the results in vivo.
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| Causes of Carryover |
Although the purchase of a color camera for stereo microscope was planned, it was not required because the experiment was performed and the images were taken without any incoviences in the joint-use university facilities. Therefore, the incurring amout to be used next ficscal year is expected to be used in the purchase of a laptop for the analysis of the obtained data and reagents required for the experiments, and to cover travel expenses.
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Research Products
(2 results)
