Project/Area Number |
22K06291
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Research Category |
Grant-in-Aid for Scientific Research (C)
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Allocation Type | Multi-year Fund |
Section | 一般 |
Review Section |
Basic Section 44040:Morphology and anatomical structure-related
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Research Institution | Toho University |
Principal Investigator |
黄 國成 東邦大学, 理学部, 博士研究員 (40526901)
|
Project Period (FY) |
2022-04-01 – 2025-03-31
|
Project Status |
Granted (Fiscal Year 2022)
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Budget Amount *help |
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2024: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2023: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2022: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
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Keywords | osmoregulation / IGFBP5 / medaka / chum salmon / ISH chain reaction / ionocyte / 成長ホルモン / 体液調節 / IGF結合タンパク質 / 塩類細胞 |
Outline of Research at the Start |
これまでの研究により、IGF結合タンパク質5a(IGFBP5a)が鰓の塩類細胞においてGH/IGFシグナルを仲介し、海水適応を可能にすることが示唆された。本研究は、IGFBP5a-KOメダカを用いて海水移行ならびにIGF受容体阻害を行うことにより、生存率、体液バランス、塩類細胞など浸透圧調節器官への影響を調べ、IGFBP5aの役割を解明する。また、自然界の海水適応時に起こるサケのスモルト化におけるIGFBP5の関与も調べる。遺伝子操作(KOメダカ)と自然現象(サケのスモルト化)双方の知見を俯瞰することで、GH/IGF/IGFBP5aの役割を明らかにする。
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Outline of Annual Research Achievements |
I have made IGFBP5a knockout (KO) medaka using CRISPR-Cas9 system. I have introduced a 5-nucleotide deletion in the IGFBP5a gene in drR medaka and established a heterologous KO line. With crossing between the heterologous line, it is in the process to breed homologous IGFBP5a KO medaka line. The salinity tolerance and transporter expression in the ionocytes in heterologous KO medaka was examined and no apparent difference was observed compared with the WT drR medaka. I have made two custom antibodies for the IGFBP5a in medaka and chum salmon respectively, but the antibodies for IGFBP5a were not desirable as the immunohistochemistry and Western blot results showed non-specific signals at the mucus. I have established a novel method to detect the transcript localization using in situ hybridization chain reaction (ISHCR). The method can distinguish closely resemble gene isoforms and I have demonstrated the distinct localization of Na-K-ATPase (NKA) alpha-1a and alpha-1b in chum salmon gill, which cannot be distinguished by conventional in situ hybridization method using cRNA probes. I have used the same method to study the localization of IGFBP5a in chum salmon ionocytes, and initial result suggested that the IGFBP5a is not colocalized with NKA, an observation different from that of zebrafish. I found a sequential increase in IGFBP5a expression during embryonic stages, in which the smoltification occurs in chum salmon. However, seawater treatment downregulated the IGFBP5a expression significantly in smolting chum salmon, an observation that is different from that of medaka.
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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
The making of the IGFBP5a-KO line in medaka was more challenging than expected as it is likely that the loss of IGFBP5a may have impacted the the growth and reproduction of the mutants. So far, few homologous individuals could be obtain and their fertility was poor, which contributing to the major obstacle in the progress of the project.
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Strategy for Future Research Activity |
I will continue to establish 5a-KO medaka and study the physiological characteristics of the 5a-KO on the osmoregulatory performance. After the establishment of the KO-line, I will perform RNA-seq analysis on the gills of wild-type and KO medaka in freshwater and seawater to examine the global influence of losing of IGFBP5a in the body system. I will perform STRING analysis to examine the protein-protein interaction to deduce what protein networks are influenced most by the 5a-KO during salinity acclimation. I consider remaking the chum salmon IGFBP5a antibody using a different peptide region or to purify the antibodies using affinity purification. I will extend the use of ISHCR to examine the localization and expression levels of IGFBP5a and other ionocyte markers in medaka and chum salmon.
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