2017 Fiscal Year Research-status Report
Identification of novel testis-specific long noncoding RNAs: a new molecular basis of infertility
| Project/Area Number |
17K16818
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| Research Institution | Nippon Medical School |
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Principal Investigator |
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| Project Period (FY) |
2017-04-01 – 2019-03-31
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| Keywords | testis-specific lncRNA / mouse spermatogenesis / in situ hybridization |
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| Outline of Annual Research Achievements |
Long non-protein-coding RNAs (lncRNAs) play as key regulators in gene expression. However, there is little report about the functions of testis-specific lncRNAs. The purpose of my research is to discover the testis-specific lncRNAs and their possible functions in mouse spermatogenesis, which is essential for reproduction. First, bioinformatics analysis was performed to detect gene expression level and I found some lncRNAs were highly expressed in adult mouse testis. I selected the two candidates of testis-specific lncRNAs and performed in situ hybridization to observe the subcellular localization. As results, these two testis-specific lncRNAs were expressed in mouse spermatogenesis process, suggesting that these data is useful to investigate the functional studies of mouse spermatogenesis.
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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
Research is progressing because I finished the first half of my research plan i.e. identification of testis-specific lncRNAs. Bioinformatics analysis showed that most of the lncRNAs were highly expressed in mouse testis. Among them, I selected 1700108J01Rik and 17001011O22Rik as highly potential candidates that are confirmed by Real-time PCR analysis. ISH analysis showed that these lncRNAs were expressed in testicular germ cells during spermatogenesis. Recently, I am trying to examine functional analysis of the newly found testis-specific lncRNAs. For in vivo specific gene analysis, I am practicing of microinjection of mouse testis. For in vitro specific gene analysis, I am preparing for siRNAs to detect the loss of function, reporter vectors, and transfecting cells etc.
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| Strategy for Future Research Activity |
Functional analysis of identified testis-specific lncRNAs by in vivo and in vitro specific gene manipulation are going to be performed.
For in vivo analysis, I will prepare the DsRed vector-mediated testis-specific lncRNAs induced cells. Then, I will design siRNAs and generate GFP-tagged retroviral vector to knockdown of specific lncRNAs. After retroviral introduction in testis-specific lncRNAs induced cells, I will perform gene expression analysis.
For in vitro analysis, I will prepare required apparatus and perform microinjection testis: injection of viral mediated GFP-tagged siRNA into the efferent duct of mouse testis so that to enter into the seminiferous tubule for knockdown of testis-specific lncRNAs. Then, I will perform morphological analysis of adult mouse testis.
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